Patent Grant
11319353
The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 25, 2019, is named 423-002US_SL.txt and is 5,388 bytes in size.
The invention belongs to the field of bioengineering genes, and relates to a recombinant Dermatophagoides pteronyssinus type 2 allergen, and its coding gene and expression and purification method.
There are many kinds of dust mites, which are widely present in human living and working environments. The excreta, metabolites and mite bodies of dust mites have strong allergenicity. According to statistics, about 10% of the world's population is allergic to dust mites, and about 80% of extrinsic asthma is caused by dust mites.
At present, a crude extract of dust mite allergens is mainly used clinically to treat allergic diseases caused by dust mites. Allergens of dust mites mainly exist in excreta and mite bodies; therefore, the extraction method takes a long time with a cumbersome process and a high cost. In addition, the composition of a natural allergen extract is very complicated, it is very difficult to make its components constant, and the natural allergen extract is easy to be contaminated by exogenous toxic substances and pathogenic microorganisms. Long-term use of a crude extract of dust mite allergens can lead to local reactions such as flush, swelling, induration and necrosis; and systemic reactions such as shock, edema, bronchospasm, urticaria, angioedema and systemic erythema. In addition, in the case that the crude extract is used for diagnosis, it is impossible to specifically determine the extent of the patient's response to each component of the allergens, which may lead to misdiagnosis.
The quality of the allergen is essential for the diagnosis and treatment of allergic diseases, and the allergen used for immunodiagnosis and immunotherapy should be a pure product rather than a crude extract. Recombinant allergens have the following advantages over crude extracts: (1) the recombinant allergens have a higher purity and contain no non-allergenic components, enzymes, enzyme inhibitors and toxic proteins as compared with the crude extracts; (2) the recombinant protein has a single composition, has good specificity, while the components in the crude extract are complex, the patient may only have reactions with some of the components of the crude extract, and the specificity is poor; (3) as compared with the natural extract, the recombinant allergen reduces IgE-bound antigenic epitopes and thus reduces IgE-mediated allergic reactions effectively, at the same time the domains of allergen necessary for T cell recognition are retained to result in better immunogenicity, thereby reducing the risk of immunotherapy and improving the desensitization therapy effect.
Allergens of dust mites are complex in composition, with more than 30 types, of which type 1 and type 2 allergens are the most important allergen components. In the serum of dust mite allergic patients, 70-80% of the patients had IgE binding to type 2 allergens, and showed strong positive reaction. The precursor of Der p2 was composed of 146 amino acids, and 129 amino acids remained after signal peptide removal. The molecular weight of Der p2 was 14 KD and there was no glycosylation site. At present, Stallergenes' patent in 2011 (European patent No. EP2388268 (A1)) is more representative of the research on Der p2 recombinant expression using eukaryotic expression system. They recombined and purified Der p2 using Pichia pastoris expression system. The patent did not optimize the Der p2 gene and molecule construction for Pichia pastoris system. It had low yield, complex purification process and was difficult to meet the clinical dosage.
In order to overcome the above-mentioned shortcomings, the inventors optimize the Der p2 gene in the Pichia pastoris expression system, and add an acting element to increase the expression of Der p2 in molecular level, and the inventors surprisingly find that Der p2 after gene optimization is expressed at a higher level as compared with the prior art, and has a similar biological activity as the natural protein.
One object of the present invention is to provide a DNA sequence encoding Der p2 protein, having a base sequence as shown in SEQ ID NO: 1. This sequence has been codon-optimized for the Pichia pastoris expression system, which is more conducive to expressing Der p2 in Pichia pastoris.
Another object of the present invention is to provide Der p2 protein having an amino acid sequence as shown in SEQ ID NO:3.
Another object of the present invention is to provide a vector comprising the above-mentioned optimized gene encoding Der p2, preferably, the vector is pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZ A, B, C or pGAPZ A, B, C, more preferably pPIC3.5K, pPICZ A or pGAPZ A.
Another object of the present invention is to provide a Pichia pastoris strain comprising the above-mentioned vector, preferably, the Pichia pastoris strain is SMD1168, GS115, KM71, X33 or KM71H, more preferably strain KM71 or X33.
Preferably, there is 242 bp interval between the DNA sequence encoding the Der p2 protein and the ATG of AOX1 of Pichia pastoris; the DNA sequence encoding the Der p2 protein is preceded by Kozak sequence GCCACCATGG (SEQ ID NO: 10).
Another object of the present invention is to provide a method for expressing the Der p2 protein, comprising the steps of:
A constructing a vector comprising the above-mentioned gene encoding Der p2;
B linearizing the vector of step A, transferring it into a Pichia pastoris strain, and culturing under a suitable condition;
C recovering and purifying the protein.
The above-mentioned vector is preferably pPIC3.5K, pPICZ A or pGAPZ A.
The above-mentioned Pichia pastoris strain is preferably a KM71 or X33 strain.
More preferably, the above-mentioned vector is pPICZ A, and the above-mentioned Pichia pastoris strain is strain X33.
Another object of the present invention is to provide a method for purifying a recombinant Der p2 protein, comprising the steps of:
A centrifuging the Der p2 fermentation broth at a low temperature and a high speed to collect a supernatant, ultrafiltering the supernatant against a 50 mM sodium acetate buffer at pH 4.0, and filtering through a 0.45 μm filter membrane;
B the first step, cation chromatography, comprising equilibrating a chromatographic column with an equilibration buffer, passing the Der p2 fermentation broth in step A through a separation packing using a purification system, and then eluting with a gradient of an elution buffer to collect an elution peak, wherein the equilibration buffer is 50 mM sodium acetate at pH 4.0, and the elution buffer is 50 mM sodium acetate and 1.0 M sodium chloride at pH 4.0;
C the second step, comprising ultra-filtrating the Der p2 protein peak collected in step B with a 20 mM phosphate solution at pH 6.0, equilibrating a chromatographic column with an equilibration buffer, loading the ultra-filtrated Der p2 protein solution on an anion chromatography packing, and collecting a flow-through peak, wherein the equilibration buffer is 20 mM phosphate at pH 6.0;
D the third step, comprising adding ammonium sulfate to the flow-through peak in step C to the final concentration of 1.5 M, pH 6.0, equilibrating a chromatographic column with an equilibration buffer, loading a Der p2 sample on a hydrophobic chromatography packing, eluting with a gradient of an elution buffer, wherein equilibration buffer is 1.5 M ammonium sulfate and 20 mM phosphate at pH 6.0, and the elution buffer is 20 mM phosphate at pH 6.0.
Another object of the present invention is to provide the use of the recombinant Der p2 protein in the preparation of a medicament for treating a dust mite allergic disease. The allergic disease is allergic rhinitis, allergic asthma, and the like.
The recombinant Der p2 protein of the present invention has a high expression level and has similar biological activity as the natural protein.
The sequence before optimization corresponds to the nucleotide sequence of the natural Der p2 gene; the sequence after optimization corresponds to the nucleotide sequence of the recombinant Der p2 gene of the present invention, that is, the codon-optimized sequence.
Lane 1 represents 500 bp DNA ladder; lane 2 represents a PCR product of the recombinant Der p2 gene containing EcoRI and XhoI restriction sites at both ends.
The invention is further illustrated below in conjunction with specific examples. It should be understood that the examples referred to are merely illustrative of the invention and are not intended to limit the scope of the present invention.
Based on the DNA sequence of Der p2 disclosed in GenBank (GenBank accession no. AAF86462), as shown in SEQ ID No: 2, the inventors performed codon optimization of the gene to obtain the Der p2 gene of the present invention of which the nucleotide sequence is as shown in SEQ ID No: 1 and the amino acid sequence is as shown in SEQ ID No: 3. Comparison of each parameter before and after codon optimization of the Der p2 is as follows:
1. Codon Adaptation Index (CAI)
As can be seen from
2. Optimal Codon Usage Frequency (POP)
As can be seen from
3. GC Base Content (GC Curve)
The ideal distribution region of GC content is 30%-70%, and any peak outside this region will affect transcription and translation efficiency to varying degrees. As can be seen from the comparison of the average GC base content distribution region plots of the Der p2 gene in
A sequence of EcoRI restriction site was introduced at the 5′ end, and a sequence of XhoI restriction site was introduced at the 3′ end of the codon-optimized Der p2, and then full gene synthesis was performed. The synthesized gene fragment was constructed into the pUC57 plasmid supplied by GenScript (Nanjing) Co., Ltd., thereby obtaining a plasmid for long-term preservation, denoted as pUC57-Der p2 plasmid.
PCR amplification was performed using the pUC57-Der p2 plasmid as a template, and primers of following sequences:
The total volume of the reaction was 50 μL, in which 2.5 μL of each primer at a concentration of 10 μmol/L was added, 1 μL of dNTP at a concentration of 10 mmol/L was added, and 0.5 μL DNA polymerase being Q5 (#M0491L, purchased from New England BioLabs) at 2 U/μL was added. The reaction conditions were 98° C. for 5 seconds, 55° C. for 45 seconds, and 72° C. for 30 seconds. After 25 cycles, the product was analyzed by 1.0% agarose gel electrophoresis. The results showed that the product size was consistent with the expected size (500 bp) (results as shown in
Formulation of YPDS solid medium: the medium was formulated according to the instructions of Easy SelectPichia Expression Kit, Invitrogen, comprising 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, 15 g/L agarose, and 182 g/L sorbitol.
1. Construction of a Host Engineering Strain Containing Codon-Optimized Der p2
Electrocompetent cells were prepared according to the method of instructions of Easy SelectPichia Expression Kit, Invitrogen. The plasmid pPICZ-Der p2 obtained in Example 2 was linearized with Sac I restriction endonuclease (#R0156S, purchased from New England Biolabs), and precipitated with ethanol. The linearized vector was electrotransformed into competent cells of Pichia pastoris X33. The cells were plated on YPDS solid media and cultured at 30° C. until the transformants grew.
Formulation of BMGY medium: the medium was formulated according to the instructions of Easy SelectPichia Expression Kit, Invitrogen, comprising 10 g/L yeast extract, 20 g/L peptone, 3 g/L K2HPO4, 11.8 g/L KH2PO4, 13.4 g/L YNB, 4×10−4 g/L biotin, and 10 g/L glycerin.
Formulation of BMMY medium: the medium was formulated according to the instructions of Easy SelectPichia Expression Kit, Invitrogen, comprising 10 g/L yeast extract, 20 g/L peptone, 3 g/L K2HPO4, 11.8 g/L KH2PO4, 13.4 g/L YNB, 4×10−4 g/L biotin, and 5 mL/L methanol.
1. Methanol-Induced Expression of an Engineering Strain of Codon-Optimized Der p2
The host monoclonal engineering strain obtained in Example 3 was picked into a 5 mL BMGY medium and cultured in a 50 mL sterile centrifuge tube at 30° C. and 220 rpm until OD600 reaches 1.0-2.0. 1 mL of the culture was stored, and the remaining strain solution was resuspended and transferred to BMMY for induced expression at a small scale, and methanol was supplemented every 24 hours to a final concentration of 1%. One week later, the supernatant of the strain solution was collected by centrifugation, and analyzed by SDS-PAGE gel electrophoresis and Western blotting. Brightness of expressed product bands was observed.
The Der p2 constructed in this invention is obtained mainly by ion exchange and hydrophobic chromatography purification methods. HiTrap SP FF, HiTrap Q FF, and HiTrap Phenyl HP were selected as the chromatographic packings. The specific steps are as follows:
1. Pretreatment of the Fermentation Broth by Impurity Removal
The fermentation broth of host engineering strain containing Der p2 obtained according to Example 4 was centrifuged at a low temperature at 12000 rpm for 15 minutes to collect a supernatant, and the supernatant was ultrafiltered against a 50 mM sodium acetate buffer at pH 4.0 for 48 h, and filtered through a 0.45 μm filter membrane to obtain a supernatant of the treated fermentation broth.
2. Cation Exchange Chromatography
The treated fermentation broth of the previous step was loaded on a SPFP cation exchange chromatographic column, wherein the equilibration buffer was 50 mM NaAc at pH 4.0, the elution buffer was 50 mM NaAc and 1.0 M NaCl at pH 4.0, isocratic elution was performed at 12%, 25% and 100%, and the sample peaks were mainly concentrated at the 25% elution peak.
3. Anion Exchange Chromatography
The Der p2 protein peak purified in the previous step was collected, and the sample was ultrafiltered with a 20 mM Tris-HCl solution at pH 8.0, and loaded on a HiTrap Q FF chromatography packing. The equilibration buffer was 20 mM Tris-HCl at pH 8.0, and the elution buffer was 20 mM Tris-HCl and 1.0 M NaCl at pH 8.0. Equivalent elution was carried out according to 25%, 50% and 100%. Der p2 protein is mainly concentrated in the penetration peak.
4. Hydrophobic Chromatography
The flow-through peak of Der p2 from the anion chromatography was collected, and ammonium sulfate was added to a final concentration of 1.5 M. The fermentation broth supernatant treated as above was loaded on a Phenyl HP chromatographic column. The equilibration buffer was 20 mM NaH2PO4 and 1.5 M (NH4)2SO4 at pH 6.0; the elution buffer was 20 mM NaH2PO4 at pH 6.0, isocratic elution was performed at 25%, 50%, 70%, and 100%, and the Der p2 protein is mainly concentrated at the 75% elution peak.
The determination of N-terminal sequence of proteins and polypeptides is one of the important links in the quality control of pharmaceutical industry. In this experiment, N-terminal sequence analysis based on classical Edman degradation method was used.
The N-terminal sequence of Der p2 protein purified from Example 5 was analyzed by Shimadzu Automatic Protein Peptide Sequencing Instrument (PPSQ-33A, SHIMADZU). The results showed in
The purified Der p2 protein was dialyzed against a PBS buffer at pH 7.4, and the protein concentration was determined by a BCA protein concentration assay kit (Cat No: 23225, purchased from Pierce), and fold-diluted to 250 ng, 125 ng, 62.5 ng, 31.25 ng, and 15.625 ng. Using the dot immunoblotting method, the obtained solution was detected for the reactivity with sera of patients allergic to Dermatophagoides pteronyssinus by comparing with natural Der p2 as the control.
1. Inoculation X33 strain: the strains were cultured in YPD media for 24 h, the X33 genome was extracted by a genomic extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.), and GAP gene was amplified using the X33 genome as a template, and GAP-1 and GAP-2 as primers of which the sequences are as follows:
The total volume of the reaction was 50 μL, in which 2.5 μL of each primer at a concentration of 10 μmol/L was added, 1 μL of dNTP at a concentration of 10 mmol/L was added, and 0.5 μL DNA polymerase being Taq DNA Polymerase (M0267S, purchased from New England BioLabs) at 2 U/μL was added. The reaction conditions were 94° C. for 10 minutes, 94° C. for 30 seconds, 55° C. for 30 seconds, 68° C. for 30 seconds, and 68° C. for 5 minutes. After 30 cycles, the product was analyzed by 1.0% agarose gel electrophoresis. The results showed that the product size was consistent with the expected size (400 bp) (results as shown in
2. The Der p2 gene was amplified using the pPICZα-Der p2 plasmid of Example 2 as a template, and 5′ AOX and 3′ AOX as primers with the following sequences:
The total volume of the reaction was 50 μL, in which 2.5 μL of each primer at a concentration of 10 μmol/L was added, 1 μL of dNTP at a concentration of 10 mmol/L was added, and 0.5 μL DNA polymerase being Taq DNA Polymerase (#M0267S, purchased from New England BioLabs) at 2 U/μL was added. The reaction conditions were 94° C. for 10 minutes, 94° C. for 30 seconds, 49° C. for 30 seconds, and 68° C. for 60 seconds, and 68° C. for 5 minutes. After 30 cycles, the product was analyzed by 1.0% agarose gel electrophoresis. The results showed that the product size was consistent with the expected size (750 bp) (results as shown in
3. Calculation of gene copy number:
The concentration (ng/μL) of the standard plasmid was determined by a nucleic acid microanalyzer (Nanodrop2000, ThermoFisher). Copy numbers of GAP and Der p2 were calculated according to the following formula:
Copies/u=(6.02×1023)3 pies μL×3p−9)/(DNA length02×10
4. Processing samples to be tested
The pPICZ-Der p2-X33 engineering strain was inoculated in YPD liquid media at 30° C. overnight; and the genome was extracted the next day, and its concentration (ng/μL) and purity were determined by a nucleic acid quantitative microanalyzer.
5. Establishment of a standard curve
The standard plasmids of T-GAP and T-Der p2 with known copy numbers were gradiently diluted to 108, 107, 106, 105, 104, and 103 copies/μl, respectively. The fluorescent quantitative PCR were performed using GAP-1 and GAP-2, 5′ AOX and 3′ AOX as primers, respectively.
6. Determination of Copy Number of Der p2 Gene in Recombinant Strains
The genome sample of extracted pPICZ-Der p2-X33 was serially 10-fold-diluted to obtain four gradients of stock solution, 10−1, 10−2, and 10−3. Fluorescent quantitative PCR was performed using GAP-1 and GAP-2, 5′ AOX and 3′ AOX as primers, and each gradient was assayed three times.
There is no stable additional plasmid in Pichia pastoris, the expression vector is homologously recombined with the host chromosome, and the exogenous gene expression framework is fully integrated into the chromosome to realize the expression of the exogenous gene; the typical Pichia pastoris expression vector contains a regulatory sequence of alcohol oxidase gene, and contains the main structures comprising AOX promoter, multiple cloning site, transcription termination and polyA formation gene sequence (TT), screening markers and the like. The promoter is a cis-element for gene expression regulation and an important element for the genetically engineered expression vector. The important role of the promoter at the transcriptional level determines the gene expression level.
The Der p2 genome was extracted according to the method of Example 8, and the Der p2 gene was amplified from the genome using 5′ AOX and 3′ AOX as primers according to the method in Step 2 of Example 8. The obtained samples were sent to GenScript (Nanjing) Co., Ltd. to detect the acting element before and after the Der p2 gene which was inserted into the genome. The results of genome sequencing indicated that the Der p2 gene expression framework was integrated into the chromosome of Pichia pastoris by a single cross-insertion, which enabled the Der p2 gene to express the gene using the AOX promoter on the yeast chromosome, and thus the expression level was higher.
Generally, the closer the first ATG of the exogenous coding sequence to the ATG of AOX1, the better the expression effect. In the gene construction, the inventors chose an enzyme cleavage site closest to the ATG of AOX1, and found that the Der p2 gene was away from ATG of AOX1 only by 242 bp. In addition, Kozak sequence GCCACCATGG (SEQ ID NO: 10) was added in front of Der p2 gene, and the signal peptide and the sequence can greatly improve transcription and translation efficiency and increase expression efficiency of Der p2 gene in eukaryotes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201611267033.8 | Dec 2016 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/119173 | 12/28/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/121633 | 7/5/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5968526 | Garman | Oct 1999 | A |
| 20110293665 | Bordas et al. | Dec 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| 1385528 | Dec 2002 | CN |
| 102732541 | Oct 2012 | CN |
| 105307679 | Feb 2016 | CN |
| WO 199934826 | Jul 1999 | WO |
| WO 2003047618 | Jun 2003 | WO |
| Entry |
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| Cui,Y.-B., “Registration No. EU346693.1, Dermatophagoides pteronyssinus Der p 2 allergen mRNA, complete cds” GenBank Database, Jan. 7, 2008 (Jan. 7, 2008), see nucleotide. |
| International Search Report and Written Opinion in International Application No. PCT/CN2017/119173 dated Apr. 3, 2018. |
| Number | Date | Country | |
|---|---|---|---|
| 20200131235 A1 | Apr 2020 | US |
