CN105755035B - A kind of building of Hansenula yeast specific expression vector and hepatitis B virus surface antigen is being improved in the method for expressed by Hansenula yeast amount - Google Patents

Abstract

The present invention discloses a kind of building of Hansenula yeast specific expression vector and is improving hepatitis B virus surface antigen in the method for expressed by Hansenula yeast amount, belong to technical field of bioengineering, building: step 1, from eukaryon Hansenula yeast strains A TCC34438 or its derivative strain genomic DNA, design specific primer, PCR amplification transfers MOX gene promoter MOXp and terminator MOX-TT respectively；Step 2, by the promoter AOX1 and terminator AOX-TT on yeast expression vector pPICZC, MOX gene promoter MOXp and terminator MOX-TT are substituted for respectively to get polymorpha expression vector pHPZF1.0.Using the method for the invention, the hepatitis B surface antibody Hansenula yeast Yeast engineering bacterium strain that can obtain can in a manner of methanol evoked stably and efficiently HBsAg expression recombinant protein, be suitable for large-scale production HBsAg recombinant protein.

Description

Construction of a Hansenula specific expression vector and its effect on improving the surface of hepatitis B virus The method of antigen expression in Hansenula

technical field

The invention relates to the construction of a Hansenula specific expression vector and a method for increasing the expression level of hepatitis B virus surface antigen in Hansenula, belonging to the technical field of bioengineering.

Background technique

Hepatitis B (referred to as hepatitis B) vaccine has been widely used for more than 20 years, but hepatitis B virus (HepatitisBvirus, HBV) infection is still one of the most serious global health problems. According to the report of the World Health Organization, about 2 billion people in the world have current or past signs of hepatitis B virus infection, and about 600,000 people die every year from liver failure, cirrhosis and hepatocellular carcinoma caused by HBV infection. There are more than 100 million hepatitis B virus carriers in my country, and more than 20 million existing patients. The epidemic in the south is heavier than that in the north, and the rural areas are heavier than the cities. Currently, there is no specific drug for the treatment of hepatitis B, and the most effective means is vaccination against hepatitis B.

The first-generation hepatitis B vaccine is a blood-derived vaccine, which is prepared from the plasma of asymptomatic carriers (HBsAg positive). Due to different extraction methods, the obtained components are also different, but all contain purified 22nm small particle hepatitis B surface antigen. Since the 1980s, the second-generation hepatitis B vaccine has been developed. It is an antigen particle assembled from the 226 amino acid products encoded by the S gene. It can be expressed in mammalian cells and recombinant yeast by genetic engineering methods. Recombinant Hepatitis B Vaccine (Hansenula) Hepatitis B Vaccine is the latest generation of genetically engineered Hepatitis B vaccine and the third generation vaccine after blood-derived vaccine and Saccharomyces cerevisiae vaccine. The seeder verified its safety. For newborns born to HBsAg-positive mothers, the city has incorporated the recombinant (Hansenula) hepatitis B vaccine, which has a high mother-to-baby blocking rate, into the immunization program, giving free vaccinations within 12 hours of birth; in local clinical observations, healthy One month after the full vaccination in adults, 97.46% of the recipients became positive for antibodies; half a year after the full vaccination, 59.8% of the recipients had antibody levels above 1000mIU/mL, so the protection period was longer. According to the World Health Organization, the relative effectiveness of different hepatitis B vaccines is not judged by the HBsAg content. The 10ug recombinant (Hansenula) vaccine is suitable for susceptible people of any age and can provide a comprehensive, efficient and durable immune protection barrier. Focusing on immune prevention, effectively curbing the high prevalence of hepatitis B, genetic recombination (Hansenula) technology provides a new option for hepatitis B immune prevention.

Hansenula Polymorpha, also known as Pichia augusta, is currently recognized as one of the most ideal exogenous gene expression systems. Hansenula is also a methanolotrophic yeast, similar to Pichia pastoris. There are two main pathways for methanol metabolism in Hansenula yeast (LodeboerA.M., et al., 1985): one is carried out in peroxisomes, and methanol generates formaldehyde under the action of methanol oxidase (methanoloxidase, MOX) and H ₂ O ₂ , formaldehyde generates CO ₂ under the action of formaldehyde dehydrogenase (formaldehyde dehydrogenase) and formate dehydrogenase (formate dehydrogenase, FMD), and H ₂ O ₂ in the action of catalase (CAT) H ₂ O and O ₂ are generated under the environment; another metabolic pathway of methanol occurs outside the peroxisome, and methanol is finally transformed into sugars through a series of enzyme catalysis in the cytoplasm, among which dihydroxyacetone synthase (dihydroxyacetone synthase , DHAS) is the key enzyme in this process. The expression of various key enzymes in the process of methanol metabolism, including MOX, DHAS and CAT, etc. are regulated at the transcriptional level, they are induced by methanol, glycerol and sorbitol, and repressed by glucose and ethanol, but when the concentration of glucose When it is lower than 0.1%, the repression is released. Under methanol fully induced conditions, peroxisomes can account for 80% of the total cell volume, MOX and DHAS can account for 15% of the total cell protein, and their promoters have a strong function of initiating the expression of downstream genes. Used as a common promoter for exogenous gene expression in yeast. As a single-cell eukaryotic microorganism, Hansenula not only has the characteristics of fast growth and easy genetic manipulation of prokaryotes, but also has the functions of post-translational processing and modification of eukaryotic cells. In addition, Hansenula has the advantages of good safety, easy cultivation, low cost, high expression level and genetic stability, and can overcome the instability of Saccharomyces cerevisiae strain, low yield and long glycosylation side chain And the low copy number of exogenous gene integration in Pichia Pastoris. At present, drugs (such as insulin, trade name Wosulin) and HBV vaccines (trade name Hepavax-Gene) produced using the Hansenula expression system have been marketed.

Contents of the invention

Aiming at the above-mentioned problems in the prior art, the present invention provides a method for constructing a Hansenula-specific expression vector and increasing the expression level of hepatitis B virus surface antigen in Hansenula, suitable for large-scale production of HBsAg recombinant protein.

In order to achieve the above object, the construction method of a Hansenula specific expression vector pHPZF1.0 adopted in the present invention is carried out according to the following steps:

Step 1, extract the genomic DNA of the eukaryotic Hansenula strain ATCC34438 or its derivatives, design specific primers, perform PCR amplification, and transfer the MOX gene promoter MOXp and terminator MOX-TT respectively;

In step 2, the promoter AOX1 and the terminator AOX-TT on the Pichia pastoris expression vector pPICZC were respectively replaced with the MOX gene promoter MOXp and the terminator MOX-TT to obtain the Hansenula expression vector pHPZF1.0.

As an improvement, the sequence of the MOX gene promoter MOXp is the nucleotide sequence shown in SEQ ID No.3.

As an improvement, the sequence of the MOX gene terminator MOX-TT is the nucleotide sequence shown in SEQ ID No.4.

The present invention also provides a method for increasing the expression of hepatitis B virus surface antigen HBsAg in Hansenula by obtaining the expression vector pHPZF1.0 through the construction method described in any one of the above methods. The method adopts the optimization of HBsAg gene.

As an improvement, the optimized HBsAg gene is based on the amino acid sequence described in the sequence listing SEQ ID No.1, combined with the codon preference of the host strain Hansenula genome, to synthesize and optimize the HBsAg gene nucleotide sequence, which has the SEQ ID No. 1 in the sequence listing. Nucleotide sequence described in ID No.2.

By adopting the method of the present invention, the HBsAg Hansenula yeast engineering strain that can be obtained can stably and efficiently express HBsAg recombinant protein in a methanol-inducible manner, and is suitable for large-scale production of HBsAg recombinant protein.

Description of drawings

Figure 1 shows the whole process of constructing the methanol-inducible Hansenula high-efficiency expression vector pHPZF1.0;

Fig. 2 is the whole process of constructing the recombinant vector pHPZF1.0-ZS for high-efficiency expression of hepatitis B virus surface antigen HBsAg;

Figure 3 is the expression of different recombinant HBsAg recombinant proteins detected by SDS-PAGE electrophoresis:

M is standard molecular weight protein (PageRulerTM Plus Prestained Protein Ladder partNo.26616 10～170KDa, product of Thermo Scientific Company);

1 is the negative control, and the recombinant yeast strain transformed with pHPZF1.0 empty vector is cultured for 120 hours;

2-9 are respectively derived from the 120-hour crushing solution of different recombinants of pHPZF1.0-ZS transformed yeast;

Figure 4 is Western-Blot electrophoresis detection of different recombinant HBsAg recombinant protein expression;

M is standard molecular weight protein (PageRulerTM Plus Prestained Protein Ladder partNo.26616 10～170KDa, product of Thermo Scientific Company);

1 is the negative control, and the recombinant yeast strain transformed with pHPZF1.0 empty vector is cultured for 120 hours;

2-9 are respectively derived from the 120-hour crushing solution of different recombinants of pHPZF1.0-ZS transformed yeast;

The primary antibody used in Western-Blot was the mouse-derived primary antibody against HBsAg (Hep B HBsAg(1023): sc-53299, product of SANTA CRUZ Company).

Fig. 5 is the changing situation of the bacterial cell wet weight and SDS-PAGE electrophoresis detection antigen expression in the fermentation process:

M is a standard molecular weight protein (PierceTM unstained Protein Molecular Weight Markerpart No.26610 14.4～116KDa, product of Thermo Scientific Company);

1 to 10 are the broken liquids derived from the engineering bacteria HBsAg1.0-38G1 at different fermentation time points, and the corresponding time points are 24, 48, 54, 60, 66, 72, 78, 84, 90 and 96 hours , the molecular weight of HBsAg recombinant protein is 20-25kD;

Fig. 6 is the protein detection after recombinant protein HBsAg chromatographic purification:

M is a standard molecular weight protein (PierceTM unstained Protein Molecular Weight Markerpart No.26610 14.4～116KDa, product of Thermo Scientific Company);

Among them, 1 is the supernatant of the broken liquid; 2 is the product of anion exchange chromatography; 3 is the product of cation exchange chromatography; 4 is the product concentrated by the 300K hollow fiber column; 5 is the product of molecular sieve;

Fig. 7 is the HPSEC detection spectrum of the purified recombinant protein HBsAg.

Detailed ways

Taking Hansenula constitutively expressing HBsAg protein as an example, some preferred embodiments are described below, but the application of the present invention is not limited thereto. The further description of the present invention is only some specific preferred embodiments, in accordance with the requirements of the patent application and for the purpose of explaining and illustrating the content of this patent. Obviously, further improvements and changes can be made on this basis without departing from the spirit and scope of the present invention.

Using conventional molecular biology techniques (endonuclease and ligase treatment), insert the MOX promoter and terminator into pPICZ C and replace the inducible promoter-alcohol oxidase promoter and terminator on the original position of the vector, Obtain the Hansenula expression vector pHPZF1.0;

Example 1: Cloning of Hansenula methanol oxidase promoter and terminator

According to the known sequences (GenBank: A11156, AR363832 and E00783), primers MOX_P-F and MOX_P-R located at both ends of the MOX gene promoter were synthesized, respectively,

Wherein MOX_P-F is 5'-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3',

MOX_P-R1 is 5'-GGAACCTCCACCAACAACAATGATATCGAAT-3', and the primers MOX_TT-F1 and MOX_TT-R1 located at both ends of the MOX gene terminator are synthesized,

Where MOX_TT-F1 is 5'-TCGGAACTTACGAGGAGACCGGACTTGCCAG-3',

MOX_TT-R1 is 5′-CTTGTGTCTCACACCCATAATGATCCCGTT-3′, using Hansenula yeast genomic DNA as a template (for the genome extraction method, see page 485 of "Molecular Cloning Experiment Guide, Third Edition"), the MOX promoter and terminator were amplified by PCR , the amplified fragment was directly inserted into the pEASY-Blunt-Zero plasmid, and according to the method provided by the company, the bacterial clone containing the intermediate plasmid vector pMOX-P and the bacterial clone of pMOX-TT were respectively obtained (see accompanying drawing 3, Fig. 4), and then, through nucleotide sequencing analysis, it was confirmed that the MOX promoter (MOX-P) and MOX terminator (MOX-TT) were correct and complete, see SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

Embodiment 2: Construction of expression vector pHPZF1.0

The MOX-P promoter was amplified by PCR from the pMOX-P plasmid, and cloned into the vector pPICZC using the BglII-EcoRI site to obtain the plasmid pPICZC-MOXP; according to the sequence of the MOX promoter obtained by sequencing verification, the primer MOX_P-R2 was redesigned. With MOX_P-F and MOX_P-R2 as primers,

Where MOX_P-F is 5'-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3' (the underlined part is the BglII site), MOX_P-R2 is 5'-CACGTGAATTCCTCGTTTCGAAGCTTTGTTTTTGTACTTTAGATT-3' (the underlined part is the EcoRI site), and the intermediate plasmid vector pMOX-P DNA was used as a template (see page 485 of "Molecular Cloning Experiment Guide, Third Edition" for the plasmid extraction method), and the MOX promoter was amplified by PCR, and BglII and EcoRI sites were introduced at both ends of the PCR product. Double digestion was performed with restriction endonuclease BglII-EcoRI, and the DNA fragment was separated and recovered by agarose gel electrophoresis. Treat the plasmid pPICZ C (product of Invitrogen, USA) with the same restriction endonuclease, separate and recover the linearized pPICZ C plasmid DNA by agarose gel electrophoresis, then mix the above two DNA fragments and connect them with ligase Together, the intermediate plasmid vector pPICZC-MOXP (see accompanying drawing 1) is obtained, and then the Escherichia coli cell DH5α (product of Invitrogen, USA) is transformed with the above-mentioned plasmid vector to facilitate the replication and preservation of the plasmid, and finally, through nucleotide sequencing Analysis to determine the correct and complete MOX promoter sequence and insertion site.

The MOX-TT terminator was amplified by PCR from the pMOX-TT plasmid, and cloned into the above intermediate plasmid pPICZC-MOXP using the SalI-BamHI site to obtain the Hansenula expression vector pHPZF1.0; the obtained MOX terminator sequence was verified by sequencing, Primers MOX_TT-F2 and MOX_TT-R2 were redesigned.

Where MOX_TT-F2 is 5'-CCAATGTCGACCATCATCATCATCATCATTGAGGAGACGTGGAAGGACATAC-3' (the underlined part is the SalI site),

MOX_TT-R2 is 5′-CAATTAGATCTGCTAGCATTGGGGATCCGGGATATCACCACAACGTCCG-3′ (the underlined part is the BglII site), and the intermediate plasmid vector pPICZC-MOXP is used as a template (for plasmid extraction, refer to the plasmid small DNA extraction kit AP-MN-P-250, Axygen Company product), the MOX terminator was amplified by PCR, and SalI and BglII sites were introduced into both ends of the PCR product, wherein BglII and BamHI are homologous enzymes. Double digestion was performed with restriction endonuclease SalI-BglII, and the DNA fragment was separated and recovered by agarose gel electrophoresis. Treat the plasmid pPICZC-MOXP with SalI-BamHI restriction endonuclease, separate and recover the linearized pPICZC-MOXP plasmid DNA by agarose gel electrophoresis, then mix the above two DNA fragments and connect them together with ligase Obtain the Hansenula expression vector pHPZF1.0 (see accompanying drawing 1), then use the above-mentioned plasmid vector to transform Escherichia coli cells DH5α (product of Invitrogen, USA) to facilitate the replication and preservation of the plasmid, and finally, through nucleotide sequencing Analysis to determine the correct and complete MOX promoter sequence and insertion site.

Example 3: Analysis of the consensus amino acid sequence of adr subtype HBsAg

Since genotype C includes all adr serotypes, we used the standard genome sequence AY123041 of hepatitis B virus type C reported in Japan as the retrieval sequence, and Blast NCBI nucleic acid database (similarity parameters were set to 93-100%) to retrieve the HBV genome sequence Nearly 2000 entries. After investigation, 617 HBV genome sequences of genotype C reported in China were sequenced. Excluding sequencing errors and HBsAg nonsense mutation sequences, a total of 479 HBsAg protein sequences of adr serotype reported in China were obtained (25 of which were from Hong Kong sequences and 4 of them were from Hong Kong). from Taiwan). Use the function of BioEdit software to perform amino acid sequence comparison analysis, and obtain the most representative HBsAg consensus amino acid sequence (consensus amino acid sequence, that is, the sequence of amino acid residues with the highest probability of occurrence at each amino acid position of HBsAg), the sequence is as follows: Shown in SEQ ID NO:1.

Embodiment 4: Artificial synthesis of HBsAg gene after codon optimization

The HBsAg gene is derived from hepatitis B virus, and its codons are preferred by mammals, while Hansenula belongs to fungi, so there are certain differences in their gene codon preferences, and this difference is likely to affect HBsAg Stability and expression efficiency of genes and their transcripts in Hansenula cells. In order to improve the biological yield of HBsAg, under the premise of not changing its amino acid sequence (see SEQ ID NO: 1), according to the codon preferred by yeast (SharpPM, et al., 1986), the DNA of a new HBsAg protein was artificially designed and synthesized Coding sequence HBsAg-ZS (see SEQ ID NO: 2). At the same time, in order to facilitate subsequent gene cloning and recombination, the synthetic DNA sequence needs to avoid the following restriction sites SphI, ScaI, HindIII, BamHI, NheI, BstBI, EcoRI and KpnI.

Embodiment 5: Construction of recombinant plasmid pHPZF1.0-ZS

Carry out double digestion with restriction endonucleases HindIII and SalI, and link the new HBsAg gene obtained by optimization synthesis with the vector pHPZF1.0 with a ligase to obtain the Hansenula expression vector pHPZF1.0-ZS (see accompanying drawing 2 ), and then transform Escherichia coli cell JM109 (purchased from GIBCO, USA) with the above-mentioned plasmid vector to facilitate the replication and preservation of the plasmid.

Example 6: Preparation of linearized plasmid expression vector DNA

First, use a kit to extract the plasmid (for detailed steps, refer to the plasmid small DNA extraction kit AP-MN-P-250, a product of Axygen Company), and prepare and extract pHPZF1.0- ZS plasmid DNA is then digested with 1-2 times excess restriction endonuclease NsiI to make it completely linearized, and agarose gel electrophoresis can be used to detect whether the digestion is complete; then recover the kit (see gel for detailed steps). The recovery kit (Wizard SV Gel and PCR Clean-up System, product of Promega Company) was used for elution with sterile deionized water, stored at -20°C, and used for later use.

Example 7: Transformation of Yeast Cells

Inoculate Hansenula saccharomyces (purchased from the American ATCC Culture Collection Center) stored at -80°C into 5mL LYPD (1% yeast extract, 2% tryptone, 2% glucose), shake culture at 37°C for about 1 day, and culture The good bacterial solution was re-inoculated in 100mLYPD with 1% inoculum, cultured overnight at 37°C with shaking (8h) until the OD600 was 1.3-1.5, centrifuged at 4000rpm for 5 minutes, discarded the supernatant, and resuspended the precipitated yeast cells in 40mL solution In A (50mM potassium phosphate buffer, pH 7.5, 25mM DTT), incubate at 37°C for 15 minutes, centrifuge at 4000rpm for 5 minutes, discard the supernatant, and resuspend the precipitated yeast cells in 200mL of ice-cold solution B (270mM sucrose , 10mM Tris-HCl, pH7.5, 1mMMgCl2), centrifuged at 4000rpm for 5 minutes, poured off the supernatant, resuspended the precipitated yeast cells in 100mL pre-cooled solution B, centrifuged at 4000rpm for 5 minutes, poured off the supernatant, and Resuspend the precipitated yeast cells in 1mL of pre-cooled solution B, pipette 80uL into a 1.5mL centrifuge tube, mix thoroughly with the above-mentioned linearized expression vector pHPZF1.0-ZS plasmid DNA (4~5ug), and then transfer to In a 0.2 cm sterile electric shock cup after ice bath, use an electric shock instrument (product of Bio-Rad Company) to introduce the linearized expression vector plasmid DNA into yeast competent cells. The electric shock parameters used are voltage 1.5kV, capacitance 50uF, resistance 125Ω. Immediately after the electric shock is completed, add 1 mL of YPD medium (1% yeast extract, 2% casein peptone, 2% glucose) to the electric shock cup, mix well, let stand at 35°C for 1 hour, and then spread on the solid On the YPD medium (2% agar powder, 0.1 mg/mL Zeocin added to the liquid medium), the plate was inverted and placed in a constant temperature incubator at 35° C. for 2 to 3 days until transformed recombinants appeared.

Example 8: Screening of Highly Expressed Yeast Strains

Single yeast colonies (transformants) grown on YPD medium (0.1 mg/mL Zeocin) were picked one by one with a sterile toothpick into YPD medium containing gradient Zeocin (containing 1 mg/mL, 2 mg/mL, 4 mg/mL, respectively mL, 6mg/mL Zeocin), and place the plate upside down in a constant temperature incubator at 35°C for 1-2 days. As the number of copies of the foreign plasmid carrying the Zeocin resistance gene integrated into the yeast genome increases, the resistance of the transformants to Zeocin increases. Pick the transformant that can grow on the YPD plate containing 6mg/mL Zeocin, and inoculate it in 100mL of BMMY medium [1% yeast extract, 2% casein, 100mM potassium phosphate buffer (pH7.0), 1.34%YNB, 0.00004 %Biotin, 0.64% methanol (v/v)], shake culture at 35°C for 120 hours, add 0.64ml (v/v) methanol once every 24 hours, centrifuge the fermentation broth at 10,000rmp for 5 minutes, collect the cells, and after crushing, Carry out SDS-PAGE electrophoresis detection.

It can be seen from accompanying drawings 3 and 4 that the target protein band is contained in the culture supernatant lane of the recombinant yeast strain obtained through the transformation of the expression vector pHPZF1.0-ZS, while the recombinant yeast obtained through the transformation of the expression vector pHPZF1.0 There is no target protein band in the culture supernatant lane of the strain. The results indicated that the HBsAg gene was optimized according to the codons preferred by yeast and placed under the operation of a Hansenula MOX promoter, together with the MOX terminator, was integrated into the Hansenula genome. The HBsAg gene can be highly expressed under the induction of methanol under the action of MOX promoter. One of them was selected and preserved as an engineering strain, and it was named HBsAg1.0-38G1Hansenula polymorpha HBsAg1.0-38G1, and was preserved in the Chinese Type Culture Collection Center located in Wuhan University, Wuhan, China on January 15, 2016 , deposit number CCTCC No: M2016039.

Embodiment 9: High-density fermentation of recombinant yeast in 30L fermenter

1) Seed liquid preparation: Take 1 tube of working seeds of the eukaryotic Hansenula engineering strain HBsAg1.0-38G1 (1ml/tube), transfer all of them to 1.2LBMGY medium, shake and culture at 35°C for 48 hours, and the OD600 value reaches 2 ~6, then use it as seed solution;

2) Initial culture stage: 15L basic fermentation medium (4.29% potassium dihydrogen phosphate, 0.5% ammonium sulfate, 1.43% potassium sulfate, 1.17% magnesium sulfate heptahydrate, 0.10% calcium sulfate dihydrate) was placed in a 30L fermenter , 0.5882% sodium citrate dihydrate, 3.00% glycerol), the pH was adjusted and maintained to 5.5 with 14% ammonia water. Then add 4.37mL trace salt solution PTM1 (0.6% copper sulfate, 0.008% sodium iodide, 0.3% manganese sulfate, 0.02% sodium molybdate, 0.002% boric acid, 0.05% Cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate, 0.025% biotin, 0.5% sulfuric acid), at 35°C, automatically control dissolved oxygen not less than 30%;

3) Glycerol fed-batch culture stage: when glycerol is exhausted, dissolved oxygen (DO) rises sharply, at this time, feed glycerol. Add 50% glycerol (which contains 12mLPTM1/L) through a peristaltic pump, and the speed of glycerol supplementation is 7G/min, until the wet weight of the bacteria is about 250-300g/L, adjust the speed and ventilation, and the dissolved oxygen is not less than 20%. At this stage, the pH was slowly adjusted to 6.0 with 14% ammonia.

4) Methanol induction stage: After stopping the feeding of glycerin and letting the cells starve for 30 minutes, feed the feed liquid through a peristaltic pump. 6.0-6.4% (v/v). The pH was adjusted and maintained to 6.0 with 14% aqueous ammonia. Adjust the rotation speed and air flow, control the dissolved oxygen amount to not less than 20% until the wet weight of the bacteria is about 300g/L, then adjust the rotation speed to greater than 1300rpm, the air flux is the largest, and the dissolved oxygen amount is not under control, until the total fermentation time 100h tank;

5) Take a few milliliters of fermentation broth every 24 hours and centrifuge at 5000rpm at 4°C for 10 minutes, and take the supernatant of the crushed solution for SDS-PAGE detection. It is found that the concentration of the target protein band increases significantly with the prolongation of methanol induction time, and the molecular weight is about It is 24kDa, which is basically consistent with the estimated molecular weight. In addition, it was found from the electropherogram that the recombinant protein expression reached the highest peak after 100 hours of culture (see Figure 5).

Example 10: Detection of HBsAg recombinant protein expression after fermentation

1) Three batches of continuous fermentation, each batch of 500mL fermentation broth was centrifuged and washed, and then homogeneously crushed under high pressure, and the cells were broken under the same conditions. Surfactant Tween 20 was added to the crushing solution to adjust the pH value, and stirred overnight at 2-8°C; the crushed cells were centrifuged at a speed of 12,000 g to remove cell debris, and the supernatant was microfiltered at 0.45 μm and 0.2 μm.

2) ELISA was used to measure HBsAg. The HBsAg enzyme-linked immunoassay kit was purchased from Kehua Company, and the HBsAg standard product (purity 99%) was purchased from China Institute for the Control of Pharmaceutical and Biological Products. For detailed steps, please refer to the kit instruction manual.

3) It was found that the average expression level of the recombinant protein was about 1.5g/L, and the biomass, growth rate and expression level of the recombinant protein remained basically stable during the three batches of continuous fermentation, confirming that the recombinant Hansenula strain and fermentation The processes all have good stability, see Table 1 for details.

Table 1. Determination of bacterial cell concentration and exogenous protein expression during HBsAg1.0-38G1 fermentation

Fermentation batch Cell concentration (wet weight) Total weight of bacteria Antigen expression 20150119 466.7g/ml 7.686kg 1.4±0.8g/L 20150130 488.0g/ml 7.325kg 1.5±0.6g/L 20150403 455.0g/ml 8.035kg 1.5±0.7g/L

Embodiment 11: Purification of recombinant protein

After one fermentation cycle was over, except for leaving 500 mL of fermentation broth as seed liquid, the rest of the fermentation broth was used for the purification of recombinant protein. The fermented liquid is centrifuged and washed, and then placed under high pressure for homogeneous disruption until the cell disruption rate reaches 90%. Add 0.05-1.0% (v/v) surfactant Tween 20 to the crushing solution, adjust the pH value to 7.0-9.0, stir at 2-8°C for 4-16 hours; centrifuge the broken cells at a speed of 12000g to remove cell debris , use 1.0M NaOH and 1.0M HCI to adjust the pH value to 7.5-9.0, and the conductivity is less than 5.0mS/cm, 0.45μm or 0.2μm microfiltration, and then perform anion exchange chromatography. The chromatography medium is DEAE sepherose FF. The flow-through of recombinant HBsAg activity expressed by Sylvia sinensis; the flow-through liquid was subjected to cationic ion exchange chromatography, and the chromatographic medium was POROS 50HS, and the flow-through containing the activity of recombinant HBsAg expressed by Hansenula was collected, and the flow-through containing Hansenula was collected. The eluate of the ultraviolet absorption peak whose activity recovery rate of the recombinant HBsAg expressed by Scutellaria subtilis is greater than 20%; for the components obtained by hydrophobic chromatography, the sample is repeated one or more times with an ultrafiltration membrane with a molecular weight cut-off of 100-500KD Concentrate until the protein concentration in the sample is 1.0-2.0 mg/ml; the obtained concentrated solution is gel-filtered using a gel filtration column to remove some aggregates formed by HBsAg virus-like particles, and detected by SDS-PAGE electrophoresis and HPLC The recovered purity of the recombinant protein is above 99% (see accompanying drawings 6 and 7).

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modification, equivalent replacement or improvement made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

SEQUENCE LISTING

<110> Anhui Zhifeilong Koma Biopharmaceutical Co., Ltd.

<120> Construction of a Hansenula-specific expression vector and its method for increasing the expression of hepatitis B virus surface antigen in Hansenula

<160> 4

<210>1

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<212>PRT

<213> Hepatitis B virus

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Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln

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Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu

20 25 30

Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys

35 40 45

Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser

50 55 60

Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe

65 70 75 80

Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val

85 90 95

Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly

100 105 110

Thr Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala

115 120 125

Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp

130 135 140

Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg

145 150 155 160

Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu

165 170 175

Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu

180 185 190

Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile

195 200 205

Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val

210 215 220

Tyr Ile

225

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ctgttgacca gaatcctcac tattcctcag tctctggact cgtggtggac gtccttgaac 120

ttcctcggag gtgctccaac ctgccctggc cagaactcgc aatctccaac ctccaatcac 180

tctcctacct cgtgcccacc tatctgccca ggctacagat ggatgtgcct gagaagattc 240

atcattttcc tgtttatctt gctgctctgc ctgatcttct tgctggtcct cctggactac 300

cagggtatgc tgcctgtttg tccattgctg cctggaacct ccactacttc taccggtcca 360

tgcaagacgt gtaccatccc tgcccagggc acttcgatgt tcccatcctg ctgttgcacc 420

aagccttctg acggcaactg cacctgtatc cctattccat cgtcctgggc tttcgccaga 480

tttctgtggg agtgggcctc ggtgagattc tcctggttgt cgctgctcgt tccattcgtc 540

cagtggtttg tgggattgtc ccctaccgtt tggctgtcgg tcatctggat gatgtggtat 600

tggggtcctt ctctgtacaa catcttgtcc ccattcctgc ctctcttgcc aatcttcttt 660

tgcctgtggg tttacatcta atag 684

<210>3

<211>1511

<212> DNA

<213> Artificial sequence

<400>3

tcgacgcgga gaacgatctc ctcgagctgc tcgcggatca gcttgtggcc cggtaatgga 60

accaggccga cggcacgctc cttgcggacc acggtggctg gcgagcccag tttgtgaacg 120

aggtcgttta gaacgtcctg cgcaaagtcc agtgtcagat gaatgtcctc ctcggaccaa 180

ttcagcatgt tctcgagcag ccatctgtct ttggagtagaga agcgtaatct ctgctcctcg 240

ttactgtacc ggaagaggta gtttgcctcg ccgcccataa tgaacaggtt ctctttctgg 300

tggcctgtga gcagcgggga cgtctggacg gcgtcgatga ggcccttgag gcgctcgtag 360

tacttgttcg cgtcgctgta gccggccgcg gtgacgatac ccacatagag gtccttggcc 420

attagtttga tgaggtgggg caggatgggc gactcggcat cgaaattttt gccgtcgtcg 480

tacagtgtga tgtcaccatc gaatgtaatg agctgcagct tgcgatctcg gatggttttg 540

gaatggaaga accgcgacat ctccaacagc tgggccgtgt tgagaatgag ccggacgtcg 600

ttgaacgagg gggccacaag ccggcgtttg ctgatggcgc ggcgctcgtc ctcgatgtag 660

aaggcctttt ccagaggcag tctcgtgaag aagctgccaa cgctcggaac cagctgcacg 720

agccgagaca attcgggggt gccggctttg gtcatttcaa tgttgtcgtc gatgaggagt 780

tcgaggtcgt ggaagatttc cgcgtagcgg cgttttgcct cagagtttac catgaggtcg 840

tccactgcag agatgccgtt gctcttcacc gcgtacagga cgaacggcgt ggccagcagg 900

cccttgatcc attctatgag gccatctcga cggtgttcct tgagtgcgta ctccactctg 960

tagcgactgg acatctcgag actgggcttg ctgtgctgga tgcaccaatt aattgttgcc 1020

gcatgcatcc ttgcaccgca agtttttaaa accactcgc tttagccgtc gcgtaaaact 1080

tgtgaatctg gcaactgagg gggttctgca gccgcaaccg aacttttcgc ttcgaggacg 1140

cagctggatg gtgtcatgtg aggctctgtt tgctggcgta gcctacaacg tgaccttgcc 1200

taaccggacg gcgctaccca ctgctgtctg tgcctgctac cagaaaatca ccagagcagc 1260

agagggccga tgtggcaact ggtggggtgt cggacaggct gtttctccac agtgcaaatg 1320

cgggtgaacc ggccagaaag taaattctta tgctaccgtg cagtgactcc gacatcccca 1380

gtttttgccc tacttgatca cagatggggt cagcgctgcc gctaagtgta cccaaccgtc 1440

cccacacggt ccatctataa atactgctgc cagtgcacgg tggtgacatc aatctaaagt 1500

acaaaaacaa a 1511

<210>4

<211>335

<212> DNA

<213> Artificial sequence

<400>4

gagacgtgga aggacatacc gcttttgaga agcgtgtttg aaaatagttc tttttctggt 60

ttatatcgtt tatgaagtga tgagatgaaa agctgaaata gcgagtatag gaaaatttaa 120

tgaaaattaa attaaatatt ttcttaggct attagtcacc ttcaaaatgc cggccgcttc 180

taagaacgtt gtcatgatcg acaactacga ctcgtttacc tggaacctgt acgagtacct 240

gtgtcaggag ggagccaatg tcgaggtttt caggaacgat cagatcacca ttccggagat 300

tgagcagctc aagccggacg ttgtggtgat atccc 335

Claims (2)

1. a kind of method for improving hepatitis b virus s antigen HBsAg expression quantity, which is characterized in that be using deposit number The Hansenula yeast bacterial strain of CCTCC No:M2016039 ferments, containing as shown by seqid no.2 in the Hansenula yeast bacterial strain Nucleotide sequence.

2. improving the method for hepatitis b virus s antigen HBsAg expression quantity as described in claim 1, which is characterized in that The nucleotide sequence as shown in SEQ ID No.2 is based on amino acid sequence as shown in SEQIDNo.1, in conjunction with host Bacterium Hansenula yeast genome codon Preference, the nucleotide sequence of the HBsAg of synthesis.