Patent Application
20240309050
This application includes a sequence listing in computer readable form (a “txt” file) that is submitted herewith on ASCII text file named P23GZINW00065US-SEQ_ST25.txt, created on Jul. 11, 2023 and 49,255 bytes in size. This sequence listing is incorporated by reference herein.
This present disclosure belongs to the field of biotechnology and particularly relates to a self-assembled nanoparticle containing a gHgL protein of an EB virus, a preparation method and the use thereof.
EB virus (Epstein-Barr virus, EBV) belongs to y herpes viruses, which is a double-stranded DNA virus with an envelope. EB virus mainly infects tissues derived from ectoderm, such as skins, membranes and nerves. The EB virus infection is very common in human populations, with a prevalence rate as high as 95%, and it very easily incubates in the body for a lifelong time. In addition to infectious mononucleosis, EB virus may also cause post-transplant lymphoproliferative diseases and some malignant tumors of B cells and epithelial cells. Lymphocytic proliferative diseases include Burkitt lymphoma, diffuse large B-cell lymphoma, NK/T-cell lymphoma, and the like; and malignant tumors include nasopharyngeal carcinoma, gastric cancer, and the like.
A fusion protein complex of EBV comprises both gB and gHgL, which are conserved in the herpesvirus family, and gp350 and gp42, which are specific to EBV. It has been found by research that gHL, as an important receptor-binding protein in the process of membrane fusion, can not only play an independent role in mediating epithelial cell infection, but can also form a common complex with gp42 to participate in B cell infection. In addition, gHgL neutralizing antibodies have very strong EB virus neutralizing effects in both epithelial cell infection and B cell infection, which proves that gHL is an ideal immunogen for EBV vaccines.
In order to overcome the shortcomings of the prior art, a first aspect of the present disclosure aims to provide a self-assembled nanoparticle containing a gHgL protein.
A second aspect of the present disclosure aims to provide a method for preparing the self-assembled nanoparticle of the first aspect.
A third aspect of the present disclosure aims to provide use of the self-assembled nanoparticle of the first aspect in the preparation of a drug for preventing EB virus infection.
A fourth aspect of the present disclosure aims to provide a vaccine comprising the self-assembled nanoparticle of the first aspect as mentioned above.
A fifth aspect of the present disclosure aims to provide use of the self-assembled nanoparticle of the first aspect in the preparation of a drug for treating diseases caused by EB virus infection.
In order to achieve the above objects, the technical solutions used in the present disclosure are as follows.
In a first aspect of the present disclosure, there is provided a self-assembled nanoparticle containing a gHgL protein, which comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a gHgL protein and a first vector subunit, and the second polypeptide comprises a second vector subunit; the first vector subunit is 153-50A1 and the second vector subunit is 153-50B.4 PT1; and the gHgL protein is linked to the first vector subunit through a linker, so that the gHgL protein is displayed outside the assembled nanoparticle, and an immune response of a body is better stimulated.
Preferably, the linker comprises a flexible sequence and a rigid connector, and the linker is used for linking the gHL protein to the vector protein, without affecting the immunogenicity of the gHgL protein and the correct folding of the protein. The vector protein is composed of a first vector subunit and a second vector subunit.
Preferably, the flexible sequence is a polypeptide comprising 5 to 9 amino acids. Furthermore, the flexible sequence is a polypeptide of any one of SEQ ID NO: 12 to SEQ ID NO: 16; moreover, the flexible sequence is a polypeptide as shown in SEQ ID NO: 15.
Preferably, an amino acid sequence of the rigid connector is EKAAKAEEAA (SEQ ID NO: 31).
Preferably, the first vector subunit and the second vector subunit are self-assembled to form a nanostructure by a non-covalent interaction, the first vector subunit is coated on a surface of the second vector subunit, and the gHgL protein is displayed on a surface of the nanostructure.
Preferably, the gHgL protein comprises a gH protein (SEQ ID NO: 28) and a gL protein (SEQ ID NO: 29).
Preferably, the gHgL protein further comprises a linking sequence (SEQ ID NO: 30) for linking the gH protein to the gL protein.
Preferably, an amino acid sequence of the 153-50A1 is shown in SEQ ID NO: 26.
Preferably, an amino acid sequence of the 153-50B.4 PT1 is shown in SEQ ID NO: 27.
Preferably, the first polypeptide further comprises a stabilizing protein.
Preferably, the stabilizing protein is located between the linker and the gHgL protein.
Preferably, the stabilizing protein is a T4 fibritin (SEQ ID NO: 32) or a GCN4 peptide fragment (SEQ ID NO: 33); furthermore, the stabilizing protein is a T4 fibritin.
Preferably, the first polypeptide is a first polypeptide trimer.
Preferably, the second polypeptide is a second polypeptide pentamer.
Preferably, a copy number of the first polypeptide trimer is 18-22, and a copy number of the second polypeptide pentamer is 10-14; preferably, the copy number of the first polypeptide trimer is 20, and the copy number of the second polypeptide pentamer is 12.
Preferably, the self-assembled nanoparticle containing the gHgL protein has an icosahedral symmetry.
In a second aspect of the present disclosure, there is provided a method for preparing the self-assembled nanoparticle containing the gHgL protein, comprising incubating the first polypeptide with the second polypeptide to obtain the self-assembled nanoparticle containing the gHgL protein. The first polypeptide comprises a gHgL protein and a first vector subunit, and the second polypeptide comprises a second vector subunit; the first vector subunit is 153-50A1, and the second vector subunit is 153-50B.4 PT1; and the gHgL protein is linked to the first vector subunit through a linker, so that the gHgL protein is displayed outside the assembled nanoparticle, and an immune response of a body is better stimulated.
Preferably, the amino acid sequence of the 153-50A1 is shown in SEQ ID NO: 26.
Preferably, the amino acid sequence of the 153-50B.4 PT1 is shown in SEQ ID NO: 27.
Preferably, a molar ratio of the first polypeptide to the second polypeptide is 1:(3-6), preferably 1:5.
Preferably, the incubation is carried out in an assembly buffer for 0.5-2 h.
Preferably, the assembly buffer comprises 250 mM NaCl, 50 mM Tris-HCl with pH 8.0, and 5% glycerol (mass fraction).
Preferably, the gHgL protein comprises a gH protein (SEQ ID NO: 28) and a gL protein (SEQ ID NO: 29).
Preferably, the gHgL protein further comprises a linking sequence (SEQ ID NO: 30) for linking the gH protein to the gL protein.
Preferably, the linker comprises a flexible sequence and a rigid connector, and the linker is used for linking the gHgL protein to the vector protein, without affecting the immunogenicity of the gHgL protein and the correct folding of the protein. The vector protein is composed of a first vector subunit and a second vector subunit.
Preferably, the flexible sequence is a polypeptide comprising 5 to 9 amino acids. Furthermore, the flexible sequence is a polypeptide of any one of SEQ ID NO: 12 to SEQ ID NO: 16; moreover, the flexible sequence is a polypeptide shown in SEQ ID NO: 15.
Preferably, the amino acid sequence of the rigid connector is EKAAKAEEAA (SEQ ID NO: 31).
Preferably, the first polypeptide further comprises a stabilizing protein.
Preferably, the stabilizing protein is located between the linker and the gHgL protein.
The stabilizing protein is preferably a T4 fibritin (SEQ ID NO: 32) or a GCN4 peptide fragment (SEQ ID NO: 33); more preferably, a T4 fibritin.
Preferably, the first polypeptide and the second polypeptide further comprise a purification tag.
The purification tag is preferably at least one selected from the group consisting of histidine tag (His-tag), streptavidin tag (Strep-tag) and maltose binding protein (MBP); more preferably, the purification tag is a histidine tag (His-tag); and most preferably, the purification tag is a histidine tag with an amino acid sequence as shown in SEQ ID NO: 34 or SEQ ID NO: 35.
Preferably, the purification tag of the first polypeptide is located between the stabilizing protein and the linker.
Preferably, the first polypeptide further comprises a linking sequence.
Preferably, the linking sequence is located between the stabilizing protein and the purification tag.
Preferably, the linking sequence is shown in SEQ ID NO: 37.
Preferably, the purification tag of the second polypeptide is located at an end of the second vector subunit.
The first polypeptide further comprises a signal peptide, so that a target protein can be secreted into a supernatant after expression.
The signal peptide is a CD5 signal peptide as shown in SEQ ID NO: 36.
The first polypeptide is preferably obtained by the following steps: introducing a nucleic acid expressing the first polypeptide into a first host cell; and incubating the first host cell to express the first polypeptide.
The first host cell is preferably a eukaryotic cell; more preferably, the first host cell is at least one selected from the group consisting of human embryonic kidney 293 cell (HEK293F), a Madin-Daby canine kidney cell (MDCK), Chlorocebus sabaeus kidney cell (VERO), SF9 (Spodoptera frugiperda 9) cell, HighFive cell, a CHO (Chinese Hamster Ovary) cell, and yeast cell; more preferably, the first host cell is a human embryonic kidney 293 cell.
The second polypeptide is preferably obtained by the following steps: introducing a nucleic acid expressing the second polypeptide into a second host cell; and incubating the second host cell to express the second polypeptide.
The second host cell is preferably a prokaryotic cell; more preferably, the second host cell is Escherichia coli; and most preferably, the second host cell is Rosetta (DE3).
In a third aspect of the present disclosure, there is provided use of the self-assembled nanoparticle of the first aspect in the preparation of a drug for preventing EB virus infection.
In a fourth aspect of the present disclosure, there is provided a vaccine comprising the self-assembled nanoparticle of the first aspect.
A vaccine comprising the above-mentioned self-assembled nanoparticle containing the gHgL protein is provided.
The vaccine further comprises an adjuvant.
The adjuvant is at least one selected from the group consisting of an aluminum adjuvant, an oil emulsion adjuvant such as oil-in-water emulsion, water-in-oil emulsion and bidirectional emulsion, a microorganism-originated adjuvant such as peptidoglycan (PG), lipopolysccharide (LPS) of Gram-negative bacterial outer membrane, mycobacteria and components thereof, GpG oligonucleotide (GpG ODN) and cholera toxin (CT), a microsomal antigen delivery system such as liposome, polymeric microsphere, inert nanosphere, nano aluminum adjuvant, immunostimulating complex (IS-COM), cytokine, a polysaccharide such as inulin (MPI), and a natural source such as propolis and sapoin. More preferably, the adjuvant is a MF59 adjuvant.
In a fifth aspect of the present disclosure, there is provided use of the self-assembled nanoparticle of the first aspect in the preparation of a drug for treating diseases caused by EB virus infection.
The disease is preferably at least one selected from the group consisting of infectious disease, malignant tumor, chronic disease, and autoimmune disease. More preferably, the disease is at least one selected from the group consisting of mononucleosis, nasopharyngeal carcinoma, gastric cancer, epithelial tumors, Burkitt lymphoma, Hodgkin lymphoma, chronic fatigue syndrome, multiple sclerosis, and ankylosing myelitis.
The drug further comprises a pharmaceutically acceptable vector.
The present disclosure has the beneficial effects as follows.
In the self-assembled nanoparticle provided by the present disclosure, the gHgL protein of the EB virus is displayed on the surface of the nanoparticle for the first time. The self-assembled nanoparticle has a larger particle size than the antigen (gHgL), a better antigen residence volume, a thermal stability comparable to the antigen (gHgL). Moreover, since a larger number of gHgLs are displayed, the self-assembled nanoparticle can strongly stimulate more B cells and induce higher antibody titer. The self-assembled nanoparticle can be used for preventing EB virus infection and treating diseases caused by EB virus infection.
Although a heterologous gene is introduced into the self-assembled nanoparticle provided by the present disclosure, since the heterologous gene is derived from a protein of bacteria, which can avoid causing autoimmune diseases, thus having an advantage of high safety without affecting an immune effects.
The content of the present disclosure will be further illustrated in detail in conjunction with specific examples and drawings.
It should be understood that these examples are only used to illustrate the present disclosure and are not used to limit the scope of the present disclosure.
In the following examples, the experimental methods in which no specific conditions are specified are usually in accordance with conventional conditions. The common chemical reagents used in the examples are all commercially available products.
The method for preparing the nanoparticle vaccine of the present disclosure includes the following steps.
The nanoparticle vaccine of the present application is further described in detail hereinafter.
By means of a computer software-aided design such as Rosetta, domain insertion design was carried out by means of rosetta remodel software, and a trimer stabilizing protein was structurally linked to gHgL antigen (SEQ ID NO: 1), so as to judge whether it was necessary to insert a linker. Finally, structure visualization was carried out by means of PyMol for visual judgment, whereby such linkers that were composed of a flexible sequence and a rigid connector were eventually selected. The various linkers were only different in the flexible sequence. The amino acid sequences and nucleotide sequences of the flexible sequences of the various linkers were as shown in Table 1, where the amino acid sequence of the rigid connector was shown in SEQ ID NO: 31, and the nucleotide sequence thereof was shown in SEQ ID NO: 17.
Softwares used in the design:
We had tried various linkers, which were composed of a flexible sequence and a rigid connector. The various linkers were only different in the flexible sequence. Similarly, after an expression vector was constructed and transfected for expression, the protein concentration was determined after purification and concentration. The specific steps were as follows: (1) a gH gene of EB virus (SEQ ID NO: 2), a linking sequence (SEQ ID NO: 3), a gL gene (SEQ ID NO: 4), a T4 fibritin (SEQ ID NO: 5), a 153-50A1 (SEQ ID NO: 6) and a linker (the amino acid sequences and nucleotide sequences of the flexible sequences of various linkers were as shown in Table 1, and the nucleotide sequence of the rigid connector was shown in SEQ ID NO: 17) were inserted into the vector PCDN3.1(+) by PCR amplification and enzyme digestion recombination, so as to obtain the target gene gHgL-I53-50A1 expressed by the expression vector. Wherein, the front end of the vector pcDNA3.1(+) was provided with a CD5 signal peptide (SEQ ID NO: 18) for secreting the expressed polypeptide outside cells, there was an eight-histidine His-tag (SEQ ID NO: 19) between T4 fibritin and the linker for convenient purification, and the front end of the His-tag was connected to a linking sequence (SEQ ID NO: 20). (2) The recombinant vector gene in pcDNA3.1 was transformed into DH5α competent bacteria, and positive clones were screened by ampicillin resistance. Then, the positive clones were picked into a TB culture medium containing 0.1% ampicillin (0.1 mg/mL) for amplification, and then extracted by using the midiprep kit. The specific method could be referred to the instruction of product. (3) 293F cells were subjected to suspension culture and amplification in in a 293F medium (Union), and were ready for transient transfection after being amplified to a certain quantity. The cells were diluted to 1 L with a density of 1*106/mL, and then, a transfection system of 1 mg of pcDNA3.1-target protein vector 5 mg PEI was prepared with a fresh medium, added into the diluted 293F cells after standing for 30 min, and cultured at 37° C., 30% humidity, 5% CO2 concentration for 7 days under shaking at 120 rpm. The cell precipitate was removed by centrifugation. The supernatant was filtered with a 0.22 μm filter membrane, and then purified by protein affinity chromatography and molecular sieve to obtain a high-purity target protein gHgL-I53-50A1 subunit.
The results were as shown in Table 1. When the flexible sequence of the linker was GGSGGSGS (SEQ ID NO: 15), the yield of the gHgL-I53-50A1 subunit was the highest.
As shown in
The gHgL self-assembled nanoparticle (gHgL-I53-50 NP), gHgL-I53-50A1 subunit and antigen protein (gHgL) in Example 2 were diluted to 0.5 mg/mL. 200 μL of the samples were then added to a special sample loading slot of Uncle, and stood for 5 min, and then the particle size of the nanoparticles was detected by an Uncle instrument from Unchained Company.
The gHgL self-assembled nanoparticles (gHgL-I53-50 NP) in Example 2 was diluted to a concentration of 0.1 mg/mL. The protein was incubated on a carbon-coated copper grid, then incubated and stained with 2% uranium acetate for 2 minutes, and dried in air. Then, the particle size and morphology of the particle were observed by using 120 KV transmission electron microscope.
The gHgL self-assembled nanoparticles (gHgL-I53-50 NP), gHgL-I53-50A1 subunit and antigen (gHgL) in Example 2 were diluted to a concentration of 0.5 mg/mL. Then the heating scanning was carried out from 25° C. to 90° C. by using an Uncle instrument from Unchained Company, and the change of the full-wavelength broad-spectrum shift (BCM) was recorded.
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0.5% PBST was prepared for kinetic detection. 150 uL of PBST was added into the pre-wetted plate, and incubated in the protein A sensor for 10 minutes. The antibody AMMO1 (for the preparation method, see the reference Snijder et al., 2018, Immunity 48, 799-811) was then diluted for coupling. After equilibrium, coupling was started, and the antigens such as nanoparticle proteins (gHgL, gHgL-I53-50A1 and gHgL-I53-50 NP in Example 2) were then diluted in a gradient (3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM), and bound to the sensor. The binding signal and dissociation signal were recorded, and the sensor was regenerated by suing a glycine solution. The binding signal was fitted by using a binding model of 1:1 to calculate the dynamic parameters.
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The above examples are preferred embodiments of the present disclosure, and the embodiments of the present disclosure are not limited by the above examples. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit essence and principle of the present disclosure shall be equivalent substitutions and are all included in the scope of protection of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110117653.8 | Jan 2021 | CN | national |
The present application is a national phase entry under 35 USC § 371 of International Application PCT/CN2021/115968, filed Sep. 1, 2021, which claims the benefit of and priority to Chinese Patent Application No. 2021101176538, filed Jan. 28, 2021, the entire disclosures of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/115968 | 9/1/2021 | WO |
