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
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 Download PDFInfo
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- CN105755035B CN105755035B CN201610137206.8A CN201610137206A CN105755035B CN 105755035 B CN105755035 B CN 105755035B CN 201610137206 A CN201610137206 A CN 201610137206A CN 105755035 B CN105755035 B CN 105755035B
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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
Technical field
Exist the present invention relates to a kind of building of Hansenula yeast specific expression vector and improving hepatitis B virus surface antigen
The method of expressed by Hansenula yeast amount, belongs to technical field of bioengineering.
Background technique
Large-scale use is cured 20 years, but hepatitis type B virus (Hepatitis to hepatitis B (abbreviation hepatitis B) vaccine
Bvirus, HBV) infection be still one of global health problem the most serious.According to World Health Organization, the whole world about 20
Hundred million people have status or the past HBV mark, and there are about 600,000 people to die of hepatic failure caused by HBV infection, cirrhosis every year
And hepatocellular carcinoma.China hepatitis carrier is more than 100,000,000, and existing patient is more than 20,000,000, and southern prevalence overweights the north, rural area
Overweight city.The drug of specific treatment hepatitis B is had no at present, and most effective means are exactly HB vaccination.
First generation hepatitis B vaccine is vaccine from blood, uses asymptomatic carrier (HBsAg is positive) blood plasma for raw material preparation.By
In extracting method difference, gained ingredient also has difference, but the 22nm little particle hepatitis B surface antibody containing purification.The eighties
Since start develop second generation hepatitis B vaccine, be the antigen particles that 226 amino acid products for encoding S gene are assembled,
It can be expressed in mammalian cell and recombination yeast using gene engineering method.Recombinant hepatitis B vaccine (Hansenula yeast)
Hepatitis B vaccine is latest generation recombinant hepatitis b vaccine, is the third generation of vaccine after vaccine from blood, saccharomyces cerevisiae vaccine,
It is National 863 tackle key problems in science and technology achievement, existing 20,000,000 are demonstrated its safety by kind of a person.To HBsAg Positive Mothers institute
Higher recombination (Hansenula yeast) hepatitis B vaccine of Mother-infant block rate is included in the Immune Programming by raw newborn, this city, in birth 12
Free vaccination is given in hour；In local clinical observation, 1 month after the inoculation of health adult's whole process, 97.46% by kind of a person
Antibody male rotary；Whole process inoculation later six months, 59.8% by kind of person's antibody level in 1000mIU/mL or more, thus the protection period is more
It is long.According to World Health Organization's data, the relative effectivenes of different hepatitis B vaccines are not judged with HBsAg content, and 10ug recombinates the (Chinese
Inferior yeast) Susceptible population of the vaccine suitable for any age, the immunoprotection barrier of comprehensive high-efficient and lasting can be provided.Immunoprophylaxis
Based on, effectively contain that the popular state of the height of hepatitis B, genetic recombination (Hansenula yeast) technology provide for the immunoprophylaxis of hepatitis B
A kind of completely new selection.
Multiple-shaped nuohan inferior yeast (Hansenula Polymorpha), also referred to as Pichia augusta are currently generally acknowledged
One of ideal heterologous gene expression system.Hansenula yeast is also a kind of methanotrophic yeast, similar with Pichia pastoris.
There are two types of (LodeboerA.M., etal., 1985) for the main path that methanol is metabolized in Hansenula yeast: one is in peroxide
It is carried out in enzyme body, methanol acts on lower generation formaldehyde and H at methanol oxidase (methanoloxidase, MOX)2O2, formaldehyde passes through again
Formaldehyde dehydrogenase (formaldehyde dehydrogenase) and hydrogenlyase (formate dehydrogenase, FMD)
Effect is lower to generate CO2, H2O2H is generated under the action of catalase (catalase, CAT)2O and O2；Another generation of methanol
It thanks to approach and occurs external in peroxidase, methanol is finally changed into sugar by the catalytic action of enzymes a series of in cytoplasm
Class, wherein dihydroxyacetone synthase (dihydroxy acetone synthase, DHAS) is the key enzyme of this process.Methanol
Various key enzymes in metabolic process, the expression including MOX, DHAS and CAT etc. is mediated in transcriptional level, they
It by the induction of methanol, glycerol and sorbierite, is checked by glucose and ethyl alcohol, but when concentration of glucose is lower than 0.1%, is checked
Effect is released from.Under the complete inductive condition of methanol, peroxisome can account for 80%, the MOX and DHAS of cell total volume
The 15% of total protein of cell can be accounted for, their promoter has the function of extremely strong starting downstream gene expression, at present by with
Make the common promoter that foreign gene is expressed in yeast.It is raw both to have protokaryon as single celled eukaryotic microorganism for Hansenula yeast
Object fast growing is easy to the features such as genetic manipulation, and has the functions such as eukaryocyte post translational processing and modification.In addition, the inferior ferment of the Chinese
Mother is also equipped with that safety is good, be easy to cultivate, low in cost, expression quantity is high and the advantages such as inheritance stability, and can overcome such as
Saccharomyces cerevisiae (Saccharomy cescerevisiae) bacterial strain is unstable, low output and glycosylation side chain are too long and finish red ferment
The lower problem of female (Pichia Pastoris) exogenous origin gene integrator copy number.Currently, using expressed by Hansenula yeast system production
Drug (such as insulin, trade name Wosulin) and HBV vaccine (trade name Hepavax-Gene) list marketing.
Summary of the invention
In view of the above existing problems in the prior art, the present invention provides a kind of buildings of Hansenula yeast specific expression vector
And hepatitis B virus surface antigen is being improved in the method for expressed by Hansenula yeast amount, it is suitable for large-scale production HBsAg recombinant protein.
To achieve the goals above, a kind of building for Hansenula yeast specific expression vector pHPZF1.0 that the present invention uses
Method carries out as steps described below:
Step 1, it extracts eukaryon Hansenula yeast strains A TCC34438 or it derives strain genomic DNA, design specificity is drawn
Object, PCR amplification transfer MOX gene promoter MOXp and terminator MOX-TT respectively；
Step 2, it by the promoter AOX1 and terminator AOX-TT on yeast expression vector pPICZC, is substituted for respectively
MOX gene promoter MOXp and terminator MOX-TT are to get polymorpha expression vector pHPZF1.0.
As an improvement, the sequence of the MOX gene promoter MOXp is nucleotide sequence shown in SEQ ID No.3.
As an improvement, the sequence of the MOX gene terminator MOX-TT is nucleotide sequence shown in SEQ ID No.4.
Expression vector pHPZF1.0, which is obtained, the present invention also provides construction method described in a kind of any of the above-described is improving second
HBsAg B HBsAg is in the method for expressed by Hansenula yeast amount, and this method is using optimization HBsAg gene.
As an improvement, the optimization HBsAg gene based on amino acid sequence described in sequence table SEQ ID No.1, is tied
Close host strain Hansenula yeast genome codon Preference, synthesis optimizing HBsAg gene nucleotide series, in sequence table
Nucleotide sequence described in SEQ ID No.2.
Using the method for the invention, the hepatitis B surface antibody Hansenula yeast Yeast engineering bacterium strain that can be obtained can
The stably and efficiently HBsAg expression recombinant protein in a manner of methanol evoked is suitable for large-scale production HBsAg recombinant protein.
Detailed description of the invention
Fig. 1 is the building overall process of methanol evoked Hansenula yeast efficient expression vector pHPZF1.0；
Fig. 2 is the full mistake of building of hepatitis b virus s antigen HBsAg high efficient expression recombinant vector pHPZF1.0-ZS
Journey；
Fig. 3 is SDS-PAGE electrophoresis detection difference recon HBsAg recombinant protein expression:
M is standard molecular weight albumen (PageRulerTM Plus Prestained Protein Ladder part
No.26616 10~170KDa, Thermo Scientific Products)；
1 is negative control, the 120 hours broken liquid of restructuring yeast strains culture of pHPZF1.0 empty carrier conversion；
2~9 are originated from pHPZF1.0-ZS transformed yeast difference recon 120 hours broken liquid of culture；
Fig. 4 is Western-Blot electrophoresis detection difference recon HBsAg recombinant protein expression；
M is standard molecular weight albumen (PageRulerTM Plus Prestained Protein Ladder part
No.26616 10~170KDa, Thermo Scientific Products)；
1 is negative control, the 120 hours broken liquid of restructuring yeast strains culture of pHPZF1.0 empty carrier conversion；
2~9 are originated from pHPZF1.0-ZS transformed yeast difference recon 120 hours broken liquid of culture；
The primary antibody that Western-Blot is used be the source of mouse original of anti-HBsAg it is anti-(Hep B HBsAg (1023): sc-53299,
SANTA CRUZ Products).
Fig. 5 is the situation of change of thallus weight in wet base and SDS-PAGE electrophoresis detection antigen presentation in fermentation process:
M is standard molecular weight albumen (PierceTM unstained Protein Molecular Weight Marker
Part No.26610 14.4~116KDa, Thermo Scientific Products)；
1~10 is respectively the broken liquid from engineering bacteria HBsAg1.0-38G1 different fermentations time point thallus, when corresponding
Between point be respectively 24,48,54,60,66,72,78,84,90 and 96 hours, 20~25kD of molecular weight of HBsAg recombinant protein；
Fig. 6 is the Protein Detection after recombinant protein HBsAg chromatographic purifying:
M is standard molecular weight albumen (PierceTM unstained Protein Molecular Weight Marker
Part No.26610 14.4~116KDa, Thermo Scientific Products)；
Wherein, 1 is broken liquid supernatant；2 be anion-exchange chromatography product；3 be cation-exchange chromatography product；4 are
Product after the concentration of 300K hollow fiber column；5 be molecular sieve product；
Fig. 7 is the HPSEC test map of recombinant protein HBsAg after purification.
Specific embodiment
Below by taking Hansenula yeast constitutive expression HBsAg albumen as an example, some preferential embodiments, but the present invention are described
Application be not limited only to this.Of the invention further describes only some special advantageous embodiments, is according to patent application
It is required that and in order to explanation and illustration this patent content.It, can be it will be apparent that within without departing substantially from the spirit and scope of the present invention
On the basis of this, further improvements and changes are done.
Using conventional Protocols in Molecular Biology (restriction endonuclease and connection enzymatic treatment), MOX promoter and terminator are inserted into
Inducible promoter-alcohol oxidase promoter and terminator into pPICZ C and on replacement vector original position, it is inferior to obtain the Chinese
Yeast expression carrier pHPZF1.0；
Embodiment 1: the clone of Hansenula yeast methanol oxidase promoter and terminator
According to known array (GenBank:A11156, AR363832 and E00783), it is respectively synthesized positioned at MOX gene promoter
The primer MOX_P-F and MOX_P-R at sub- both ends,
Wherein MOX_P-F is 5 '-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3 ',
MOX_P-R1 is 5 '-GGAACCTCCACCAACAACAATGATATCGAAT-3 ', and synthesis is located at MOX gene end
The primer MOX_TT-F1 and MOX_TT-R1 at sub- both ends,
Wherein MOX_TT-F1 is 5 '-TCGGAACTTACGAGGAGACCGGACTTGCCAG-3 ',
MOX_TT-R1 is 5 '-CTTGTGTCTCACACCCATAATGATCCCGTT-3 ', is with Hansenula yeast genomic DNA
Template (Extraction Methods of Genome referring to " the Molecular Cloning:A Laboratory guide third edition " page 485), by PCR, amplification obtains MOX and opens
Amplified fragments are directly inserted into pEASY-Blunt-Zero plasmid by mover and terminator, according to side provided by the said firm
Method respectively obtains the bacterial clone containing intermediate plasmid vector pMOX-P and the bacterial clone (see attached drawing 3, Fig. 4) of pMOX-TT,
Then, it is analyzed by nucleotide sequencing, determines that MOX promoter (MOX-P) and MOX terminator (MOX-TT) are correct and complete
, SEQ ID NO:3 and SEQ ID NO:4 is seen respectively.
Embodiment 2: the building of expression vector pHPZF1.0
From PCR amplification MOX-P promoter on pMOX-P plasmid, carrier pPICZ is cloned into using the site BglII-EcoRI
C obtains plasmid pPICZC-MOXP；The MOX promoter sequence obtained according to sequence verification redesigns primer MOX_P-R2.With
MOX_P-F and MOX_P-R2 is primer,
Wherein MOX_P-F is 5 '-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3 ' (dashed part BglII
Site), MOX_P-R2 is 5 '-CACGTGAATTCCTCGTTTCGAAGCTTTGTTTTTGTACTTTAGATT-3 ' (dashed part
For the site EcoRI), using intermediate plasmid vector pMOX-P DNA as template, (plasmid extracting method is referring to " Molecular Cloning:A Laboratory guide
The third edition " page 485), pass through PCR, amplification obtains MOX promoter, and PCR product both ends introduce the site BglII and EcoRI.With limit
Property restriction endonuclease BglII-EcoRI processed carries out double digestion, separates and recycle the DNA fragmentation by agarose gel electrophoresis.With same
Restriction enzyme enzymatic treatment plasmid pPICZ C (U.S.'s Invitrogen Products), separated by agarose gel electrophoresis
PPICZ C Plasmid DNA with after recycling linearisation, is connected to one after then mixing above-mentioned two DNA fragmentation and with ligase
It rises and just obtains intermediate plasmid vector pPICZC-MOXP (see attached drawing 1), then convert Bacillus coli cells with above-mentioned plasmid vector
DH5 α (U.S.'s Invitrogen Products) is to facilitate the duplication and preservation that carry out the plasmid, finally, passing through nucleotide sequencing
Analysis determines the correct and complete of MOX promoter sequence and insertion point.
From PCR amplification MOX-TT terminator on pMOX-TT plasmid, interstitial in above is cloned into using the site SalI-BamHI
Grain pPICZC-MOXP, obtains polymorpha expression vector pHPZF1.0；The MOX terminator sequence obtained according to sequence verification, weight
New design primer MOX_TT-F2 and MOX_TT-R2.
Wherein MOX_TT-F2 is 5 '-CCAATGTCGACCATCATCATCATCATCATTGAGGAGACGTGGAAGGACAT
AC-3 ' (dashed part is the site SalI),
MOX_TT-R2 is that 5 '-CAATTAGATCTGCTAGCATTGGGGATCCGGGATATCACCACAACGTCCG-3 ' (are drawn
Line part is the site BglII), using intermediate plasmid vector pPICZC-MOXP as template, (plasmid is extracted to be mentioned referring to small amount plasmid DNA
Take kit AP-MN-P-250, Axygen Products), by PCR, amplification obtains MOX terminator, and PCR product both ends introduce
The site SalI and BglII, wherein BglII and BamHI is isocaudarner.Double digestion is carried out with restriction enzyme SalI-BglII,
The DNA fragmentation is separated and recycled by agarose gel electrophoresis.With SalI-BamHI restriction enzyme enzymatic treatment plasmid
PPICZC-MOXP is separated and is recycled the pPICZC-MOXP Plasmid DNA after linearisation by agarose gel electrophoresis, then will be upper
State after the mixing of two DNA fragmentations and linked together with ligase just obtain polymorpha expression vector pHPZF1.0 (see
Attached drawing 1), then with above-mentioned plasmid vector conversion Bacillus coli cells DH5 α (U.S.'s Invitrogen Products) with facilitate into
The duplication and preservation of the row plasmid are determining MOX promoter sequence and insertion point just finally, analyzing by nucleotide sequencing
It is really and complete.
The analysis of embodiment 3:adr hypotype HBsAg consensus amino acid sequences
Since genotype C contains all adr serotypes, we are with the hepatitis B c-type standard base of Japan Report
Because group sequence AY123041 is retrieval sequence, Blast NCBI nucleic acid database (Similarity Parameter is set as 93~100%) is total
Retrieval HBV gene group sequence nearly 2000.Through checking, the wherein HBV gene group sequence 617 of China Report sequence genotypes C,
Sequencing mistake and HBsAg nonsense mutation sequence are excluded, obtains adr serotype HBsAg protein sequence 479 of China Report altogether
(wherein 25 come from Hong Kong sequence, and 4 come from Taiwan).Amino acid alignment is carried out using BioEdit software function
Analysis, obtaining most representative HBsAg consensus amino acid sequences, (consensus amino acid sequence, that is, exist
The each amino acid position of HBsAg is all made of the sequence of the highest amino acid residue of probability of occurrence), sequence such as SEQ ID NO:1
It is shown.
Embodiment 4: HBsAg gene after codon optimization it is artificial synthesized
HBsAg gene is derived from hepatitis type B virus, and codon is that mammalian biological is preferred, and Hansenula yeast belongs to
Fungi, thus they there is certain differences in terms of gene codon preference, and this species diversity is likely to influence HBsAg
The stability and expression efficiency of gene and its transcription product in Hansenula yeast cell.For improve HBsAg biological yield, not
(see SEQIDNO:1) under the premise of changing its amino acid sequence, the codon had a preference for according to yeast (SharpPM, etal.,
1986), engineer and the DNA encoding sequence HBsAg-ZS for having synthesized new HBsAg albumen (see SEQ ID NO:2).With this
Meanwhile it is convenient for the ease of hereafter gene cloning and recombination, the DNA sequence dna of synthesis need to avoid following restriction enzyme site SphI,
ScaI, HindIII, BamHI, NheI, BstBI, EcoRI and KpnI.
Embodiment 5: the building of recombinant plasmid pHPZF1.0-ZS
Double digestion is carried out with restriction enzyme HindIII and SalI, the new HBsAg gene that optimum synthesis is obtained and load
Body pHPZF1.0 is linked together with ligase just obtains polymorpha expression vector pHPZF1.0-ZS (see attached drawing 2), then uses
Above-mentioned plasmid vector conversion Bacillus coli cells JM109 (being purchased from U.S. GIBCO company) with facilitate carry out the plasmid duplication and
It saves.
Embodiment 6: the preparation of linearization plasmid expression vector dna
First with kit extract plasmid (detailed step referring to small amount plasmid DNA extraction kit AP-MN-P-250,
Axygen Products), it prepares and extracts from above-mentioned bacillus coli DH 5 alpha (U.S.'s Invitrogen Products) cell
Then pHPZF1.0-ZS Plasmid DNA carries out digestion with 1~2 times of excessive restriction enzyme NsiI, is allowed to total Linearization,
It is whether complete using agarose gel electrophoresis detection digestion；Then (detailed step is referring to plastic recovery kit for QIAquick Gel Extraction Kit
Wizard SV Gel and PCR Clean-up System, Promega Products), it is washed using sterile deionization
De-, -20 DEG C of preservations are spare.
Embodiment 7: the conversion of yeast cells
The Hansenula yeast (being purchased from U.S. ATCC Culture Collection Center) that -80 DEG C save is inoculated into 5mLYPD (1% yeast
Extract, 2% tryptone, 2% glucose), 37 DEG C shake culture 1 day or so, by cultured bacterium solution with 1% inoculum concentration weight
Newly be inoculated in 100mLYPD, 37 DEG C of concussions be incubated overnight (8h) to OD600 be 1.3~1.5,4000rpm be centrifuged 5 minutes,
Fall supernatant, the yeast cells of precipitating is resuspended in 40mL solution A (50mM kaliumphosphate buffer, pH7.5,25mM DTT), 37
DEG C warm bath 15min, 4000rpm are centrifuged 5 minutes, outwell supernatant, the yeast cells of precipitating is resuspended in the solution of 200mL ice pre-cooling
In B (270mM sucrose, 10mMTris-HCl, pH7.5,1mMMgCl2), 4000rpm is centrifuged 5 minutes, supernatant is outwelled, by precipitating
Yeast cells is resuspended in the solution B of 100mL pre-cooling, and 4000rpm is centrifuged 5 minutes, supernatant is outwelled, by the yeast cells weight of precipitating
It is suspended in the solution B of 1mL pre-cooling, 80uL is drawn in 1.5mL centrifuge tube, with above-mentioned linearisation expression vector pHPZF1.0-ZS
Plasmid DNA (4~5ug) mixes well respectively, in the sterile electric shock cup of the 0.2cm after being then transferred into ice bath, utilizes electric shock instrument
(Bio-Rad Products) import the expression vector Plasmid DNA of linearisation in competent yeast cells, the shock parameters used
For voltage 1.5kV, capacitor 50uF, 125 Ω of resistance.After the completion of electric shock, the YPD culture of 1mL room temperature is added into electric shock cup immediately
Base (1% yeast extract, 2% casein peptone, 2% glucose), after mixing well, 35 DEG C stand 1 hour, are then coated on solid
On body YPD culture medium (joined 2% agar powder, 0.1mg/mL Zeocin in fluid nutrient medium), plate is inverted in 35 DEG C of constant temperature
2~3 days in incubator, until conversion recon occurs.
Embodiment 8: the screening of height expression yeast strain
The yeast single colonie (transformant) that will be grown on YPD culture medium (0.1mg/mL Zeocin) with sterile toothpick by
One picking is to the YPD culture medium containing gradient Zeocin (respectively containing 1mg/mL, 2mg/mL, 4mg/mL, 6mg/mL Zeocin)
On, plate is inverted in 35 DEG C of constant incubators 1~2 day.As the exogenous plasmid for carrying Zeocin resistant gene is integrated into
The increase of copy number in Yeast genome, transformant enhance the resistance of Zeocin.Picking can contain 6mg/mL
The transformant grown on the YPD plate of Zeocin is inoculated in 100mLBMMY culture medium [1% yeast extract, 2% casein
Peptone, 100mM kaliumphosphate buffer (pH7.0), 1.34%YNB, 0.00004%Biotin, 0.64% methanol (v/v)] in, 35 DEG C
Shake culture 120 hours, primary every 24 hours supplement 0.64ml (v/v) methanol, fermentation liquid 10000rmp is centrifuged 5 minutes, is received
Collect thallus, after being crushed, carries out SDS-PAGE electrophoresis detection.
From attached drawing 3, Fig. 4 it is found that being converted in restructuring yeast strains culture obtained through expression vector pHPZF1.0-ZS
Contain target protein band in clear liquid swimming lane, and is converted in restructuring yeast strains culture obtained through expression vector pHPZF1.0
There is no target protein band in clear liquid swimming lane then.Should the result shows that: HBsAg gene is optimized by the codon being had a preference for according to yeast
It is placed under a Hansenula yeast MOX promoter manipulation, together with MOX terminator, is integrated into Hansenula yeast genome together
In, with the growth of recombination yeast engineering bacteria, HBsAg gene can obtain under the action of MOX promoter under the induction of methanol
To high efficient expression.It selects one plant to be saved as engineering bacteria, and is named as HBsAg1.0-38G1
Hansenula ploymorpha HBsAg1.0-38G1, and be preserved on January 15th, 2016 big positioned at Wuhan, China Wuhan
China typical culture collection center, deposit number CCTCC No:M2016039.
Embodiment 9: high density fermentation of the recombination yeast in 30L fermentor
1) prepared by seed liquor: taking 1 pipe eukaryon Hansenula yeast engineering bacteria HBsAg1.0-38G1 to work seed (1ml/ pipe), to the greatest extent
Number switching in 1.2LBMGY culture medium, 35 DEG C shaken cultivation 48 hours, OD600 value reaches 2~6, then as seed
Liquid；
2) first cultivation stage: held in 30L fermentor 15L basal fermentation medium (4.29% potassium dihydrogen phosphate,
0.5% ammonium sulfate, 1.43% potassium sulfate, 1.17% epsom salt, 0.10% calcium sulphate dihydrate, 0.5882% citrate dihydrate
Sodium, 3.00% glycerol), pH is adjusted with 14% ammonium hydroxide and is maintained to 5.5.According still further to following ratios, in every liter of basal fermentation culture
4.37mL Trace salts solution PTM1 (0.6% copper sulphate, 0.008% sodium iodide, 0.3% manganese sulfate, 0.02% molybdic acid are added in base
Sodium, 0.002% boric acid, 0.05% cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate, 0.025% biotin, 0.5% sulfuric acid),
Under the conditions of 35 DEG C, automatically controls dissolved oxygen and be not less than 30%；
3) glycerol feeding cultivation stage: after glycerol depletion, dissolved oxygen (DO) suddenly rises, at this point, stream glycerol adding.By compacted
Dynamic pump stream plus 50% glycerol (wherein containing 12mLPTM1/L), it is 7G/min that glycerol, which adds speed, until thallus weight in wet base about 250~
300g/L adjusts revolving speed and ventilatory capacity, and dissolved oxygen is not less than 20%, and pH value is slowly adjusted to 6.0 with 14% ammonium hydroxide by this stage.
4) methanol induction phase: allowing after thallus starvation 30min after stopping glycerol feeding, by peristaltic pump flow feeding liquid,
Feed supplement liquid is 100% methanol (wherein containing 12mLPTM1/L), and methanol concentration On-line Control, concentration maintains 6.0~6.4%
(v/v).PH is adjusted with 14% ammonium hydroxide and is maintained to 6.0.Revolving speed and ventilatory capacity are adjusted, control dissolved oxygen amount is not less than 20% to thallus
Then revolving speed is transferred to greater than 1300rpm by weight in wet base about 300g/L, air flux is maximum, and dissolved oxygen amount is not in control, until fermentation is total
When 100h under tank；
5) it is centrifuged 10 minutes at 5000rpm4 DEG C every 24 hours access milliliter fermentation liquids, broken liquid supernatant is taken to carry out
SDS-PAGE detection, find destination protein band with the extension of methanol induction time and concentration dramatically increases, molecular weight is about
24kDa coincide substantially with middle-molecular-weihydroxyethyl is speculated.In addition, found from electrophoretogram, after culture 100 hours, recombinant protein expression
Amount reaches top (see attached drawing 5).
Embodiment 10: HBsAg recombinant protein detection of expression after fermentation
1) three batches continuously ferment, and every batch of takes 500mL fermentation liquid to be centrifuged and washed, and then high pressure is even broken, in identical item
Smudge cells under part.The surface active agent tween 20 into broken liquid adjusts pH value, and 2~8 DEG C are stirred overnight；It will broken rear cell
The revolving speed of 12000g is centrifuged off cell fragment, and supernatant passes through 0.45 μm and 0.2 μm of micro-filtration.
2) HBsAg is measured using ELISA method, HBsAg enzyme-linked immunologic detecting kit is purchased from Xiamen Kehua, HBsAg mark
Quasi- product (purity 99%) are purchased from Chinese pharmaceutical biological product and examine and determine institute, and detailed step is referring to kit specification.
3) as a result, it has been found that, the average expression amount of recombinant protein about 1.5g/L, biomass, the speed of growth and the recombination of thallus
The expression quantity of albumen kept stable in three batches of Continuous Fermentation Processes, it was demonstrated that recombination Hansenula yeast bacterial strain and zymotechnique are equal
With good stability, referring specifically to table 1.
Cell concentration and exogenous protein expression measurement in table 1.HBsAg1.0-38G1 fermentation process
| Batch fermentation | Cell concentration (weight in wet base) | Thallus total weight | Antigen presentation amount |
| 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: the purifying of recombinant protein
After a fermentation period terminates, in addition to leaving 500mL fermentation liquid as seed liquor, remaining fermentation liquid is used for
The purifying of recombinant protein.Fermentation liquid is centrifuged and is washed, and it is even broken until cell crashing ratio reaches 90% to be subsequently placed in high pressure.To
The surface active agent tween 20 of 0.05~1.0% (v/v) in broken liquid adjusts pH value to 7.0~9.0,2~8 DEG C of stirrings 4~16
Hour；The revolving speed of cell 12000g is centrifuged off cell fragment after will be broken, with 1.0MNaOH and 1.0MHCI adjust pH value to
7.5~9.0, and conductivity is less than 5.0mS/cm, then 0.45 μm or 0.2 μm of micro-filtration carry out anion-exchange chromatography, chromatography is situated between
Matter is DEAE sepherose FF, collects that the recombination hepatitis B surface antigen containing expressed by Hansenula yeast is active flows through；Liquid will be flowed through
Cation exchange chromatography is carried out, chromatography media is POROS 50HS, collects the recombination hepatitis B surface containing expressed by Hansenula yeast
Antigen active flows through, and collects the purple that the recombination hepatitis B surface antigen activity recovery containing expressed by Hansenula yeast is greater than 20% or more
The eluent of outer absorption peak；To hydrophobic chromatography obtained component, using molecular cut off is the ultrafiltration membrane of 100~500KD by sample
It is one or many to repeat to be concentrated, until protein concentration is 1.0~2.0mg/ml in sample；Obtained concentrate uses gel
Filter column carries out gel filtration, the aggregation that removal part hepatitis B surface antigen virus-like particle is formed, in SDS-PAGE electrophoresis
Recycle purity, 99% or more purity with HPLC detection recombinant protein (see attached drawing 6 and 7).
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modification, equivalent replacement or improvement etc., should all be included in the protection scope of the present invention within mind and principle.
SEQUENCE LISTING
<110>Anhui intelligence flying dragon Ke Ma Biology Pharmacy Co., Ltd
<120>a kind of building of Hansenula yeast specific expression vector and improve hepatitis B virus surface antigen in the inferior ferment of the Chinese
The method of female expression quantity
<160> 4
<210>1
<211>226
<212>PRT
<213>hepatitis type B virus
<400>1
Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln
1 5 10 15
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 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
<210>2
<211>684
<212>DNA
<213>artificial sequence
<400>2
atggagaaca ccacttcggg attcctgggt cctttgctgg ttctccaggc cggattcttc 60
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 ttggagtaga 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 acccactcgc 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.
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