Patent Application
20240032514
The sequence listing provided in the file entitled C6160-035_SQL_Rev2.xml, which is an Extensible Markup Language (XML) file that was created on Sep. 12, 2023, and which comprises 158,883 bytes, is hereby incorporated by reference in its entirety.
The present application relates to the field of biotechnology, in particular to a method for preparing a non-human mammal or progeny thereof, wherein the non-human mammal or progeny thereof can be used to produce a heavy chain antibody.
An antibody is a four-peptide-chain structure formed by connecting two identical heavy chains (H chains) and two identical light chains (L chains) by means of interchain non-covalent bonds or disulfide bonds. The heavy chain of antibody includes a heavy chain constant region (CH) and a heavy chain variable region (VH), wherein the heavy chain constant region of each of IgD, IgG and IgA includes four domains: CH1, a hinge region Hinge, CH2 and CH3; each of IgM and IgE includes fours domains: CH1, CH2, CH3 and CH4 (Janeway's Immunobiology, 9th Edition); the CH1 domain of the heavy chain constant region is connected to a light chain constant domain by means of a disulfide bond; the heavy chain variable region includes a hypervariable region of a complementary determining region (CDR) and a relatively conservative region called a framework region (FR); and the heavy chain variable region includes 3 CDRs and 4 FRs, which are arranged in the following order from an amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
According to differences in antigen specificity of the heavy chain constant regions of antibodies, the antibodies include the following five types: IgM, IgD, IgG, IgE and IgA. Since antibodies of the same class still have certain differences in antigen specificity in their heavy chain constant regions, they further can be divided into several isotypes, and for example, human IgG includes IgG1, IgG2, IgG3, IgG4, etc., and human IgA includes IgA1, IgA2, etc. The isotypes of antibodies of different mammals (such as humans, alpacas and mice) and of different varieties of mice (such as C57BL/6 mice and BALB/c mice) may differ.
A gene related to a heavy chain of an antibody includes five types of gene segments, i.e., L, V, D, J and C, wherein the heavy chain variable region is encoded by three types of gene segments, i.e., V, D and J, and the heavy chain constant region is encoded by the gene segment C. Antibody heavy chain genes of a human include about 40 V genes, 23 D genes, 6 J genes, and 9 C genes (source: Janeway's Immunobiology, 9th Edition, Page 177). A gene encoding an IgM heavy chain constant region is Ighm gene (Immunoglobulin heavy constant mu), a gene encoding an IgD heavy chain constant region is Ighd gene (Immunoglobulin heavy constant delta), a gene encoding an IgG heavy chain constant region is Ighg (Immunoglobulin heavy constant gamma), and a gene encoding an IgA heavy chain constant region is Igha (Immunoglobulin heavy constant alpha), etc. Correspondingly, a gene encoding an IgG1 heavy chain constant region is Ighg1 (Immunoglobulin heavy constant gamma 1), a gene encoding an IgG3 heavy chain constant region is Ighg3 (Immunoglobulin heavy constant gamma 3), and a gene encoding an IgG2b heavy chain constant region is Ighg2b (Immunoglobulin heavy constant gamma 2b). Diverse antibodies are prepared by means of the rearrangement of the heavy chain variable region. Mature B cells produce a secretory antibody IgM after being stimulated by an antigen, and second rearrangement occurs after antigen stimulation is performed again, and the class of immunoglobulin expressed and secreted on the membrane is switched from IgM with low affinity to IgG, IgA, IgE and other classes or isotypes of immunoglobulins. This phenomenon is called class switch or isotype switch.
A heavy chain antibody, also called a heavy chain only antibody, refers to an immunoglobulin antibody composed of only two heavy chains. Naturally occurring heavy chain antibodies are presented in camelids and sharks in the natural world. A heavy chain locus in a camelid germline contains a gene segment encoding a heavy chain constant region, and during maturation, and the rearranged VDJ-binding region is spliced to the 5′ terminus of a gene segment encoding an IgG hinge region, so that the heavy chain constant region lacks a CH1 region that mediates binding to a light chain and cannot bind to the light chain, resulting in the preparation of IgG2 and IgG3 heavy chain antibodies. Heavy chain antibodies can also be produced by using genetically modified animals, and the produced heavy chain antibodies are also classified according to the difference in antigen specificity of the heavy chain constant regions of antibodies.
Compared with the molecular weight (150-160 kDa) of a traditional antibody, the heavy chain antibody is much smaller. One heavy chain of an IgG2c heavy chain antibody is about 40 kDa, and its heavy chain variable region that determines the antigen recognition specificity is only about 15 kDa. The heavy chain antibody is characterized by small molecular weight, can bind to some hidden epitopes, and are particularly suitable for targets of antibodies which are difficult to obtain.
The information disclosed in the background art is only intended to enhance the understanding of the general background of the present application, and should not be considered to recognize or imply in any form that the information constitutes the prior art that is well known to a person skilled in the art.
An object of the present application is to provide a method for preparing a non-human mammal or progeny thereof, and the use thereof.
In order to achieve the object of the present application, the present application provides the following technical solutions.
In a first aspect, the present application provides a method for preparing a non-human mammal or progeny thereof, wherein the method comprises the following steps:
In a possible embodiment, according to the above-mentioned preparation method, making a CH1 domain of an IgM heavy chain constant region not expressed or incorrectly expressed in the non-human mammal is
In a possible embodiment, according to the above-mentioned preparing method, making one, two, three, four or more genes encoding an IgG heavy chain constant region in the non-human mammal not express or incorrectly express a CH1 domain during expression is
The present application further provides a non-human mammal in which a CH1 domain of an IgM heavy chain constant region is not expressed or incorrectly expressed and in which one, two, three, four or more genes encoding an IgG heavy chain constant region do not express or incorrectly express a CH1 domain.
In a possible embodiment, according to the above-mentioned non-human mammal, making a CH1 domain of an IgM heavy chain constant region not expressed or incorrectly expressed in the non-human mammal is
In a possible embodiment, according to the above-mentioned non-human mammal, making one, two, three, four or more genes encoding an IgG heavy chain constant region in the non-human mammal not express or incorrectly express a CH1 domain is
In a second aspect, the present application provides a method for preparing a non-human mammal or progeny thereof, wherein the method comprises the following steps:
The present application further provides a non-human mammal, wherein a nucleotide sequence, comprising a nucleotide sequence encoding a CH1 domain of an IgM heavy chain constant region, and a target gene on a genome of the non-human mammal are knocked out. The target gene comprises:
In a possible embodiment, according to the above-mentioned preparation method or non-human mammal, knocking out a nucleotide sequence, comprising a nucleotide sequence encoding a CH1 domain of an IgM heavy chain constant region, on the genome of the non-human mammal is
In a possible embodiment, according to the above-mentioned preparation method or non-human mammal, the target gene is
In a possible embodiment, according to the above-mentioned preparation method, the following steps are completed in the same operation step or in different operation steps:
In a possible embodiment, according to the above-mentioned preparation method or non-human mammal, the non-human mammal is a rodent; optionally, the rodent is a rat or a mouse; further optionally, the rodent is a mouse; and still further, the mouse is a C57BL/6 mouse or a BALB/c mouse.
In a possible embodiment, according to the above-mentioned preparation method or non-human mammal, the first gene encoding the IgG heavy chain constant region is Ighg3.
In a possible embodiment, according to the above-mentioned preparation method or non-human mammal, when the non-human mammal is a C57BL/6 mouse, the first gene encoding the IgG heavy chain constant region is Ighg3, the second gene encoding the IgG heavy chain constant region is Ighg1, the third gene encoding the IgG heavy chain constant region is Ighg2b, and the fourth gene encoding the IgG heavy chain constant region is Ighg2c; and
In a possible embodiment, according to the above-mentioned preparation method or non-human mammal, the genome of the non-human mammal comprises a complete gene encoding a κ light chain and/or a λ light chain; and optionally, the κ light chain and/or the λ light chain can be normally expressed in the non-human mammal.
In a possible embodiment, according to the above-mentioned preparation method or non-human mammal, a gene knockout method comprises one or more of the followings: a gene targeting technology, a CRISPR/Cas9 method, a zinc finger nuclease method and a transcription activator-like effector nuclease method.
In a possible embodiment, according to the above-mentioned preparation method or non-human mammal, the non-human mammal or progeny thereof is used to produce a heavy chain antibody.
In a third aspect, the present application provides a method for preparing a C57BL/6 mouse or progeny thereof, wherein the method comprises the following steps:
The present application further provides a C57BL/6 mouse, wherein nucleotide sequences, which encode heavy chain constant regions of antibodies IgM, IgD, IgG1, IgG2b and IgG3, in a genome of the mouse and a nucleotide sequence, encoding a CH1 domain, on a gene encoding an antibody IgG2c heavy chain constant region, in the genome of the mouse are knocked out.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, the genome of the C57BL/6 mouse comprises a complete gene encoding a κ light chain and/or an λ light chain; and optionally, the C57BL/6 mouse can normally express the κ light chain and/or the λ light chain.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, a gene knockout method comprises one or more of the followings: a gene targeting technology, a CRISPR/Cas9 method, a zinc finger nuclease method and a transcription activator-like effector nuclease method.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, all nucleotide sequences from the first exon of the gene encoding the IgM heavy chain constant region to the first exon of the gene encoding the IgG2c heavy chain constant region in the mouse are knocked out.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, in the gene knockout step, sgRNA targeting the upstream portion of the first exon of the gene encoding the IgM heavy chain constant region in the mouse and sgRNA targeting the downstream portion of the first exon of the gene encoding the IgG2c heavy chain constant region in the mouse are a small a step.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, a targeting sequence, which is targeted by sgRNA, on the upstream portion of the first exon of the gene encoding the IgM heavy chain constant region in the mouse comprises SEQ ID NO. 1 and SEQ ID NO. 2; and/or a targeting sequence, which is targeted by sgRNA, on the downstream portion of the first exon of the gene encoding the IgG2c heavy chain constant region in the mouse comprises SEQ ID NO. 3 and SEQ ID NO. 4.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, the sgRNA targeting the upstream portion of the first exon of the gene encoding the IgM heavy chain constant region in the mouse is SEQ ID NO. 9 and SEQ ID NO. 10; and/or the sgRNA targeting the downstream portion of the first exon of the gene encoding the IgG2c heavy chain constant region in the mouse is SEQ ID NO. 11 and SEQ ID NO. 12.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, the C57BL/6 mouse or progeny thereof is used to produce a heavy chain IgG2c antibody.
In a fourth aspect, the present application provides a method for preparing a C57BL/6 mouse or progeny thereof, wherein the method comprises the following steps:
The present application further provides a C57BL/6 mouse, a nucleotide sequence, which encodes the CH1 domain, on a gene encoding an IgM heavy chain constant region in a genome in the mouse and a nucleotide sequence, encoding a CH1 domain, on a region starting from a gene encoding an IgG3 heavy chain constant region to a gene encoding an IgG2c heavy chain constant region in a genome in the mouse are knocked out.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, the genome of the C57BL/6 mouse comprises a complete gene encoding a κ light chain and/or an λ light chain; and optionally, the C57BL/6 mouse can normally express the κ light chain and/or the λ light chain.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, a gene knockout method comprises one or more of the followings: a gene targeting technology, a CRISPR/Cas9 method, a zinc finger nuclease method and a transcription activator-like effector nuclease method.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, in the gene knockout step, the first exon of the gene encoding the IgM heavy chain constant region in the mouse is knocked out, and all nucleotide sequences from the first exon of the gene encoding the IgG3 heavy chain constant region to the first exon of the gene encoding the IgG2c heavy chain constant region in the mouse are further knocked out.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, in the gene knockout step, sgRNA targeting the upstream and downstream portions of the first exon of the gene encoding the IgM heavy chain constant region in the mouse is used, and sgRNA targeting the upstream portion of a first exon of a gene encoding an IgG3 heavy chain constant region in a mouse and sgRNA targeting the downstream portion of the first exon of the gene encoding IgG2c in the mouse are further used.
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, a targeting sequence, which is targeted by sgRNA, on the upstream portion of the first exon of the gene encoding the IgM heavy chain constant region in the mouse comprises SEQ ID NO. 1 and SEQ ID NO. 2; and/or
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, the sgRNA targeting the upstream portion of the first exon of the gene encoding the IgM heavy chain constant region in the mouse is SEQ ID NO. 9 and SEQ ID NO. 10; and/or
In a possible embodiment, according to the above-mentioned preparation method or C57BL/6 mouse, the C57BL/6 mouse or progeny thereof is used to produce a heavy chain IgG2c antibody.
In a fifth aspect, the present application provides a method for preparing a non-human mammal or progeny thereof, wherein the method comprises knocking out a nucleotide sequence, encoding a CH1 domain of an IgM heavy chain constant region, on a genome of the non-human mammal.
The present application further provides a non-human mammal, wherein a nucleotide sequence, encoding a CH1 domain of an IgM heavy chain constant region, on a genome of the non-human mammal is knocked out.
In a possible embodiment, according to the above-mentioned preparation method or non-human mammal, the non-human mammal is a rodent; optionally, the rodent is a rat or a mouse; further optionally, the rodent is a mouse; and still further, the mouse is a C57BL/6 mouse or a BALB/c mouse.
In a possible embodiment, according to the above-mentioned preparation method or non-human mammal, the non-human mammal or progeny thereof is used to construct the above-mentioned non-human mammal or progeny thereof.
In a sixth aspect, the present application provides the use of a non-human mammal or offspring thereof constructed by using the above-mentioned preparation method, a C57BL/6 mouse or offspring thereof constructed by using the above-mentioned preparation method, the above-mentioned non-human mammal and the above-mentioned C57BL/6 mouse in the screening of a target heavy chain antibody.
In a possible embodiment, according to the use, a phage display method is used when the target heavy chain antibody is screened.
In a possible embodiment, according to the use, the screening of the target heavy chain antibody is the screening of a C-reactive coronavirus S protein or coronavirus N protein antigen-specific IgG2c heavy chain antibody.
In a seventh aspect, the present application provides a method for screening a target heavy chain antibody, wherein the method comprises performing screening by using a non-human mammal or progeny thereof constructed by using the preparation method derived from the above-mentioned first aspect, a non-human mammal or progeny thereof constructed by using the preparation method derived from the above-mentioned the second aspect, a C57BL/6 mouse or progeny thereof constructed by the preparation method derived from the above-mentioned third aspect, or a C57BL/6 mouse or progeny thereof constructed by using the preparation method derived from the above-mentioned fourth aspect as an immune animal.
In a possible embodiment, according to the above-mentioned method, a phage display method is used when the target heavy chain antibody is screened.
In a possible embodiment, according to the method, the screening of the target heavy chain antibody is the screening of a C-reactive coronavirus S protein or coronavirus N protein antigen-specific IgG2c heavy chain antibody.
In an eighth aspect, the present application provides a non-human mammal cell or cell line or primary cell culture, wherein the non-human mammal cell or cell line or primary cell culture is derived from a non-human mammal or progeny thereof constructed by using the above-mentioned preparation method, or the C57BL/6 mouse or progeny thereof constructed by using the above-mentioned preparation method.
In a ninth aspect, the present application provides an in-vitro tissue or in-vitro organ or a culture thereof, the in-vitro tissue or in-vitro organ or the culture thereof is derived from the non-human mammal or progeny thereof constructed by using the above-mentioned preparation method, or the C57BL/6 mouse or progeny thereof constructed by using the above-mentioned preparation method.
In the tenth aspect, the present application provides an sgRNA composition, wherein the sgRNA composition comprises: sgRNA targeting the upstream portion of the first exon of the gene encoding the IgM heavy chain constant region in the mouse and sgRNA targeting the downstream portion of the first exon of the gene encoding the IgG2c heavy chain constant region in the mouse;
In a possible embodiment, according to the sgRNA composition, a targeting sequence, which is targeted by sgRNA, on the upstream portion of the first exon of the gene encoding the IgM heavy chain constant region in the mouse comprises SEQ ID NO. 1 and SEQ ID NO. 2; and/or
In a possible embodiment, according to the sgRNA composition, the sgRNA targeting the upstream portion of the first exon of the gene encoding the IgM heavy chain constant region in the mouse is SEQ ID NO. 9 and SEQ ID NO. 10; and/or
In an eleventh aspect, the present application provides a knockout vector. The knockout vector comprises one or more segments of DNA sequences encoding sgRNA, wherein the targeting sequence targeted by the sgRNA is selected from one of the followings:
In a possible embodiment, according to the above-mentioned knockout vector, a backbone of the knockout vector is an sgRNA expression vector.
In a twelfth aspect, the present application provides a cell containing the above-mentioned knockout vector.
Incorrect expression: incorrect expression is opposite to correct expression, and both correct expression and incorrect expression in the present application actually have meanings commonly understood in the art. For example, incorrectly expressing a CH1 domain refers to that mutation or deletion at the genomic level causes incorrect expression of a CH1 domain of a heavy chain constant region, which leads to the CH1 domain losses the ability to bind to a light chain; and for example, mutation or deletion occurs in a range of 10 nucleotides on each of the upstream and downstream portion of an exon encoding a CH1 domain of a heavy chain constant region, which leads to the CH1 domain losses the ability to bind to a light chain. For another example, a gene encoding an IgG heavy chain constant region incorrectly expresses the IgG heavy chain constant region encoded thereby during expression, which means that the immunoglobulin antibody expressed by the gene does not have antibody effects; and by contrast, a gene encoding an IgG heavy chain constant region correctly expresses the encoded IgG heavy chain constant region during expression, which means that the immunoglobulin antibody expressed by the gene has antibody effects;
In the present application, by means of making a CH1 domain of an IgM heavy chain constant region not expressed or incorrectly expressed in a non-human mammal and making one or more genes encoding an IgG heavy chain constant region not express or incorrectly express a CH1 domain during expression, the non-human mammal or progeny thereof is prepared and can be used for preparing a heavy chain antibody. The non-human mammal obtained by using the preparation method of the present application does not introduce any exogenous gene encoding heavy chain variable and constant regions of an antibody, and can directly use all VDJ genes encoding the heavy chain variable region of the antibody in its own genome, so as to produce more diverse heavy chain antibodies by means of the rearrangement of the heavy chain variable region.
In immunology, it is generally believed that IgM is extremely important for the growth of B cells, whereas the growth of B cells is also extremely important for the preparation of the antibody. In the present application, it is verified through experiments that even if a CH1 domain of an IgM heavy chain constant region is not expressed and encoded or even a gene encoding an IgM heavy chain constant region and a gene encoding an IgD heavy chain constant region are deleted, the immunologic maturation of a non-human mammal is not significantly affected, all mice survive normally, high-titer immune response still can be produced, and a heavy chain antibody with strong specificity and high affinity can be obtained therefrom by means of screening.
By selecting a genetically modified gene encoding an IgG heavy chain constant region, a specific isotype of heavy chain IgG antibody can be obtained.
One or more examples are illustrated by means of figures in the drawings corresponding to the examples, and these illustrations are not intended to limit the examples. The word “exemplary” used exclusively herein means “serving as an example, embodiment or illustration”. Any embodiment illustrated herein as “exemplary” need not be construed as being superior or better than other embodiments.
In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Obviously, the embodiments described are some of, rather than all of, the embodiments of the present application. All the other examples obtained by a person of ordinary skill in the art on the basis of the examples of the present application without any creative effort shall fall within the scope of protection of the present application.
In addition, in order to better illustrate the present application, numerous specific details are given in the detailed description of embodiments below. It should be understood by a person skilled in the art that the present application is also implementable, without certain specific details. In some embodiments, materials, components, methods, means, etc. that are well known to a person skilled in the art are not described in detail, so as to highlight the gist of the present application.
Unless expressly stated otherwise, throughout the description and claims, the term “comprise” or variants thereof such as “contains” or “comprises” will be understood as including the stated elements or components, but not excluding other elements or components.
The experimental materials used in the following examples and their sources are as follows:
Part I. Ighm-d-g Homozygous Mice and Immunization Results Thereof
Ighm-d-g mice referred to C57BL/6 mice in which Ighm genes, Ighd genes, Ighg3 genes, Ighg1 genes, Ighg2b genes and first exons on Ighg2c gene were knocked out, wherein the first exons were responsible for encoding CH1 domains of IgG2c heavy chains. Ighm-d-g homozygous mice were produced by means of the following steps:
(1) Acquisition of Nucleic Acid Molecules:
A schematic diagram of gene loci of heavy chain variable and constant regions of a C57BL/6 mouse antibody was as shown in
(2) Construction of Nucleic Acid Molecule into Backbone Plasmid and In-Vitro Transcription to Obtain sgRNA:
The synthesized forward and reverse DNA oligos encoding an sgRNA sequence formed complementary double strands by means of annealing, the double strands were connected to an sgRNA expression vector (px330) by using T4 ligase, and after the connection, the resulting product was sequenced and verified by a professional sequencing company. The results showed that a target plasmid was obtained, and sgRNA was further obtained by in-vitro transcription.
(3) Introduction of sgRNA and Cas9 Proteins into Fertilized Eggs of Host Animals:
The mice were subjected to ovulation induction and in-vitro fertilization, and the obtained fertilized eggs were cultivated. Then, the sgRNA and Cas9 proteins (or Cas9 mRNA, commercially available) were mixed and electroporated the fertilized eggs of the mice or injected together into the fertilized eggs of the mice by means of a microinjection method.
(4) Implantation of Cells Containing Above-Mentioned sgRNA and Cas9 Proteins into Host Animals:
The above-mentioned fertilized egg cells were implanted into surrogate mother mice, and F0-generation chimeric mice can be produced. Individuals in which knockout occurred among the F0-generation mice were detected by extracting mouse tail genomic DNA and performing PCR detection. The knockout mice were sequenced so as to confirm that target sequences were deleted. F0-generation chimeric mice in which genes were correctly knocked out were selected for subsequent propagation and identification.
The genes which are knocked out can be detected by using PCR primers Ighm-d-g-1F and Ighm-d-g-1R (as indicated by arrows in
(5) Propagation of Heterozygous and Homozygous Knockout Mice:
Ighm-d-g homozygous mice were immunized with a human C-reactive protein (CRP).
An immunization method was as follows: 6- to 8-week-old male mice were selected, and the antigen human C-reactive protein (CRP, A-5172, Beijing Biobridge Biotechnology Co., Ltd) was emulsified with a freund's complete adjuvant (F5881, Sigma) with the equal volume to a state of no dripping and then was used for subcutaneous multi-point injection for first immunization of mice, wherein the injected dose was 100 μg/mouse in the first immunization. After the first immunization, subsequent subcutaneous immunization was performed every 2 weeks, wherein the CRP antigen was emulsified with a Freund's incomplete adjuvant (F5506, Sigma) and then was used for subcutaneous multi-point injection of mice, and the injected dose was 100 μg/mouse every time.
A method for determining a serum titer was as follows: a CRP antigen was diluted to 2 μg/mL, 100 μL of diluted antigen was taken and added to a polystyrene enzyme-linked assay plate, the plate was covered, and a specific IgG2c antibody heavy chain that specifically binds to the CRP antigen in serum was detected by using HRP-goat anti-mouse IgG-Fc (Jackson 115-035-071).
As shown in
Following example 2 above, the mice were euthanized by causing asphyxiation using carbon dioxide, and dissected to observe the thymus glands, spleens, mesenteric lymph nodes, submaxillary lymph nodes, etc. and the sizes and shapes of the heart, liver, lung, kidney and other organs. The dissection results showed that the thymus glands, mesenteric lymph nodes, submaxillary lymph nodes and major organs of unimmunized Ighm-d-g homozygous mice and antigen-immunized Ighm-d-g homozygous mice all had no obvious abnormalities.
Following example 2 above, Western blot was performed on the serum of the CRP antigen-immunized mice: 2 μL of serum was taken from the immunized mice, 100 μL of PBS was added, the resulting mixture was reacted with 10 μL of CRP antigen-Sepharose packings at room temperature for 60 min, then the resulting product was centrifuged at 6000 rpm for 30 s, and the supernatant was discarded; and the packings were washed three times with PBS, resuspended with L of PBS, boiled, electrophoresed by using 12% SDS-PAGE, transferred to a PVDF membrane, and then reacted with an HRP-goat anti-mouse IgG-Fc (Jackson 115-035-071, for detection of a heavy chain) antibody and an HRP-goat anti-mouse Ig light chain (Jackson, 115-035-174) antibody respectively, and further color development was performed.
According to the design of the present application, since the IgM, IgD, IgG1, IgG2b and IgG3 genes in the mice were knocked out, only an IgG2c isotype antibody can be produced by using antigen protein-immunized Ighm-d-g homozygous mice, and since the CH1 gene of the IgG2c heavy chain was knocked out, an IgG2c heavy chain antibody (free of a CH1 domain) can be produced by using the antigen protein-immunized Ighm-d-g homozygous mice, and the molecular weight of one IgG2c heavy chain alone was about 40 KD. The antibody produced by the antigen protein-immunized Ighm-d-g homozygous mice was subjected to separation, electrophoresis, color development and other steps, then a band that specifically binds to the antigen CRP existed around a band of about 80 KD, which is consistent with the theoretical dimer molecular weights of the IgG2c heavy chain, and a dimer not bound to an HRP-goat anti-mouse Ig light chain (Jackson, 115-035-174) antibody was included.
Following example 2 above, the Ighm-d-g homozygous mice were immunized with a CRP antigen and the serum titer was determined. Then, the mice were euthanized, splenocytes were taken and lysed with Trizol, total RNA was extracted and reversely transcribed to obtain cDNA, and a heavy chain variable region and a heavy chain constant region connected thereto were amplified by PCR by using the following IgG2c isotype antibody-specific primers.
The obtained PCR amplification product was connected with a pEASY-T5 Zero Cloning vector, transformed TOP10 competent cells and coated an LB plate. 11 clones were randomly selected, and the clones identified as positive by means of colony PCR were sent for sequencing (as shown in
The sequencing results of the positive clones showed (as shown in
I. Phage Display Library Construction
Following example 2 above, total RNA was extracted from the spleens of CRP-immunized Ighm-d-g homozygous mice, cDNA (a TAKAR 6110A cDNA synthesis kit) was prepared by using oligo (Dt), and heavy chain antibody genes were obtained by two rounds of PCR amplification using nested PCR.
The first round of PCR process was exactly the same as the PCR process mentioned in example 5; and
The reaction system was as shown in Table 4:
The PCR amplification product was digested with SfiI (FD1824, Thermo) and subjected to agarose gel electrophoresis, the target product was subjected to gel extraction, and then the target fragment subjected to gel extraction was cloned into a pComb3XSS vector digested with Sfi. R2738 electrocompetent cells were electroporated. An IgG2c heavy chain antibody library was prepared and multiple rounds of panning were performed. Multiple positive clones were selected and sent for sequencing identification, and the results were as shown in
II. Panning
1. Panning
2. Phage Amplification and Purification
3. Titer Determination
III. An IgG2c Heavy Chain Antibody Variable Region Library was Prepared and Subjected to Five Rounds of Panning, and the Results were as Shown in Table 5 Below:
A total of 5 rounds of panning were performed; as can be seen from the table above, the output phages were significantly enriched; and positive clones were selected by means of PHAGE-ELISA experiments and sent for sequencing identification.
Following example 6 above, the sequences for sequencing were analyzed, and D4-12 (the nucleotide sequence and amino acid sequence were as shown in Table 6) that appeared repeatedly in the first five rounds of panning was selected to transfect BL21 Escherichia coli, Different concentrations of IPTG were added at 16° C., prokaryotic expression was induced, ultrasonication was performed, and the supernatant was collected and purified by means of a nickel affinity column.
A specific antigen CRP and an unrelated protein OVA (2 μg/mL) were coated, gradient dilution was performed by using purified D4-12, and ELISA detection was performed. The results showed that a variable region of the purified D4-12 heavy chain antibody can specifically recognize and bind to the antigen CRP and showed a good concentration dependence, as shown in
Following example 6 above, the sequences for sequencing were further analyzed, and the sequence D5S-12 (the nucleotide sequence and amino acid sequence were as shown in Table 7) with the most repetitions was selected for eukaryotic expression and biological activity identification. KOP293 cells (Zhuhai Kairui) were transfected, the supermatant of cultured cells was collected on day 6, and a target protein was purified by means of a nickel affinity column.
1. A specific antigen CRP and an unrelated protein OVA or BSA (2 μg/mL) were coated, and the purified D5S-12 antibody was subjected to gradient dilution and detected by means of ELISA. The results showed that the purified D5S-12 heavy chain antibody can specifically recognize and bind to the antigen CRP and showed a good concentration dependence, as shown in
2. Determination of affinity of D5S-12 heavy chain antibody: The affinity of the purified D5S-12 heavy chain antibody to the antigen CRP was determined by using FORTEBIO-OCTET, the antibody was diluted to 10 μg/mL and the antigen CRP was diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.13 nM. The affinity constants of the antibody were determined as KD(M)=9.87E-10, Kon(1/Ms)=8.55E+04 and Koff(1/s)=8.44E-05, and the heavy chain antibody D5S-12 had high affinity to the antigen CRP. The results were as shown in
Part II. Preparation of IghM-3G3 Homozygous Mice and Immunization Results Thereof
IghM-3G3 mice were C57BL/6 mice in which the first exon on the Ighm gene was knocked out, and the Ighg3 gene, the Ighg1 gene, the Ighg2b gene and the first exon located on the Ighg2c gene were knocked out. IghM homozygous mice (i.e., C57BL/6 mice where the first exon on the Ighm gene was knocked out) were produced first, and then IghM-3G3 homozygous mice were produced on the basis of the IghM homozygous mice.
Preparation of IghM Homozygous Mice Comprises the Following Steps:
(1) Acquisition of Nucleic Acid Molecules:
A schematic diagram of the genetic loci of the heavy chain variable and constant regions of a C57BL/6 mouse antibody was as shown in
In order to knock out the nucleotide sequence encoding the CH1 domain in the mouse Ighm gene, targets were selected on the upstream and downstream portions of the first exon in the mouse Ighm gene. Targeting sequences (SEQ ID NO: 1 and SEQ ID NO: 2) for sgRNA were selected on the upstream portion of the first exon of the Ighm gene in the mouse, targeting sequences (SEQ ID NO: 5 and SEQ ID NO: 6) for sgRNA were selected on the downstream portion of the first exon of Ighm in the mouse, and sgRNA sequences were designed according to the targeting sequences. The details were as shown in Table 8.
(2) Construction of Nucleic Acid Molecule into Backbone Plasmid and In-Vitro Transcription to Obtain RNA:
The synthesized forward and reverse DNA oligos for an sgRNA sequence formed complementary double strands by means of annealing, the double strands were connected to an sgRNA expression vector (px330) by using T4 ligase, and after the connection, the resulting product was sequenced and verified by a professional sequencing company. The results showed that a target plasmid was obtained, and sgRNA was further obtained by in-vitro transcription.
(3) Introduction of sgRNA and Cas9 Proteins into Fertilized Eggs of Host Animals:
The mice were subjected to ovulation induction and in-vitro fertilization, and the obtained fertilized eggs were cultivated. Then, the sgRNA and Cas9 proteins (or Cas9 mRNA, commercially available) were mixed and electroporated the fertilized eggs of the mice or injected together into the fertilized eggs of the mice by means of a microinjection method.
(4) Implantation of Cells Containing Above-Mentioned sgRNA and Cas9 Proteins into Surrogate Animals:
The above-mentioned fertilized egg cells were implanted into surrogate mother mice, and F0-generation chimeric mice can be produced. Individuals in which knockout occurred among the F0-generation mice were detected by extracting mouse tail genomic DNA and performing PCR detection. The knockout mice were sequenced so as to confirm that target sequences were deleted. F0-generation chimeric mice in which genes were correctly knocked out were selected for subsequent propagation and identification.
The genes which are knocked out (as indicated by arrows in
The test results were as shown in
(5) Propagation of Heterozygous and Homozygous Knockout Mice:
the F0-generation mice in which target genes were knocked out were mated with wild-type mice to obtain F1-generation mice, and by extracting genomes of mouse tails and performing PCR detection, F1-generation positive heterozygous knockout mice that can be stably inherited were selected. Then, the F1-generation heterozygous mice were mated with each other to obtain F2-generation positive homozygous knockout mice, i.e., IghM homozygous mice. The method of identifying the genotypes of the obtained F1-generation heterozygous or F2-generation homozygous mice were the same as that in step 4.
Following example 2 above, IghM homozygous mice were immunized with a CRP antigen and then euthanized, splenocytes were taken and lysed with Trizol, total RNA was extracted and reversely transcribed to obtain cDNA, and a heavy chain variable region and a heavy chain constant region connected thereto were amplified by PCR by using the IgM isotype antibody-specific primers. The primers used and sequences thereof were as follows: See example 5 for MHV1, MHV2, MHV3, MHV4, MHV5, MHV6, MHV7, MHV8, MHV9, MHV10, MHV11 and MHV12; and
The PCR reaction system was as shown in Table 10:
The PCR amplification product was connected to a pEASY-T5 Zero Cloning vector and transfected TOP10 competent cells, and the transfected cells were used to coat an LB plate (amphicillin-resistant), and the clones were picked for colony PCR. The positive clones were sent for sequencing. As can be ween from the sequencing results, FR4 in the heavy chain variable region of the IgM antibody expressed in the IghM homozygous mice was directly connected to CH2 (starting amino acid sequence AVAEMN). The results as were shown in
A method for preparing IghM-3G3 homozygous mice, comprises the following steps:
(1) Acquisition of Nucleic Acid Molecules:
A schematic diagram of the gene loci of the heavy chain variable region and heavy chain constant region of a C57BL/6 mouse antibody was as shown in
In order to knock out the first exon encoding a CH1 domain on the mouse Ighm, the Ighg3, Ighg1 and Ighg2b genes and the first exon encoding a CH1 domain on Ighg2c, on the basis of the IghM homozygous mice, targeting sequences (SEQ ID NO: 7 and SEQ ID NO: 8) for sgRNA were selected on the upstream portion of the first exon of the Ighg3 gene in the mouse, targeting sequences (SEQ ID NO: 3 and SEQ ID NO: 4) for sgRNA were selected on the downstream portion of the first exon of Ighg2c in the mouse, and sgRNA sequences were designed according to the targeting sequences. The details were as shown in Table 11.
(2) Construction of Nucleic Acid Molecule into Backbone Plasmid and In-Vitro Transcription to Obtain RNA:
The synthesized forward and reverse DNA oligos for an sgRNA sequence formed complementary double strands by means of annealing, the double strands were connected to an sgRNA expression vector (px330) by using T4 ligase, and after the connection, the resulting product was sequenced and verified by a professional sequencing company. The results showed that a target plasmid was obtained, and sgRNA was further obtained by in-vitro transcription.
(3) Introduction of sgRNA and Cas9 Proteins into Fertilized Eggs of Host Animals:
The mice were subjected to ovulation induction and in-vitro fertilization, and the obtained fertilized eggs were cultivated. Then, the sgRNA and Cas9 proteins (or Cas9 mRNA, commercially available) were mixed and electroporated the fertilized eggs of the mice or injected together into the fertilized eggs of the mice by means of a microinjection method.
(4) Implantation of Cells Containing Above-Mentioned sgRNA and Cas9 Proteins into Host Animals:
The above-mentioned fertilized egg cells were implanted into surrogate mother mice, and F0-generation chimeric mice can be produced. Individuals in which knockout occurred among the F0-generation mice were detected by extracting mouse tail genomic DNA and performing PCR detection. The knockout mice were sequenced so as to confirm that target sequences were deleted. F0-generation chimeric mice in which genes were correctly knocked out were selected for subsequent propagation and identification.
The genes which are knocked out can be detected by using PCR primers Ighg-1F and Ighg-1R, and the wild-type genes can be detected by using primers Ighg-2R and Ighg-1F (arrows in
IghM-3G3 mice were subjected to genotype identification by means of PCR (
(5) Propagation of Heterozygous and Homozygous Knockout Mice:
the F0-generation mice in which target genes were knocked out were mated with wild-type mice to obtain F1-generation mice, and by extracting genomes of mouse tails and performing PCR detection, F1-generation positive heterozygous knockout mice that can be stably inherited were selected. Then, the F1-generation heterozygous mice were mated with each other to obtain F2-generation positive homozygous knockout mice, i.e., IghM-3G3 homozygous mice. The method of identifying the genotypes of the obtained F1-generation heterozygous or F2-generation homozygous mice were the same as that in step 4.
IghM-3G3 homozygous mice were immunized with a human C-reactive protein (CRP).
An immunization method was as follows: 6- to 8-week-old male mice were selected, and the antigen human C-reactive protein (CRP, A-5172, Beijing Biobridge Biotechnology Co., Ltd) was emulsified with a freund's complete adjuvant (F5881, Sigma) with made a state of no dripping and then was used for subcutaneous multi-point injection for first immunization of mice, wherein the injected dose was 100 μg/mouse in the first immunization. After the first immunization, subsequent subcutaneous immunization was performed every 2 weeks, wherein the CRP antigen was emulsified with a Freund's incomplete adjuvant (F5506, Sigma) and then was used for subcutaneous multi-point injection of mice, and the injected dose was 100 μg/mouse every time.
Serum Titer Determination:
A method for determining a serum titer was as follows: a CRP antigen was diluted to 2 μg/mL, 100 μL of diluted antigen was taken and added to a polystyrene enzyme-linked assay plate, the plate was covered, and a specific IgG antibody that specifically binds to the CRP antigen in serum was detected by using HRP-goat anti-mouse IgG-Fc (Jackson, 115-035-071).
As shown in
The IghM-3G3 homozygous mice were immunized with a CRP antigen and the serum titer was determined. Then, the mice were euthanized, splenocytes were taken and lysed with Trizol, total RNA was extracted and reversely transcribed to obtain cDNA, and a heavy chain variable region and a heavy chain constant region connected thereto were amplified by PCR by using the following IgG2c isotype antibody-specific primers.
See example 5 for MHV1, MHV2, MHV3, MHV4, MHV5, MHV6, MHV7, MHV8, MHV9, MHV10, MHV11, MHV12 and B6_IgG2c_CH2R1.
The PCR reaction system and the PCR reaction process were as shown in example 5.
The obtained PCR amplification product was connected with a pEASY-T5 Zero Cloning vector, transformed TOP10 competent cells and coated an LB plate. 7 clones were randomly selected, and the clones identified as positive by means of colony PCR were sent for sequencing (as shown in
The sequencing results of the positive clones showed (as shown in
Following example 13 above, the steps for phage display library construction and antibody panning were exactly the same as those in example 6, only the parameters in Table 13 were adjusted, and the results obtained after 5 rounds of panning were as shown in Table 13 below:
A total of 5 rounds of panning were performed; as can be seen from Table 13, the output phages were significantly enriched; and positive clones were selected by means of PHAGE-ELISA experiments and sent for sequencing identification, as shown in Table 14.
EKDYWGQGTILTVSS (SEQ ID NO: 47)
EKDYWGQGTTLTVSS (SEQ ID NO: 48)
EKDYWGQGTILTVSS (SEQ ID NO: 49)
Example 15. Antigen Immunization and Titer Determination for IghM-3G3 Homozygous Mice
In this example, IghM-3G3 homozygous mice were immunized with a coronavirus S protein antigen.
An immunization method was as follows: 6- to 8-week-old male mice were selected, and the antigen coronavirus S protein (SARS-CoV-2 (2019-nCoV) Spike S1-His Recombinant Protein, 40591-V08H, Sino Biological Inc.) was emulsified with a freund's complete adjuvant (F5881, Sigma) with made a state of no dripping and then was used for subcutaneous multi-point injection for first immunization of mice, wherein the injected dose was 100 μg/mouse in the first immunization. After the first immunization, subsequent subcutaneous immunization was performed every 2 weeks, and the injected dose was 100 μg/mouse every time.
Serum Titer Determination:
A method for determining a serum titer was as follows: a coronavirus S protein antigen was diluted to 2 μg/mL, 100 μL of diluted antigen was taken and added to a polystyrene enzyme-linked assay plate, the plate was covered, and a specific IgG antibody that specifically binds to the coronavirus S protein antigen in serum was detected by using HRP-goat anti-mouse IgG-Fc (Jackson, 115-035-071).
Blood was collected when the mice was unimmunized and one week after the second immunization, one week after the third immunization and one week after the fourth immunization. The serum was subjected to doubling dilution with PBS, starting from a dilution degree of 1:500. The serum titer was detected by ELISA, and the results were as shown in
According to example 15 above, the IghM-3G3 homozygous mice were immunized with an antigen coronavirus S protein and the serum titer was determined. Then, the mice were euthanized, splenocytes were taken and lysed with Trizol, total RNA was extracted and reversely transcribed to obtain cDNA, and a heavy chain variable region and a heavy chain constant region connected thereto were amplified by PCR by using the following IgG2c isotype antibody-specific primers.
See example 5 for MHV1, MHV2, MHV3, MHV4, MHV5, MHV6, MHV7, MHV8, MHV9, MHV10, MHV11, MHV12 and B6_IgG2c_CH2 R1.
The PCR reaction system and the PCR reaction process were as shown in example 5.
The obtained PCR amplification product was connected with a pEASY-T5 Zero Cloning vector, transformed TOP10 competent cells and coated an LB plate. 23 clones were randomly selected, and the clones identified as positive by means of colony PCR were sent for sequencing (PCR results were as shown in
Following example 16 above, the steps for phage display library construction and antibody panning were exactly the same as those in example 6. A total of 5 rounds of panning were performed; the output phages were significantly enriched every time; and positive clones were selected by means of PHAGE-ELISA experiments and sent for sequencing identification, as shown in Table 15. The similarities and differences of the CDR regions of these sequences were analyzed, and the results were as shown in
The sequences for sequencing were analyzed, and the S-9, S-19, S-27 and S-47 (amino acid sequences were as shown in Table 15) that appeared in the first 5 rounds of panning were selected to transfect BL21 Escherichia coli. Different concentrations of IPTG were added at 30° C., prokaryotic expression was induced, ultrasonication was performed, and the supernatant was collected and purified by means of a nickel affinity column.
A specific antigen coronavirus S protein and an unrelated protein OVA (2 μg/mL) were coated, and the purified S-9, S-19, S-27 and S-47 antibodies were subjected to gradient dilution and detected by means of ELISA. The results showed that the variable region of the heavy chain antibody extracted this time can specifically recognize and bind to the antigen coronavirus S protein, and showed a good concentration dependence, as shown in
In this example, IghM-DG1 homozygous mice were immunized with a coronavirus S protein antigen.
The mouse antigen immunization operation and the step for serum titer determination were exactly the same as those in example 15, and the results of the serum titer determination were as shown in
The sequences for sequencing were analyzed, and DNA sequences of the S-1, S-2, S-7, S-12, S-17, S19 and S-65 antibodies (amino acid sequences were as shown in table 16) that appeared in the first 5 rounds of panning were selected, cloned into a PET28 vector and transfected BL21 Escherichia coli. Different concentrations of IPTG were added at 16° C., prokaryotic expression was induced, ultrasonication was performed, and the supernatant was collected and purified by means of a nickel affinity column.
A specific antigen coronavirus S protein and an unrelated protein OVA (2 μg/mL) were coated, and the purified S-1, S-7, S-12, S-17, S19, S-25, S-51 and S-65 antibodies were subjected to gradient dilution and detected by means of ELISA. The results showed that the variable region of the heavy chain antibody extracted this time can specifically recognize and bind to the antigen coronavirus S protein, and showed a good concentration dependence, as shown in
In this example, MDG1 homozygous mice were immunized with an antigen coronavirus N protein.
The immunization method and the method for serum titer determination were exactly the same as those in example 15, and after immunization, the results of the serum titer determination were as shown in
The steps for phage display library construction and antibody panning were exactly the same as those in example 6. A total of 5 rounds of panning were performed; the output phages were significantly enriched every time; and positive clones were selected by means of PHAGE-ELISA experiments and sent for sequencing identification, as shown in Table 17. The similarities and differences of the CDR regions of these sequences were analyzed, as shown in
The DNA sequences of the positive clones were cloned into a PET28 vector and transfected BL21 bacteria, induced expression was performed, then a target protein was purified by using a nickel column, and the specificity and binding ability of the antibodies were detected by means of ELISA. The results were as shown in
The spleens of MDG1 mice and wild-type mice were taken, and RNA was extracted and reversely transcribed into cDNA. The cDNA was subjected to 5-fold dilution, and then 5 μL of diluent was taken for a fluorescent quantitative PCR experiment. Four individuals were selected for each genotype, and each gene of each individual was subjected to 2 technical repetitions. The lengths of primers and amplified fragments used in the experiment were as shown in Table 18, and the fluorescent quantitative PCR amplification system was as shown in Table 19.
Amplification Process:
Continuously raise the temperature to 95° C. at a rate of 0.11° C./s
After the process was completed, the degree of credibility of a numerical value was judged according to the dissolution curve, then the relative expression level of each immunoglobulin gene transcript was calculated by using a 2−ΔΔCt method, and statistical analysis was performed by using a two-tailed T test. The statistical results were as shown in
The serum of 8-week-old wild-type mice and MDG1 mice under the same genetic background were taken for Western Blot detection. The serum of the wild-type mice was subjected to 20-fold dilution with PBS, and the serum of the MDG1 mice was subjected to 100-fold dilution with PBS. Under reducing conditions, an intramolecular disulfide bond was opened by using 1 mM of DTT, and the Goat Anti-Mouse IgG2c heavy chain (HRP) antibody was subjected to 10000-fold dilution. The results of Western Blot detection were as shown in
The serum of 8-week-old wild-type mice and MDG1 mice under the same genetic background were detected by using double-antibody sandwich ELISA. For the wild-type mice, IgM was subjected to 2000-fold dilution, IgG2c was subjected to 4000-fold dilution, IgG was subjected to 10000-fold dilution, IgA was subjected to 5000-fold dilution, and IgE was subjected to 20-fold dilution; and for the MDG1 mice, IgM was subjected to 2000-fold dilution, IgG2c was subjected to 8000-fold dilution, IgG was subjected to 10000-fold dilution, IgA was subjected to 5000-fold dilution, and IgE was subjected to 20-fold dilution. The absorbance value of each sample well at 450 nm was read by using a microplate reader, a standard curve was obtained by using ELISA Calc according to four-parameter fitting, the content of immunoglobulin in each well was calculated, and statistical analysis was performed by using a two-tailed T test.
The results were as shown in
B Cells of Bone Marrow:
Six 8-week-old wild-type mice and six 8-week-old MDG1 mice under the same genetic background were prepared. Two femurs and tibiae of the lower limbs of each mouse were taken, and bone marrow cells were flushed into a centrifuge tube with FACS, centrifuged transiently and then resuspended with ACK. The cell suspension was filtered with a 70-μm filter screen and centrifuged, and then the supernatant was discarded. The cells were resuspended by adding 1 mL of FACS, 50 μL of the resuspended cells was taken and stained, and 10 μL of stained cells was taken, subjected to 40-fold dilution and calculated. A staining solution for the B cells of the bone marrow was as shown in table 20.
B Cells of Spleens:
Four 8-week-old wild-type mice and four 8-week-old MDG1 mice under the same genetic background were prepared. A 70-μm filter screen was placed in a 60 mm dish, 1 mL of ACK was added, and the spleens of the mice were gently ground on the filter screen. The cell suspension obtained after grinding was collected and centrifuged transiently, and the supernatant was discarded. The cells were resuspended by adding 1 mL of FACS, 50 μL of the resuspended cells was taken and stained, and 10 μL of stained cells was taken, subjected to 40-fold dilution and calculated. A staining solution for the B cells of the spleens was as shown in Table 20.
B Cells of Peritoneal Cavities:
Seven 8-week-old wild-type mice and seven 8-week-old 7 MDG1 mice under the same genetic background were prepared, and the abdomens of the mice were fixed upward to a foam board, and the ventral epidermis was cut to expose the peritoneum part. 5 mL of FACS was aspirated with a syringe into the peritoneal cavity, blowing-suction was performed 2-3 times by using a pipette and the cell suspension was collected into a centrifuge tube, centrifuged transiently, then resuspended by using ACK, filtered with a 70-μm filter screen and centrifuged, and then the supernatant was discarded. The cells were resuspended by adding 200 μL of FACS, and 100 μL of the resuspended cells was taken and stained. A staining solution for the B cells of the peritoneal cavities was as shown in Table 20.
The flow cytometry results of B cells in the bone marrow were as shown in
The flow cytometry results of B cells in the spleens were as shown in
The flow cytometry results of B cells in the peritoneal cavities were as shown in
Six to seven 6-week-old wild-type and six to seven 6-week-old MDG1 mice under the same genetic background were prepared, and immunization experiments were performed by using chloramphenicol and atrazine coupled with BSA, 100 μg/100 μL per mouse per immunization. A Freund's complete adjuvant was used during the first immunization, wherein 50 μL of Freund's complete adjuvant was injected subcutaneously in the back and 50 μL of Freund's complete adjuvant was injected intraperitoneally; a Freund's incomplete adjuvant was used during the booster immunizations, wherein 100 μL of Freund's incomplete adjuvant was injected intraperitoneally, and a total of four booster immunizations were performed; and 3 days before the first immunization and 3 days after each booster immunization, blood was collected from the inner canthus.
Following example 24 above, in order to avoid the interference with the detection results caused by a large amount of BSA-specific antibodies in the serum after immunostimulation, this ELISA experiment used chloramphenicol and atrazine coupled with OVA as coating antigens. The coating amount of each antigen was 200 ng/100 μL/well, the serum was subjected to 4000-fold dilution in the detection of antigen-specific IgM, IgG2c, IgG and IgA, and the serum was subjected to 100-fold dilution in the detection of IgE. In order to ensure the consistency of each sample well, a TMB chromogenic solution was added, then the reaction time was strictly controlled to 20 min, then the absorbance value at 450 nm was read with a microplate reader, the individuals without immunization were excluded, and a diagram of the change trend of the relative expression quantity of the antigen-specific antibody was plotted.
The changes in the relative expression quantities of specific antibodies chloramphenicol and atrazine were as shown in
Six wild-type mice and six MDG1 mice obtained after antigen immunization were taken. A 70-μm filter screen was placed in a 60 mm dish, 1 mL of ACK was added, and the spleens of the mice were gently ground on the filter screen. The cell suspension obtained after grinding was collected and centrifuged transiently, and the supernatant was discarded. The cells were resuspended by adding 1 mL of FACS, 20 μL of the resuspended cells was taken and stained, and L of stained cells was taken, subjected to 80-fold dilution and calculated. A staining solution for the B cells of the spleens obtained after immunization was as shown in Table 21.
The growth conditions of spleen B cells, germinal center B cells and plasma cells in the mice obtained after antigen immunization were as shown in
Finally, it should be noted that: the above examples are only used to illustrate the technical solution of the present application, rather than to limit them. Although the present application was described in detail with reference to the foregoing examples, a person skilled in the art should understand that it was still possible to amend the technical solutions described in the foregoing examples or to make equivalent replacements to some of the technical features thereof; and these amendments or replacements should not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the examples of the present application.
The non-human mammal or offspring thereof obtained by using the method for preparing the non-human mammal or offspring thereof in the present application does not introduce any exogenous gene encoding heavy chain variable and constant regions of an antibody, and can directly use all VDJ genes encoding the heavy chain variable region of the antibody in its own genome, so as to produce more diverse heavy chain antibodies by means of the rearrangement of the heavy chain variable region.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010924095.1 | Sep 2020 | CN | national |
The present application is a Continuation of International Application Number PCT/CN2021/116282 filed Sep. 2, 2021, and claims priority to the invention patent application no. 202010924095.1, titled “METHOD FOR PREPARING NON-HUMAN MAMMAL OR PROGENY THEREOF, AND USE THEREOF” and filed on Sep. 4, 2020, which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2021/116282 | Sep 2021 | US |
| Child | 18177298 | US |
