WO2018121639A1 - 一种重组粉尘螨1类变应原蛋白及其制备方法和应用 - Google Patents

一种重组粉尘螨1类变应原蛋白及其制备方法和应用 Download PDF

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WO2018121639A1
WO2018121639A1 PCT/CN2017/119190 CN2017119190W WO2018121639A1 WO 2018121639 A1 WO2018121639 A1 WO 2018121639A1 CN 2017119190 W CN2017119190 W CN 2017119190W WO 2018121639 A1 WO2018121639 A1 WO 2018121639A1
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proder
protein
gene
der
buffer
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马永
范宇
王俊
王安良
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江苏众红生物工程创药研究院有限公司
常州京森生物医药研究所有限公司
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Publication of WO2018121639A1 publication Critical patent/WO2018121639A1/zh
Priority to US16/456,357 priority Critical patent/US10975128B2/en

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Definitions

  • the invention belongs to the field of bioengineering genes, and relates to a recombinant dust mite class 1 allergen and a coding gene thereof and a method for expressing and purifying the same.
  • the dust mite allergen crude extract is mainly used to treat allergic diseases caused by dust mites.
  • the “Changdi” dust mites of Zhejiang Iwu Biological which was listed in 2006, is a metabolic culture of dust mites. Extract. Dust mites allergens mainly exist in excreta and carcass. The extraction method takes a long time, the process is cumbersome and the cost is high. In addition, the composition of natural allergen extract is very complicated, and it is very difficult to keep its components, and it is easy to be Contamination of exogenous toxic substances and pathogenic microorganisms.
  • the quality of the allergen is essential for the diagnosis and treatment of allergic diseases, and the allergen used for immunotherapy should be pure and not a crude extract.
  • Recombinant allergens have the following advantages over crude extracts: (1) recombinant allergens have higher purity and contain no non-allergenic components, enzymes, enzyme inhibitors and toxic proteins compared to crude extracts. (2) The recombinant protein has a single component, has good specificity, and the components in the crude extract are complex. The patient may only react with some of the components, and the specificity is poor; (3) the recombinant allergen is reduced compared with the natural extract. IgE-binding epitopes, effectively reducing IgE-mediated allergic reactions, while retaining the necessary domains for allergen T cell recognition, have better immunogenicity, reduce the risk of immunotherapy, and improve desensitization therapy Effect.
  • Dust mites allergens are complex in composition, with more than 30 species, of which Class 1 and Class 2 allergens are the most important allergen components.
  • class 1 allergen Der f1
  • the article indicates that Der f1 needs to be added to the Der f1 protein when expressed in the Pichia system.
  • Peptide Der f1 with propeptide, denoted as proDer f1
  • proDer f1 propeptide
  • the inventors optimized the proDer f1 gene in the Pichia pastoris expression system and added an original element to improve the expression of proDer f1 in molecular design.
  • the inventors were consciously surprised to find that proDer f1 was optimized after gene optimization.
  • the inventors have intensively studied and optimized the activation process of proDer f1, and adopted a more operational and scalable activation process, and purified mature Der f1 protein. It has similar biological activity compared to the native protein.
  • Another object of the present invention is to provide a proDer f1 protein having an amino acid sequence as shown in SEQ ID NO: 3.
  • Another object of the present invention is to provide a Der f1 protein having an amino acid sequence as shown in SEQ ID NO:
  • Another object of the present invention is to provide a vector comprising the above-described optimized proDer f1 gene, preferably 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 strain comprising the above-described vector, preferably, the Pichia strain is SMD1168, GS115, KM71, X33 or KM71H, more preferably a KM71 or X33 strain.
  • the DNA sequence encoding the proDer f1 protein differs from the ATG of AOX1 on Pichia by only 242 bp; the DNA sequence encoding the proDer f1 protein is preceded by an alpha-factor signal peptide and a Kozak sequence GCCACCATGG.
  • Another object of the present invention is to provide a method for expressing a proDer f1 protein, the method comprising the steps of:
  • step B linearizing the vector of step A, transferring it into Pichia pastoris strain, and culturing under suitable conditions;
  • the vector described above is preferably pPIC3.5K, pPICZ ⁇ A or pGAPZ ⁇ A.
  • the Pichia pastoris strain described above is preferably a KM71 or X33 strain.
  • the above vector is pPICZ ⁇ A
  • the Pichia strain described above is the X33 strain.
  • Another object of the present invention is to provide a method for purifying a recombinant Der f1 protein, which is as follows:
  • step B The first step of cation chromatography, equilibrate the column with equilibration buffer, and then use the purification system to pass the activated mature Der f1 fermentation broth in step A through the separation packing, and then use the elution buffer gradient elution to collect
  • Another object of the present invention is to provide a use of recombinant Der f1 protein for the preparation of a medicament for the treatment of dust mite allergic diseases.
  • the allergic disease is allergic rhinitis, allergic asthma, and the like.
  • the recombinant proDer f1 protein of the present invention has a high expression level and has similar biological activity as a native protein.
  • Figure 1 shows a comparison of the sequence of the proDer f1 gene before and after optimization.
  • the optimized pre-sequence corresponds to the nucleotide sequence of the native proDer f1 gene; the optimized sequence corresponds to the nucleotide sequence of the recombinant proDer f1 of the present invention, that is, the codon-optimized sequence.
  • Figure 2-a, 2-b are the CAI indices of the proDer f1 gene in the Pichia pastoris expression system before and after optimization.
  • Figure 2-a shows that the nucleotide sequence of the native proDer f1 gene was calculated by the program to be 0.76 in the Pichia expression system;
  • Figure 2-b shows the optimized expression of the proDer f1 codon of the present invention in Pichia pastoris
  • the CAI index in the system is calculated to be 0.85.
  • Figure 3-a, 3-b are regions of the optimal codon frequency distribution of the proDer f1 gene in the Pichia pastoris expression host before and after codon optimization.
  • Figure 3-a shows the optimal codon frequency distribution map of the proDer f1 natural gene nucleotide sequence in the Pichia pastoris system. It can be seen from the figure that the low utilization codon of the proDer f1 natural gene nucleotide sequence The percentage of occurrence is 10%;
  • Figure 3-b shows the optimal codon frequency distribution region map of the optimized proDer f1 codon of the present invention in the Pichia pastoris system, and the optimized utilization of the proDer f1 codon sequence of the present invention is low. The codon appears as 0.
  • Figure 4-a, 4-b are regions of the average GC base content distribution of the proDer f1 gene in the Pichia pastoris expression system before and after codon optimization.
  • Figure 4-a shows that the nucleotide sequence of the proDer f1 native gene nucleotide sequence in the Pichia pastoris expression system is: 41.85%;
  • Figure 4-b shows the optimized proDer f1 codon of the present invention in Biqi
  • the average GC base content in the yeast expression system was: 42.10%.
  • Figure 5 is an agarose gel electrophoresis pattern of the proDer f1 gene PCR product after codon optimization.
  • Lane 1 is a 200 bp DNA ladder
  • Lane 2 is a recombinant proDer f1 gene PCR product containing XhoI and NotI restriction sites at both ends.
  • Figure 6 is a diagram showing the construction process of the proDer f1 expression plasmid pPICZ ⁇ -proDerf1 after codon optimization.
  • Figure 7 is a diagram showing the expression of the proDer f1 gene in the host engineered bacteria after codon optimization.
  • Fig. 7-a is a SDS-PAGE gel electrophoresis pattern of the supernatant of the proDer f1 gene host strain after methanol expression induction for one week after codon optimization.
  • Lane 1 is a pre-stained protein loading marker in the range of 10-250 KD; the remaining lanes are the supernatants of the positive monoclonal host engineering strains of the proDer f1 gene screened by Zeocin.
  • Figure 7-b is an immunoblot of the supernatant of the supernatant of the proDer f1 gene host strain after one week of induction of expression by methanol after codon optimization.
  • Lane 1 is a 10-250 KD pre-stained protein Marker and lanes 2-10 are proDer f1 monoclonal-induced expression supernatants.
  • Figure 8 is a chromatogram and gel electrophoresis pattern of the first step of the supernatant of the proDer f1 fermentation broth.
  • Figure 8-a shows the first step cation chromatogram of the supernatant of the proDer f1 fermentation broth
  • Figure 8-b shows the first step cation chromatography purification of the proDer f1 fermentation broth.
  • the lane 1 is 11-100KD.
  • Pre-stained protein Marker lane 2 is the supernatant of the proDer f1 fermentation broth before purification
  • lane 3 is the permeate
  • lanes 4-8 are the elution tubes.
  • Figure 9 is a second step anion chromatogram and gel electrophoresis pattern of Der f1 protein.
  • Figure 9-a shows the second step anion chromatogram of Der f1 protein
  • Figure 9-b shows the second step of anion chromatography purification of Der f1 protein.
  • Lane 1 is 11-100KD non-pre-stained protein Marker
  • lane 2 is the supernatant of the Der f1 protein before purification
  • Lane 3 is the permeate
  • Lane 4 is the elution peak.
  • Figure 10 is a third step chromatogram and gel electrophoresis pattern of Der f1 protein.
  • Fig. 10-a is the third step hydrophobic chromatography chromatogram of Der f1 protein
  • Fig. 10-b is the hydrophobic chromatographic purification result of Der f1 protein
  • lane 1 is 11-100KD non-pre-stained protein Marker
  • lane 2-10 For each elution tube.
  • Figure 12 is a PCR-amplified GAP gene agarose gel electrophoresis map in which lane 1 is a 250 bp DNA ladder and lane 2 is a GAP gene.
  • Figure 13 is a PCR analysis of the GAP gene T-carrier positive clone agarose gel electrophoresis map, wherein lane 1 is a 250 bp DNA ladder, lanes 2-11 are positive clones obtained by blue-white screening, and lane 12 is a negative clone obtained by blue-white screening. .
  • Figure 14 is a PCR-amplified agarose gel electrophoresis map of proDer f1 gene, wherein lane 1 is a 500 bp DNA ladder and lane 2 is a proDer f1 gene.
  • Figure 15 is a PCR diagram of the pro-er f1 gene T-carrier positive clone agarose gel electrophoresis.
  • Lanes 1, 2 are negative clones obtained by blue-white spot screening
  • lane 3 is 500 bp DNA Ladder
  • lanes 4-13 are obtained by blue-white spot screening.
  • lane 14 is the positive control (proDer f1 gene), wherein lanes 4, 6, and 7 are positive clones containing the proDer f1 gene, and the rest are false positive clones.
  • Figure 16 is a graph showing the amplification of a standard plasmid.
  • Figure 16-a is a T-GAP standard plasmid amplification curve
  • Figure 16-b is a T-proDerf1 standard plasmid amplification curve.
  • Figure 17 is a graph showing the melting curve of a standard plasmid.
  • Figure 17-a is a T-GAP standard plasmid melting curve
  • Figure 17-b is a T-proDerf1 standard plasmid melting curve.
  • Figure 18 is a standard curve of a standard plasmid.
  • Figure 18-a is a standard curve of the T-GAP standard plasmid
  • Figure 18-b is a standard curve of the T-proDerf1 standard plasmid.
  • Figure 19 is a graph showing the amplification of the sample to be tested.
  • Figure 19-a shows the amplification curve obtained when the sample to be tested is amplified by GAP-1 and GAP-2 primers
  • Figure 19-b shows the amplification of the sample to be tested with 5'AOX and 3'AOX primers. Increase the graph.
  • Figure 20 is a graph showing the melting curve of the sample to be tested.
  • Figure 20-a shows the melting curve of the sample to be tested with GAP-1 and GAP-2 primers
  • Figure 20-b shows the melting curve of the sample with 5'AOX and 3'AOX primers.
  • the inventors have codon-optimized the gene according to the DNA sequence of proDer f1 published by GenBank (GenBank Accession No.: AB034946.1), as shown in SEQ ID No: 2, to obtain the proDer f1 gene of the present invention, nucleotide The sequence is shown in SEQ ID No: 1, and the amino acid sequence is shown in SEQ ID No: 3. The following is a comparison of the parameters before and after optimization of the proDer f1 codon:
  • CAI Codon Adaptation Index
  • the codon adaptation index (CAI) of the proDer f1 original gene in the Pichia pastoris expression system was 0.76 before the codon was optimized.
  • the proDer f1 gene has a CAI index of 0.85 in the Pichia pastoris expression system by codon optimization.
  • CAI codon adaptation index
  • the gene sequence can increase the expression level of the proDer f1 gene in the Pichia pastoris expression system.
  • the percentage of the low-utilization codon of the proDer f1 gene sequence was 10% before the codon was optimized.
  • This unoptimized gene uses tandem rare codons that may reduce translation efficiency and even disintegrate translational assemblies.
  • the proDer f1 gene has a low utilization codon frequency of 0 in the Pichia pastoris system after codon optimization.
  • the ideal distribution of GC content is 30%-70%, and any peak outside this region will affect transcription and translation efficiency to varying degrees.
  • the average content of the GC base of the proDer f1 gene shown in Fig. 4-a is 41.85%, as shown in Fig. 4-b. It shows that the peak of GC content appearing outside the 30%-70% region after optimization is optimized, and the average GC base content of proDer f1 is finally optimized to be 42.80%.
  • Example 2 Construction of an expression plasmid containing the proDer f1 gene
  • the codon-optimized proDer f1 was introduced into the XhoI restriction site sequence at the 5' end, the NotI restriction site sequence was introduced at the 3' end, and the whole gene synthesis was carried out, and the synthesized gene fragment was constructed into the pUC57 plasmid.
  • a long-term preservation plasmid was obtained, which was recorded as pUC57-proDer f1 plasmid.
  • the pUC57- pro Der f1 plasmid was used as a template for PCR amplification.
  • the primer sequences used were as follows:
  • Upstream primer (SEQ ID No: 5):
  • the total volume of the reaction was 50 ⁇ L, and the concentration of 10 ⁇ mol/L primer was added 2.5 ⁇ L, and the concentration of 10 mmol/L dNTP was added with 1 ⁇ L.
  • the DNA polymerase used was Q5 (#M0491L, purchased from New England BioLabs), 2 U/ ⁇ L, plus 0.5 ⁇ L.
  • 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 (915 bp) (results shown in Figure 5). Shown).
  • the LB solid medium containing bleomycin purchased from Invitrogen
  • the positive clones were picked and sequenced, and the sequence was identical to the expected sequence, that is, the expression plasmid optimized by proDer f1 codon was obtained, and it was recorded as pPICZ ⁇ -proDer f1 (the plasmid was constructed as shown in Fig. 6).
  • Example 3 Construction of Pichia pastoris host engineering strain containing recombinant proDer f1 gene
  • YPDS solid medium preparation Invitrogen Easy Select Pichia Expression Kit specification provided, wherein yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L, sorbitol 182g / L.
  • Electrocompetent cells were prepared according to the method of Invitrogen Easy Select Pichia Expression Kit.
  • the plasmid pPICZ ⁇ -proDer f1 obtained in Example 2 was linearized by Sac I restriction endonuclease (#R0156S, purchased from New England BioLabs), and the vector was linearized and converted into Pichia pastoris X33 after ethanol precipitation.
  • Competent cells were plated in YPDS solid medium and cultured at 30 ° C until the transformants grew.
  • Example 4 Inducible expression and identification of proDer f1 genetically engineered strains after codon optimization
  • BMGY medium Invitrogen Easy Select Pichia Expression Kit instructions, including yeast extract 10g / L, peptone 20g / L, K 2 HPO 4 3g / L, KH 2 PO 4 11.8g / L, YNB13.4g / L, Biotin 4 ⁇ 10 -4 g/L, glycerol 10 g/L.
  • BMMY medium preparation Invitrogen Easy Select Pichia Expression Kit instructions, including yeast extract 10g / L, peptone 20g / L, K 2 HPO 4 3g / L, KH 2 PO 4 11.8g / L, YNB13.4g / L, Biotin 4 ⁇ 10 -4 g/L, methanol 5 mL/L.
  • Figure 7-a Figure 7-b shows the expression of the induced strain of the engineered strain containing proDer f1. . From Fig. 7-a, Fig. 7-b, the proDer f1 protein was significantly expressed in the engineered strain.
  • Der f1 constructed in this patent is mainly obtained by ion exchange and hydrophobic chromatography.
  • the chromatographic packing was selected as HiTrap SP FF, HiTrap Q FF, HiTrap Phenyl HP. The specific steps are as follows:
  • the fermentation broth of proDer f1 host engineering strain was obtained according to Example 4.
  • the anion chromatography Der f1 breakthrough peak was collected, ammonium sulfate was added to a final concentration of 1.5 M, and the treated fermentation broth supernatant was subjected to a Phenyl HP chromatography column, and the equilibration buffer was 20 mM NaH 2 PO 4 , 1.5 M (NH 4 ).
  • Figure 10-a is the chroma chromatogram of Der f1 hydrophobic chromatography
  • Figure 10-b is the SDS-PAGE analysis of Der f1 hydrophobic chromatography. According to further calculations, the protein yield per liter of fermentation broth is as high as 200 mg or more.
  • Figure 11 shows the comparison of serum reactivity between recombinant Der f1 (rDer f1) and natural Der f1 (nDer f1, purchased from Indoor), and the results indicate that recombinant Der f1 and natural Der f1 is substantially identical to serum reactivity, indicating that recombinant Der f1 has similar biological activity compared to native protein.
  • Example 7 Determination of gene copy number of recombinant proDer f1 engineering strain
  • Upstream primer (SEQ ID No: 7)
  • GAP-1 GGTATTAACGGTTTCGGACGTATTG
  • GAP-2 GATGTTGACAGGGTCTCTCTCTTGG
  • the total volume of the reaction was 50 ⁇ L, and the concentration of 10 ⁇ mol/L primer was added 2.5 ⁇ L, and the concentration of 10 mmol/L dNTP plus 1 ⁇ L.
  • the DNA polymerase used was Taq DNA Polymerase (M0267S, New England Biolabs), 2 U/ ⁇ L, plus 0.5 ⁇ L. .
  • the reaction conditions were 94 ° C for 10 minutes, 94 ° C for 30 seconds, 55 ° C for 30 seconds, 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 the product size and expected. The size (400 bp) was consistent (results are shown in Figure 12).
  • the obtained gene product was purified by DNA gel recovery kit (DP214, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), and 2xbuffer (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.) was connected to pGM-T vector kit (VT202-01). It was purchased from Beijing Tiangen Biochemical Technology Co., Ltd., and transformed into Top10 competent cells (CB104, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), and cultured overnight at 37 ° C on blue-white spot screening medium. The next day, white clones were picked and identified by PCR. The primers used were GAP-1 and GAP-2. The PCR reaction conditions were consistent with the above conditions. The product was analyzed by 1.0% agarose gel electrophoresis.
  • the results showed the product size and expected size (400bp). Consistent (results shown in Figure 13), the positive clones were sent to Nanjing Kingsray Biotechnology Co., Ltd. for sequencing, alignment, and the expected sequence was identical, that is, the T vector clone of the GAP gene was obtained, which was recorded as T-GAP, inoculated.
  • the correctly sequenced T-GAP clone was cultured in LB liquid medium at 37 ° C overnight, and the plasmid (plasmid extract kit DP103, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.) was extracted to obtain a standard plasmid for real-time quantitative PCR.
  • the 5'AOX, 3'AOX primer amplifies the proDer f1 gene, and the primer sequences used are as follows:
  • Upstream primer (SEQ ID No: 9):
  • the total volume of the reaction was 50 ⁇ L, and the concentration of 10 ⁇ mol/L primer was added to 2.5 ⁇ L, and the concentration of 10 mmol/L dNTP plus 1 ⁇ L.
  • the DNA polymerase used was Taq DNA Polymerase (#M0267S, New England Biolabs), 2 U/ ⁇ L, plus 0.5. ⁇ L.
  • the reaction conditions were 94 ° C for 10 minutes, 94 ° C for 30 seconds, 49 ° C for 30 seconds, 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 the product size and expected.
  • the size (1500 bp) was consistent (results are shown in Figure 14).
  • the obtained gene product was purified by DNA gel recovery kit (DP214, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.) and ligated into pGM-T vector kit (VT202-01, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.).
  • the cells were transformed into Top10 competent cells (CB104, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), and cultured on a blue-white spot screening medium at 37 ° C overnight. The next day, white clones were picked and identified by PCR.
  • the primers used were 5'AOX and 3'AOX.
  • the PCR reaction conditions were consistent with the above conditions.
  • the product was analyzed by 1.0% agarose gel electrophoresis. The results showed the product size and expected size (1500 bp). Consistent (results shown in Figure 15), positive clones were sent to Nanjing Kingsray Biotechnology Co., Ltd. for sequencing, alignment, and the expected sequence was identical, that is, the T vector clone of proDer f1 was obtained, which was recorded as T-proDer f1. The correct T-proDer f1 clone was inoculated and cultured in LB liquid medium at 37 ° C overnight, and the plasmid (DNA Plasmid 103, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.) was extracted to obtain the standard plasmid for real-time quantitative PCR. .
  • a standard nucleic acid concentration (ng/ ⁇ L) was determined using a micronuclear analyzer (Nanodrop 2000, available from Thermo Fisher). Calculate the copy number of GAP and proDer f1 according to the following formula:
  • the pPICZ ⁇ -proDer f1-X33 engineered strain was inoculated in YPD liquid medium at 30 ° C overnight, and the genome was extracted the next day, and its concentration (ng/ ⁇ L) and purity were measured by a micro nucleic acid quantifier.
  • the standard copy number of the standard plasmids T-GAP and T-proDer f1 were serially diluted to 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 copies/ ⁇ L, respectively, with GAP-1 and GAP-2.
  • 5'AOX and 3'AOX are primers for real-time PCR
  • Figure 16-a is a T-GAP standard plasmid amplification curve
  • 16-b is a T-proDer f1 standard plasmid amplification curve
  • Figure 17-a is The T-GAP standard plasmid melting curve
  • 17-b is the T-proDer f1 standard plasmid melting curve
  • each gradient was repeated 3 times to verify the repeatability of the standard curve.
  • Figure 18-a is the standard curve of the T-GAP standard plasmid
  • Figure 18-b is the standard curve of the T-proDer f1 standard plasmid.
  • the extracted pPICZ ⁇ -proDer f1-X33 genomic sample was serially diluted 10-fold to obtain four gradients of stock solution, 10 -1 , 10 -2 , and 10 -3 .
  • Fluorescence quantitative PCR was performed using GAP-1 and GAP-2, 5'AOX and 3'AOX as primers, and each gradient was assayed three times.
  • Figure 19-a is an amplification curve of the sample to be tested with GAP-1 and GAP-2 as primers
  • Fig. 19-b is an amplification curve of the sample to be tested with 5'AOX and 3'AOX as primers
  • Fig. 20 -a is a melting curve of the sample to be tested with GAP-1 and GAP-2
  • the 20-b is a melting curve of the sample to be tested with 5'AOX and 3'AOX as primers.
  • the GAP gene exists in a single copy in Pichia pastoris. Therefore, the copy number of the GAP gene can be used to characterize the initial copy number of the genome in the template.
  • the copy number of the proDer f1 gene to the copy number of the GAP gene is the proDer f1 gene.
  • the copy number of the proDer f1 gene in the recombinant engineered strain was determined to be 5.
  • Example 8 Analysis of the active elements in the Der f1 genome
  • the expression vector is homologously recombined with the host chromosome, and the exogenous gene expression framework is integrated into the chromosome to realize the expression of the foreign gene; the typical Pichia expression vector contains the alcohol oxidase gene.
  • the main structures include AOX promoter, multiple cloning site, transcription termination and polyA surname gene sequence (TT), screening markers and the like.
  • the promoter is a cis-origin of gene expression regulation and an important element of the gene engineering expression vector. The important role of the promoter at the transcriptional level determines the gene expression level.
  • the proDer f1 genome was extracted according to the method of Example 7, and the proDer f1 gene was amplified, and 5'AOX and 3'AOX were used as primers.
  • the samples were sent to Nanjing Jinsrui Biotechnology Co., Ltd. to determine the position of the proDer f1 gene inserted into the genome. The original function.
  • the results of genome sequencing indicated that the proDer f1 gene expression framework was integrated into the chromosome of Pichia pastoris.
  • the proDer f1 gene can express the gene by using the AOX promoter on the yeast chromosome, so the expression level is higher.
  • the inventor chose the enzyme cleavage site closest to the ATG1 ATG, and found the proDer f1 gene by genome sequencing. It is only 242 bp away from ATX1 ATG.
  • the alpha-factor signal peptide and Kozak sequence GCCACCATGG are added in front of proDer f1 gene. This signal peptide and sequence can greatly improve transcription and translation efficiency and increase proDer f1 gene in eukaryotes. The efficiency of expression.

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Abstract

提供了一种经优化的proDer f1基因、其编码的proDer f1蛋白、包含所述基因的载体和毕赤酵母菌株。还提供了该proDer f1蛋白的表达方法和纯化方法。

Description

一种重组粉尘螨1类变应原蛋白及其制备方法和应用 技术领域
本发明属于生物工程基因领域，涉及一种重组粉尘螨1类变应原及其编码基因和表达纯化方法。
背景技术
尘螨种类繁多，广泛存在于人类生活和工作环境中，其排泄物、代谢物及螨体均具有较强的变应原型，据统计全球约10％的人口尘螨过敏，约80％的外源性哮喘由尘螨引起。
目前，临床上主要采用尘螨变应原粗提液治疗尘螨引起的变态反应性疾病，例如2006年上市的浙江我武生物的“畅迪”粉尘螨滴剂即为粉尘螨代谢培养物的提取液。尘螨变应原主要存在于排泄物和螨体中，采用提取方法耗时长，过程繁琐，成本较高；此外天然变应原提取液的组成非常复杂，恒定其组分非常困难，且容易受到外源性有毒物质、病原体微生物的污染。长期使用尘螨变应原粗提液易导致红晕、肿胀、硬结、坏死等局部反应和休克、水肿、支气管痉挛、荨麻疹、血管性水肿、全身性红斑等全身反应。此外，采用粗提液进行诊断，无法明确患者对变应原各组分的反应程度，易致误诊。
变应原的质量对变态反应疾病的诊断和治疗至关重要，用于免疫诊治的变应原应该是纯品而不宜为粗提取液。重组变应原与粗提液相比具有以下优势：(1)重组变应原具有更高的纯度，与粗提液相比不含非变应原成分、酶类、酶抑制剂和毒性蛋白等；(2)重组蛋白成分单一、具有较好的特异性，粗提液中成分复杂，患者可能只与其中部分成分反应，特异性差；(3)重组变应原与天然提取液相比减少了IgE结合的抗原表位，有效降低IgE介导的过敏反应，同时保留变应原T细胞识别所必须的结构域，具有较好的免疫原性，减少免疫治疗的危险性，提高脱敏治疗的效果。
尘螨变应原组成复杂，约有30余种，其中1类和2类变应原是最主要的变应原组分。目前对粉尘螨的1类变应原(Der f1)研究最全面的是日本学者Toshiro Takai等在2005年进行的研究，文章表明Der f1在毕赤酵母系统中表达时需要加入Der f1蛋白的前肽(带前肽的Der f1，记为proDer f1)，否则无法在真核表达系统中进行表达，然后再经过活化过程得到与天然蛋白氨基酸序列一致的成熟的Der f1蛋白；文章中未对proDer f1基因进行优化，产量较低，暂时还没有更进一步的研究报道。
发明内容
为了克服以上缺点，发明人将proDer f1基因在毕赤酵母表达系统中进行了优化，并在分 子设计时加入了提高proDer f1表达的作用原件，发明人惊喜地发现，经过基因优化后proDer f1与现有技术相比具有更高的表达量；此外，发明人对proDer f1的活化工艺进行了深入研究和优化，采用了更具有操作性和可放大的活化工艺，经过纯化后的成熟Der f1蛋白与天然蛋白相比具有相似的生物学活性。
本发明的一个目的是提供一种编码proDer f1蛋白的DNA序列，其碱基序列如SEQ ID NO:1所示。该序列针对毕赤酵母表达系统进行了密码子优化，其更利于proDer f1在毕赤酵母中表达。
本发明的另一个目的是提供一种proDer f1蛋白，其氨基酸序列如SEQ ID NO:3所示。
本发明的另一个目的是提供一种Der f1蛋白，其氨基酸序列如SEQ ID NO:4所示
本发明的另一个目的是提供一种含有上述编码优化后proDer f1基因的载体，优选的，所述的载体为pAO815，pPIC9，pPIC9K，pPIC3.5，pPIC3.5K，pPICZα A、B、C或pGAPZα A、B、C，更优选为pPIC3.5K，pPICZα A或pGAPZα A。
本发明的另一个目的是提供一种包含上述所述载体的毕赤酵母菌株，优选的，所述毕赤酵母菌株为SMD1168、GS115、KM71、X33或KM71H，更优选为KM71或X33菌株。
作为优选，编码proDer f1蛋白的DNA序列与毕赤酵母上AOX1的ATG仅相差242bp；编码proDer f1蛋白的DNA序列前有alpha-factor信号肽和Kozak序列GCCACCATGG。
本发明的另一个目的是提供一种proDer f1蛋白的表达方法，所述方法包含下述步骤：
A.构建含有上述编码proDer f1基因的载体；
B.将步骤A的载体线性化后转入毕赤酵母菌株中，并在合适的条件下培养；
C.回收纯化蛋白质。
上述所述载体优选为pPIC3.5K，pPICZα A或pGAPZα A。
上述所述毕赤酵母菌株优选为KM71或X33菌株。
更优选的，上述所述载体为pPICZαA，并且上述所述毕赤酵母菌株为X33菌株。
本发明的另一个目的是提供一种重组Der f1蛋白纯化方法，所述纯化方法如下：
A.将proDer f1发酵液低温高速离心收集上清，于5KD透析袋，25mM乙酸钠，pH＝4.5缓冲液中透析48h，0.45μm滤膜过滤。
B.第一步阳离子层析，用平衡缓冲液平衡层析柱，接着运用纯化系统将步骤A中已活化的成熟的Der f1发酵液通过分离填料，然后运用洗脱缓冲液梯度洗脱，收集洗脱峰，平衡缓冲液为50mM乙酸钠，pH＝4.5，洗脱缓冲液为50mM乙酸钠，1.0M氯化钠，pH＝4.5。
C.第二步首先将B中收集得到的Der f1蛋白峰用20mM磷酸盐pH＝6.0溶液超滤，平衡缓冲液平衡层析柱，将超滤的Der f1蛋白溶液上阴离子层析填料，收集穿透峰，平衡缓冲液为20mM 磷酸盐，pH＝6.0。
D.第三步将C中穿透峰加入硫酸铵至终浓度1.5M，pH＝6.0，平衡缓冲液平衡层析柱，Der f1样品上疏水层析填料，洗脱缓冲液梯度洗脱，平衡缓冲液为1.5M硫酸铵，20mM磷酸盐，pH＝6.0，洗脱缓冲液为20mM磷酸盐，pH＝6.0。
本发明的另一个目的是提供重组Der f1蛋白在制备治疗尘螨变态反应性疾病药物中的应用。所述变态反应性疾病为过敏性鼻炎、过敏性哮喘等。
本发明的重组proDer f1蛋白具有较高的表达量，且与天然蛋白具有相似的生物学活性。
附图说明
图1表示优化前后proDer f1基因序列对比图。
其中优化前序列对应的为天然proDer f1基因核苷酸序列；优化后序列对应的为本发明的重组proDer f1的基因核苷酸序列，即密码子优化后的序列。
图2-a，2-b为优化前后proDer f1基因在毕赤酵母表达系统中的CAI指数。
其中，图2-a表示天然proDer f1基因核苷酸序列在毕赤酵母表达系统中CAI指数经程序计算为0.76；图2-b表示优化后的本发明的proDer f1密码子在毕赤酵母表达系统中CAI指数经程序计算为0.85。
图3-a，3-b为密码子优化前后proDer f1基因在毕赤酵母表达宿主中最优密码子频率分布区域图。
其中图3-a表示proDer f1天然基因核苷酸序列在毕赤酵母系统中最优密码子频率分布区域图，从图中可以看出：proDer f1天然基因核苷酸序列的低利用率密码子出现百分比为10％；图3-b表示优化后的本发明的proDer f1密码子在毕赤酵母系统中最优密码子频率分布区域图，优化后的本发明的proDer f1密码子序列低利用率密码子出现为0。
图4-a，4-b为密码子优化前后proDer f1基因在毕赤酵母表达系统中平均GC碱基含量分布区域图。
其中，图4-a表示proDer f1天然基因核苷酸序列在毕赤酵母表达系统中平均GC碱基含量为：41.85％；图4-b表示优化后的本发明的proDer f1密码子在毕赤酵母表达系统中平均GC碱基含量为：42.10％。
图5为密码子优化后proDer f1基因PCR产物的琼脂糖凝胶电泳图。
其中，泳道1为200bp DNA Ladder；泳道2为两端含有XhoI和NotI酶切位点的重组proDer f1基因PCR产物。
图6为密码子优化后proDer f1表达质粒pPICZα-proDerf1构建过程图。
图7为密码子优化后proDer f1基因在宿主工程菌中的表达鉴定图。
其中，图7-a为密码子优化后proDer f1基因宿主工程菌株通过甲醇诱导表达一周后，菌液上清SDS-PAGE凝胶电泳图。其中泳道1为10-250KD范围的预染蛋白上样Marker；其余泳道为通过Zeocin筛选出来的proDer f1基因各阳性单克隆宿主工程菌株培养菌液上清。
图7-b为密码子优化后proDer f1基因宿主工程菌株通过甲醇诱导表达一周后，菌液上清蛋白免疫印迹图。其中泳道1为10-250KD预染蛋白Marker，泳道2-10为proDer f1单克隆诱导表达上清。
图8为proDer f1发酵液上清第一步阳离子层析色谱图和凝胶电泳图。
其中，图8-a为proDer f1发酵液上清第一步阳离子层析色谱图；图8-b为proDer f1发酵液上清第一步阳离子层析纯化鉴定结果，泳道1为11-100KD非预染蛋白Marker，泳道2为proDer f1发酵液纯化前上清，泳道3为穿透液，泳道4-8为各洗脱分管。
图9为Der f1蛋白第二步阴离子层析色谱图和凝胶电泳图。
其中，图9-a为Der f1蛋白第二步阴离子层析色谱图；图9-b为Der f1蛋白第二步阴离子层析纯化鉴定结果，泳道1为11-100KD非预染蛋白Marker，泳道2为Der f1蛋白纯化前上清，泳道3为穿透液，泳道4为洗脱峰。
图10为Der f1蛋白第三步疏层析色谱图和凝胶电泳图。
其中，图10-a为Der f1蛋白第三步疏水层析色谱图；图10-b为Der f1蛋白疏水层析纯化鉴定结果，泳道1为11-100KD非预染蛋白Marker，泳道2-10为各洗脱分管。
图11为重组Der f1与天然Der f1比较与血清反应性，其中nDerf1表示天然Der f1蛋白，rDerf1表示重组Der f1蛋白，NC为pH＝7.4PBS溶液。
图12为PCR扩增GAP基因琼脂糖凝胶电泳图，其中泳道1为250bp DNA Ladder，泳道2为GAP基因。
图13为PCR鉴定GAP基因T载体阳性克隆琼脂糖凝胶电泳图，其中泳道1为250bp DNA Ladder，泳道2-11为蓝白斑筛选获得的阳性克隆，泳道12为为蓝白斑筛选获得的阴性克隆。
图14为PCR扩增proDer f1基因琼脂糖凝胶电泳图，其中泳道1为500bp DNALadder，泳道2为proDer f1基因。
图15为PCR鉴定proDer f1基因T载体阳性克隆琼脂糖凝胶电泳图，其中泳道1，2为蓝白斑筛选获得的阴性克隆，泳道3为500bp DNA Ladder，泳道4-13为蓝白斑筛选获得的阳性克隆，泳道14为阳性对照(proDer f1基因)，其中泳道4，6，7为含有proDer f1基因的阳性克隆，其余为假阳性克隆。
图16为标准质粒扩增曲线图。
其中图16-a为T-GAP标准质粒扩增曲线图，图16-b为T-proDerf1标准质粒扩增曲线图。
图17为标准质粒熔解曲线图。
其中图17-a为T-GAP标准质粒熔解曲线图，图17-b为T-proDerf1标准质粒熔解曲线图。
图18为标准质粒标准曲线图。
其中图18-a为T-GAP标准质粒标准曲线图，图18-b为T-proDerf1标准质粒标准曲线图。
图19为待测样品扩增曲线图。
其中图19-a为待测样品以GAP-1，GAP-2引物扩增时得到的扩增曲线图，图19-b为待测样品以5’AOX，3’AOX引物扩增得到的扩增曲线图。
图20为待测样品熔解曲线图。
其中图20-a为待测样品以GAP-1，GAP-2引物扩增时得到的熔解曲线图，图20-b为待测样品以5’AOX，3’AOX引物扩增得到的熔解曲线图。
具体实施方式
下面结合具体实施例，进一步阐述本发明，应理解，引用实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1 重组proDer f1密码子优化
发明人根据GenBank已公开的proDer f1的DNA序列(GenBank登录号：AB034946.1)，如SEQ ID No：2所示，对该基因进行密码子优化后得到本发明的proDer f1基因，核苷酸序列如SEQ ID No：1所示，氨基酸序列如SEQ ID No：3所示。下面是对proDer f1密码子优化前后各参数对比如下：
1.密码子适应指数(CAI)
由图2-a可知，密码子没有优化前，proDer f1原始基因在毕赤酵母表达系统中密码子适应指数(CAI)为0.76。由图2-b可知，通过密码子优化，proDer f1基因在毕赤酵母表达系统中CAI指数为0.85。通常CAI＝1时被认为该基因在该表达系统中是最理想的高效表达状态，CAI指数越低表明该基因在该宿主中表达水平越差，因此可以看出经过了密码子优化后得到的基因序列可以提高proDer f1基因在毕赤酵母表达系统中的表达水平。
2.最优密码子使用频率(FOP)
由图3-a可知，基于毕赤酵母表达载体，密码子没有优化前，proDer f1基因序列的低利用率密码子(利用率低于40％的密码子)出现百分比为10％。这条未进行优化的基因采用串联稀有密码子，这些密码子可能降低翻译效率，甚至能够解散翻译装配物。由图3-b可知，通过密码子优化后，proDer f1基因在毕赤酵母系统中出现低利用率密码子的频率为0。
3.GC碱基含量(GC curve)
GC含量理想分布区域为30％-70％，在这个区域外的出现任何峰都会不同程度地影响转录和翻译效率。由图4-a、图4-b的proDer f1基因的GC碱基平均含量分布区域图对比可知，由图4-a中显示proDer f1基因GC碱基平均含量为41.85％，由图4-b中显示出优化后去除在30％-70％区域外出现的GC含量峰值，最终得到优化后proDer f1的GC碱基平均含量为42.80％。
实施例2：含有proDer f1基因的表达质粒构建
将密码子优化后的proDer f1在5’端引入XhoI酶切位点序列，在3’端引入NotI酶切位点序列，并进行全基因合成，将合成的基因片段，构建到pUC57质粒(由南京金斯瑞科技有限公司提供)中，得到一种长期保存质粒，记为pUC57-proDer f1质粒。
以pUC57- proDer f1质粒为模板，进行PCR扩增，所用引物序列如下：
上游引物(SEQ ID No：5)：
M13F:TGT AAA ACG ACG GCC AGT
下游引物(SEQ ID No：6)：
M13R:CAG GAA ACA GCT ATG AC
反应总体积50μL，其中浓度为10μmol/L引物各加2.5μL，浓度为10mmol/L的dNTP加1μL，所用DNA聚合酶为Q5(#M0491L，购自New England BioLabs公司)，2U/μL，加0.5μL。反应条件为98℃5秒、55℃45秒、72℃30秒，25个循环后，产物经1.0％琼脂糖凝胶电泳分析，结果显示产物大小与预期大小(915bp)一致(结果如图5所示)。分别用XhoI(#R0146S，购自New England Biolabs公司)和NotI(#R0189S，购自New England BioLabs公司)双酶切后，1％琼脂糖电泳，得到的基因产物用DNA凝胶回收试剂盒(DP214，购自北京天根生化科技有限公司)纯化。用T4连接酶(#M0202S，购自New England BioLabs公司)连接到pPICZα A质粒(V173-20，购自Invitrogen公司)中，转化到DH5α感受态细胞(CB101，购自北京天根生化科技有限公司)中，在含有博来霉素(购自Invitrogen公司)的LB固体培养基中37℃培养过夜。第二天挑取阳性克隆菌测序，比对，与预期序列完全一致，即得到proDer f1密码子优化后的表达质粒，记为pPICZα-proDer f1(质粒构建如图6所示)。
实施例3：含有重组proDer f1基因毕赤酵母宿主工程菌株的构建
YPDS固体培养基配制：Invitrogen公司Easy SelectPichia Expression Kit说明书提供，其中酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，琼脂糖15g/L，山梨醇182g/L。
1.含有密码子优化后proDer f1宿主工程菌株的构建
按照Invitrogen公司Easy SelectPichia Expression Kit说明书的方法制备成电感受态细胞。将实施例2得到的质粒pPICZα-proDer f1，用Sac I限制性内切酶(#R0156S，购自New England BioLabs公司)酶切线性化，乙醇沉淀后将线性化载体，电转化毕赤酵母X33感受态细胞，涂布于YPDS固体培养基，30℃培养直到转化子长出。
实施例4：含有密码子优化后proDer f1基因工程菌株诱导表达及鉴定
BMGY培养基配制：Invitrogen公司Easy SelectPichia Expression Kit说明书提供，其中酵母提取物10g/L，蛋白胨20g/L，K 2HPO 43g/L，KH 2PO 411.8g/L，YNB13.4g/L，生物素4×10 -4g/L，甘油10g/L。
BMMY培养基配制：Invitrogen公司Easy SelectPichia Expression Kit说明书提供，其中酵母提取物10g/L，蛋白胨20g/L，K 2HPO 43g/L，KH 2PO 411.8g/L，YNB13.4g/L，生物素4×10 -4g/L，甲醇5mL/L。
密码子优化后proDer f1工程菌株甲醇诱导表达
挑取实施例3获得的宿主单克隆工程菌于5mL BMGY培养基中，于50mL无菌离心管中30℃，220rpm培养，至OD600＝1.0-2.0时，取1mL保存菌种，并将剩余菌液重悬后转移到BMMY中小量诱导表达，每隔24h补加甲醇至终浓度为1％。一周后，离心收集菌液上清，通过SDS-PAGE凝胶电泳和蛋白免疫印迹分析，观察表达产物条带亮度，图7-a，图7-b为含有proDer f1基因工程菌株诱导表达鉴定图。由图7-a，图7-b可知，proDer f1蛋白在工程菌株中得到了显著表达。
实施例5：重组proDer f1蛋白的纯化
本专利构建的Der f1，主要通过采用离子交换，疏水层析获得。层析填料选择为HiTrap SP FF，HiTrap Q FF，HiTrap Phenyl HP，具体步骤如下：
1.发酵液的除杂预处理
按实施例4得到proDer f1宿主工程菌株发酵液，12000rpm，15min低温离心收集上清，于5KD透析袋，25mM乙酸钠，pH＝4.5缓冲液中透析48h，0.45μm滤膜过滤即得处理后发酵液上清。
2.阳离子交换层析
将上一步处理的发酵液上SP FF阳离子交换层析柱，平衡缓冲液为50mM NaAc，pH＝4.5，洗脱缓冲液为50mM NaAc，1.0M NaCl，pH＝4.5，按照12％，25％，100％等度洗脱，样品峰 主要集中在25％洗脱峰，图8-a为Der f1离子交换纯化色谱图，图8-b为Der f1离子交换层析后SDS-PAGE分析图。
3.阴离子层析纯化
收集上一步纯化的Der f1蛋白峰，样品用20mM NaH 2PO 4，pH＝6.0溶液超滤，上HiTrap Q FF层析填料，平衡缓冲液为20mM NaH 2PO 4，pH＝6.0，洗脱缓冲液为20mM NaH 2PO 4，1.0M NaCl，pH＝6.0，收集Der f1穿透峰，图9为Der f1蛋白穿透峰。
4.疏水层析纯化
收集阴离子层析Der f1穿透峰，加入硫酸铵至终浓度为1.5M，上述处理后的发酵液上清上Phenyl HP层析柱，平衡缓冲液为20mM NaH 2PO 4，1.5M(NH 4) 2SO 4，pH＝6.0，洗脱缓冲液为20mM NaH 2PO 4，pH＝6.0，按照25％，50％，70％，100％等度洗脱，Der f1蛋白主要集中在75％洗脱峰，图10-a为Der f1疏水层析纯化色谱图，图10-b为Der f1疏水层析SDS-PAGE分析图。经进一步测算可知每升发酵液目的蛋白产量高达200mg以上。
实施例6：Der f1蛋白活性的分析
将纯化得到的Der f1蛋白用pH＝7.4PBS缓冲液透析，Pierce BCA蛋白浓度测定试剂盒(Cat No:23225)测定蛋白浓度，倍比稀释至250ng，125ng，62.5ng，31.25ng，15.625ng，与天然蛋白比较与粉尘螨病人血清反应性；图11所示为重组Der f1(rDer f1)和天然Der f1(nDer f1，购自Indoor公司)与血清反应性比较，结果说明重组Der f1与天然Der f1相比与血清反应性基本一致，说明重组Der f1与天然蛋白相比具有相似的生物学活性。
实施例7：重组proDer f1工程菌株基因拷贝数的测定
1.接种X33菌株:于YPD培养基中培养24h，基因组提取试剂盒(购自北京天根生化科技有限公司)提取X33基因组，以X33基因组为模板，GAP-1，GAP-2引物扩增GAP基因，所用引物序列如下：
上游引物(SEQ ID No：7)
GAP-1：GGTATTAACGGTTTCGGACGTATTG
下游引物(SEQ ID No：8)
GAP-2：GATGTTGACAGGGTCTCTCTCTTGG
反应总体积50μL，其中浓度为10μmol/L引物各加2.5μL，浓度为10mmol/L的dNTP加1μL，所用DNA聚合酶为Taq DNA Polymerase(M0267S，New England Biolabs)，2U/μL，加0.5μL。 反应条件为94℃10分钟，94℃30秒、55℃30秒、68℃60秒，68℃5分钟，30个循环后，产物经1.0％琼脂糖凝胶电泳分析，结果显示产物大小与预期大小(400bp)一致(结果如图12所示)。得到的基因产物用DNA凝胶回收试剂盒(DP214，购自北京天根生化科技有限公司)纯化，2xbuffer(购自北京天根生化科技有限公司)连接到pGM-T载体试剂盒(VT202-01，购自北京天根生化科技有限公司)中，转化到Top10感受态细胞(CB104，购自北京天根生化科技有限公司)中，在蓝白斑筛选培养基上37℃培养过夜。第二天挑取白色克隆菌PCR鉴定，所用引物为GAP-1，GAP-2，PCR反应条件与上述条件一致，产物经1.0％琼脂糖凝胶电泳分析，结果显示产物大小与预期大小(400bp)一致(结果如图13所示)，取阳性克隆送南京金斯瑞生物科技有限公司测序，比对，与预期序列完全一致，即得到GAP基因的T载体克隆，记为T-GAP，接种测序正确的T-GAP克隆于LB液体培养基37℃培养过夜，抽提质粒(质粒小提试剂盒DP103，购自北京天根生化科技有限公司)，即得到作为实时定量PCR的标准质粒。
2.以实施例2中pPICZα-proDer f1质粒为模板，5’AOX,3’AOX引物扩增proDer f1基因，所用引物序列如下：
上游引物(SEQ ID No：9)：
5’AOX:GACTGGTTCCAATTGACAAGC
下游引物(SEQ ID No：10)：
3’AOX:GGCAAATGGCATTCTGACAT
反应总体积50μL，其中浓度为10μmol/L引物各加2.5μL，浓度为10mmol/L的dNTP加1μL，所用DNA聚合酶为Taq DNA Polymerase(#M0267S，New England Biolabs)，2U/μL，加0.5μL。反应条件为94℃10分钟，94℃30秒、49℃30秒、68℃60秒，68℃5分钟，30个循环后，产物经1.0％琼脂糖凝胶电泳分析，结果显示产物大小与预期大小(1500bp)一致(结果如图14所示)。得到的基因产物用DNA凝胶回收试剂盒(DP214，购自北京天根生化科技有限公司)纯化，连接到pGM-T载体试剂盒(VT202-01，购自北京天根生化科技有限公司)中，转化到Top10感受态细胞(CB104，购自北京天根生化科技有限公司)中，在蓝白斑筛选培养基上37℃培养过夜。第二天挑取白色克隆菌PCR鉴定，所用引物为5’AOX，3’AOX，PCR反应条件与上述条件一致，产物经1.0％琼脂糖凝胶电泳分析，结果显示产物大小与预期大小(1500bp)一致(结果如图15所示)，取阳性克隆送南京金斯瑞生物科技有限公司测序，比对，与预期序列完全一致，即得到proDer f1的T载体克隆，记为T-proDer f1，接种测序正确的T-proDer f1克隆于LB液体培养基37℃培养过夜，抽提质粒(质粒小提试剂盒DP103，购自北京天根生化科技有限公司)，即得到作为实时定量PCR的标准质粒。
3.基因拷贝数的计算：
微量核酸分析仪(Nanodrop2000，购自ThermoFisher)测定标准质粒浓度(ng/μL)。根据以下公式计算GAP和proDer f1的拷贝数：
Copies/u＝(6.02×10 23)×(ng/μl×10 -9)/(DNA length×660)
4.待测样品处理
接种pPICZα-proDer f1-X33工程菌株于YPD液体培养基中30℃培养过夜，第二天抽提基因组，并用微量核酸定量仪测定其浓度(ng/μL)及纯度。
5.标准曲线的建立
将已知拷贝数的标准质粒T-GAP和T-proDer f1分别梯度稀释至10 8，10 7，10 6，10 5，10 4，10 3copies/μL，分别以GAP-1和GAP-2、5’AOX和3’AOX为引物进行荧光定量PCR，图16-a为T-GAP标准质粒扩增曲线图，16-b为T-proDer f1标准质粒扩增曲线图，图17-a为T-GAP标准质粒熔解曲线图，17-b为T-proDer f1标准质粒熔解曲线图，每个梯度重复测定3次，以验证标准曲线的重复性。以Ct值为纵坐标、起始模板拷贝数为横坐标建立标准曲线，图18-a为T-GAP标准质粒的的标准曲线图，图18-b为T-proDer f1标准质粒的的标准曲线图。
6.proDer f1基因在重组工程菌株中的拷贝数测定
取抽提的pPICZα-proDer f1-X33基因组样品，将其依次进行10倍稀释，得到原液、10 -1、10 -2、10 -3四个梯度。分别以GAP-1和GAP-2、5’AOX和3’AOX为引物进行荧光定量PCR，每个梯度重复测定3次。图19-a为以GAP-1，GAP-2为引物待测样品的扩增曲线图，图19-b为以5’AOX和3’AOX为引物待测样品的扩增曲线图，图20-a为以GAP-1，GAP-2为引物待测样品的熔解曲线图，图20-b为以5’AOX和3’AOX为引物待测样品的熔解曲线图。GAP基因在毕赤酵母中以单拷贝的形式存在，因此用GAP基因的拷贝数可以表征模板中基因组的起始拷贝数，proDer f1基因的拷贝数于GAP基因的拷贝数比值即为proDer f1基因在毕赤酵母基因组中的拷贝数；表1所示为proDer f1基因在毕赤酵母基因工程菌株中拷贝数检测结果，测定的拷贝数在4.85-6.02之间，消除系统误差取平均值，最终确定proDer f1基因在重组工程菌株中的拷贝数为5。
表1.实时荧光定量PCR法检测proDer f1在基因组中拷贝数结果
Figure PCTCN2017119190-appb-000001
Figure PCTCN2017119190-appb-000002
实施例8：Der f1基因组中作用原件的分析
毕赤酵母没有稳定的附加质粒，表达载体与宿主染色体发生同源重组，外源基因表达框架整体整合于染色体中以实现外源基因的表达；典型的毕赤酵母表达载体含有醇氧化酶基因的调控序列，主要的结构包括AOX启动子、多克隆位点、转录终止和polyA姓曾基因序列(TT)、筛选标记等。启动子是基因表达调控的顺式原件，也是基因工程表达载体的重要原件，启动子在转录水平上的重要作用决定了基因的表达水平。
按照实施例7中方法提取proDer f1基因组，扩增得到proDer f1基因，以5’AOX和3’AOX为引物，送样到南京金斯瑞生物科技有限公司测定proDer f1基因插入基因组中前后位置的作用原件。基因组测序结果表明proDer f1基因表达框架在毕赤酵母染色体中的整合方式为单一交叉插入，使得proDer f1基因可利用酵母染色体上的AOX启动子进行基因表达，因此表达量更高。
通常外源编码序列的第一个ATG与AOX1的ATG距离越近、表达效果越好；在基因构建时发明人选用了与AOX1的ATG距离最近的酶切位点，通过基因组测序发现proDer f1基因与AOX1的ATG仅相差242bp；此外在proDer f1基因前加上了alpha-factor信号肽和Kozak序列GCCACCATGG，此信号肽和序列可在真核生物中极大地提高转录和翻译效率，提高proDer f1基因的表达效率。

Claims (9)

  1. 编码proDer f1蛋白的DNA序列，其碱基序列如SEQ ID NO:1所示。
  2. 一种重组proDer f1蛋白，其氨基酸序列如SEQ ID NO:3所示。
  3. 一种重组Der f1蛋白，其氨基酸序列如SEQ ID NO:4所示。
  4. 含有权利要求1所述编码proDer f1基因的载体，所述的载体为pAO815，pPIC9，pPIC9K，pPIC3.5，pPIC3.5K，pPICZαA、B、C或pGAPZαA、B、C。
  5. 包含权利要求3所述载体的毕赤酵母菌株，所述毕赤酵母菌株为SMD1168、GS115、KM71、X33或KM71H。
  6. 权利要求4所述毕赤酵母菌株，其特征是编码proDer f1蛋白的DNA序列与毕赤酵母上AOX1的ATG相差242bp；编码proDer f1蛋白的DNA序列前有alpha-factor信号肽和Kozak序列GCCACCATGG。
  7. 重组proDer f1蛋白的表达方法，所述方法包含下述步骤：
    A.构建含有如权利要求1所述的编码proDer f1基因的载体；
    B.将步骤A的载体线性化后转入毕赤酵母菌株中，并在合适的条件下培养；
    C.回收纯化蛋白质。
  8. 重组Der f1蛋白纯化方法，所述纯化方法如下：
    A.将如权利要求6所述的proDer f1发酵液低温高速离心收集上清，于5KD透析袋，25mM乙酸钠，pH＝4.5缓冲液中透析48h，0.45μm滤膜过滤。
    B.第一步阳离子层析，用平衡缓冲液平衡层析柱，接着运用纯化系统将步骤A中已活化的成熟Der f1发酵液通过分离填料，然后运用洗脱缓冲液梯度洗脱，收集洗脱峰，平衡缓冲液为50mM乙酸钠，pH＝4.5，洗脱缓冲液为50mM乙酸钠，1.0M氯化钠，pH＝4.5。
    C.第二步首先将B中收集得到的Der f1蛋白峰用20mM磷酸盐pH＝6.0溶液超滤，平衡缓冲液平衡层析柱，将超滤的Der f1蛋白溶液上阴离子层析填料，收集穿透峰，平衡缓冲液为20mM磷酸盐，pH＝6.0。
    D.第三步将C中穿透峰加入硫酸铵至终浓度1.5M，pH＝6.0，平衡缓冲液平衡层析柱，Der f1样品上疏水层析填料，洗脱缓冲液梯度洗脱，平衡缓冲液为1.5M硫酸铵，20mM磷酸盐，pH＝6.0，洗脱缓冲液为20mM磷酸盐，pH＝6.0。
  9. 如权利要求3所述的重组Der f1蛋白在制备治疗尘螨变态反应性疾病药物中的应用。
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