CN103361377A - Method for generating HPV6 L1 (Human Papillomavirus) proteins by using hansenula polymorpha expression system - Google Patents
Method for generating HPV6 L1 (Human Papillomavirus) proteins by using hansenula polymorpha expression system Download PDFInfo
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Abstract
The invention relates to a method for generating HPV6 L1 (Human Papillomavirus) proteins by using a hansenula polymorpha expression system, and in particular discloses a method for generating recombinant hansenula polymorpha cells for expressing HPV6 L1 proteins and recombinant hansenula polymorpha cells generated by using the method. The invention further discloses a method for generating HPV6 L1 proteins by using the recombinant hansenula polymorpha cells and application of the generated HPV6 L1 proteins in preparation of a prophylactic vaccine.
Description
Invention field
The invention belongs to the medical bioengineering technical field, relate to the method that produces HPV6L1 albumen, relate in particular to the method that produces HPV6L1 albumen with the expressed by Hansenula yeast system.
Background technology
Human papillomavirus (human papillomavirus, HPV) is a kind of nonencapsulated closed loop double-stranded DNA virus, belongs to papovaviridae polyomavirus subfamily, mainly invades the epithelium mucous membrane tissue of human body, and then brings out various optimum and neoplasm pathologies.
The different subtype HPV that has at present identified out is above 200 kinds, HPV infects and has obvious tissue specificity, other HPV of different shaped for skin and mucous membrane to have a liking for the tropism different, can bring out different papillary lesion, nearly more than 30 kinds of HPV types are relevant with genital tract infection, and kind more than 20 and Tumor-assaciated are wherein arranged.Bring out the good pernicious difference of pathology according to HPV, HPV can roughly be divided into two classes: 1) high-risk-type (such as HPV16, HPV18, HPV45, HPV31, HPV33, HPV52, HPV58, HPV35, HPV59, HPV56, HPV39, HPV51 etc.): closely related with human Various Tissues malignant tumour, mainly cause severe atypical hyperplasia and infiltrating carcinoma, especially infect with the generation of cervical cancer the closest with HPV16 and HPV18 type, these two types infect and account for more than 70% of cervical cancer infection, and wherein HPV16 accounts for more than 50%; 2) low risk (such as HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV72, HPV 81 etc.): can cause the optimum proliferative venereal disease of epidermic cell, such as pointed condyloma and condyloma latum etc., the pointed condyloma that is wherein brought out by HPV6 and HPV11 accounts for more than 90%.
HPV mainly is made of virus coat and genomic dna.Genome is about 7900bp, and 8 encoding hiv protease genes are arranged.Wherein the albumen of 6 ORF codings is called early protein at the early expression of virus replication; The albumen of 2 ORF codings was expressed in the late period of virus replication, was called late protein.Late protein comprises major cat protein L1 and less important coat protein L2, and participates in the formation of virus coat.
The HPV virus capsid protein can carry out self-assembly, the L1 albumen of single expression or all can be self-assembled into virus-like particle (virus-like particle during with L1 albumen and L2 albumen coexpression in multiple expression system, VLP), wherein with at the VLP of the generations such as Yeast system, baculovirus insect expression system and mammalian cell expression system more near the structure of natural viral.Can bring out in vivo the generation neutralizing antibody after the VLP immunity that utilizes the heterogenous expression system to produce, obtain good immune protective effect.
Multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) is called again Pichia augusta, is one of current generally acknowledged ideal heterologous gene expression system.Multiple-shaped nuohan inferior yeast had both possessed prokaryotic organism and had grown fast, has been easy to the characteristics such as genetic manipulation as unicellular eukaryotic microorganisms, and the functions such as eukaryotic cell translation post-treatment and modification are arranged again.In addition, debaryomyces hansenii also possess security good, be easy to cultivate, with low cost, expression amount is high and the advantage such as inheritance stability, and can overcome unstable such as yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain, yield poorly and the glycosylation side chain is long and pichia spp (Pichia Pastoris) problem that the exogenous origin gene integrator copy number is lower.At present, use medicine (such as Regular Insulin, trade(brand)name Wosulin) and all list marketings of HBV vaccine (trade(brand)name Hepavax-Gene) that the expressed by Hansenula yeast system produces.
Summary of the invention
Aspect first, the invention provides a kind of method that produces the restructuring debaryomyces hansenii cell of expressing HPV6L1 albumen, it comprises following steps:
A) the exogenous polynucleotide insertion vector of the nucleotide sequence by will comprising coding HPV6L1 albumen comes the construction expression construct;
B) use a) the middle expression construct conversion debaryomyces hansenii cell that obtains of step; With
C) to step b) in the debaryomyces hansenii cell that obtains screen, obtain to contain the restructuring debaryomyces hansenii cell of described exogenous polynucleotide.
Aspect second, the present invention also provides the restructuring debaryomyces hansenii cell that produces according to aforesaid method.
Aspect the 3rd, the present invention also provides a kind of method of the HPV6L1 of generation albumen, may further comprise the steps:
I) under the condition that is suitable for the HPV6L1 protein expression, cultivate restructuring debaryomyces hansenii cell of the present invention; With
Ii) from culture, reclaim and purifying HPV6L1 albumen.
Described method can also comprise the step of purified HPV6L1 albumen being carried out depolymerization and repolymerization.
Aspect in the end, the purposes of HPV6L1 albumen in the vaccine that infects for the preparation of prevention HPV6 that the present invention also provides the method according to this invention to produce.
Description of drawings
The Southern trace detected result of Fig. 1 take the MOX promoter fragment as probe.Take the ATCC26012 genomic dna as contrast.1: (applied sample amount is: 1000ng) in contrast; 2: (applied sample amount is: 500ng) in contrast; 3: (applied sample amount is: 250ng) in contrast; 4: (applied sample amount is: 125ng) in contrast; 5:HP/pRMHP2.1-6wt genomic dna (applied sample amount is: 15.625ng, its brightness is between 500ng and 1000ng contrast); 6:HP/pRMHP2.1-6sc genomic dna (applied sample amount is: 15.625ng, its brightness is between 500ng and 1000ng contrast); 7:HP/pRMHP2.1-6hp genomic dna (applied sample amount is: 15.625ng, its brightness is between 500ng and 1000ng contrast).
The SDS-PAGE result of Fig. 2 A HPV6L1 albumen abduction delivering in intestinal bacteria.1: protein marker; 2: the control sample of not inducing; 3,4,5:IPTG abduction delivering sample.
The purification result of the HPV6L1 albumen of Fig. 2 B prokaryotic expression.1: protein marker; 2: the supernatant liquor after the ultrasonication; 3: the precipitation part after the ultrasonication; 4,5: stream is worn liquid; The 6:20% elutriant; The 7:100% elutriant.
Fig. 2 C rabbit polyclonal antibody purification result.1: protein marker; 2: antiserum(antisera); 3: stream is worn liquid; 4: wash-out antibody.
The Western trace of the restructuring debaryomyces hansenii cellular expression levels that Fig. 3 is different detects.1:HP/pRMHP2.1-6wt; 2:HP/pRMHP2.1-6sc; 3,4:HP/pRMHP2.1-6hp; 5: standard substance (30 μ g/L); 6: dye in advance protein marker.
The HPV6L1 protein expression detects in Fig. 4 fermenting process.1: dye in advance protein marker; 2: standard substance (30 μ g/L); 3: induced 0 hour; 4: induced 3 hours; 5: induced 6 hours; 6: induced 9 hours; 7: induced 10 hours.
The POROS 50HS purifying electrophorogram of Fig. 5 A HPV6L1 albumen.1: the upper prop sample; 2: stream is worn liquid; 3～9:NaCl gradient elution.
The CHT purifying electrophorogram of Fig. 5 B HPV6L1 albumen.1: the upper prop sample; 2:3～6: phosphoric acid salt gradient elution.
The transmission electron microscope observing result of the HPV6L1 albumen of Fig. 6 purifying.
Detailed Description Of The Invention
The inventor has successfully set up the method for utilizing debaryomyces hansenii to produce HPV6L1 albumen, and the HPV6L1 albumen that produces can be self-assembled into virus-like particle, can be used for preparing the vaccine that prevention HPV infects.
The present invention at first provides a kind of method that produces the restructuring debaryomyces hansenii cell of expressing HPV6L1 albumen, and it comprises following steps:
A) the exogenous polynucleotide insertion vector of the nucleotide sequence by will comprising coding HPV6L1 albumen comes the construction expression construct;
B) use a) the middle expression construct conversion debaryomyces hansenii cell that obtains of step; With
C) to step b) in the debaryomyces hansenii cell that obtains screen, obtain to contain the restructuring debaryomyces hansenii cell of described exogenous polynucleotide.
The present invention also comprises the restructuring debaryomyces hansenii cell that produces according to described method.
The aminoacid sequence that derives from the HPV6L1 albumen of different HPV6 virus strain can there are differences.The inventor is by comparing to HPV6L1 protein sequences all in the database, choose the highest amino-acid residue of the frequency of occurrences at each amino acid position of HPV6L1 albumen, obtained to be shown in the aminoacid sequence of SEQ ID NO:1, this sequence is the most representative consensus sequence of HPV6L1 albumen.Therefore, the HPV6L1 albumen among the present invention preferably has the aminoacid sequence shown in the SEQ ID NO:1.
In order to utilize debaryomyces hansenii to express efficiently HPV6L1 albumen, the contriver carries out the codon optimized of nucleotide sequence according to the aminoacid sequence shown in the SEQ ID NO:1 for debaryomyces hansenii.Does optimization principles comprise: a) according to debaryomyces hansenii genetic code frequency of utilization table (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi? species=4905) select the high or the highest codon of frequency of utilization; B) avoid genetic transcription or protein translation are had the negative regulatory element of potential impact, such as PolyAT district, PolyGC district, silencer (Sliencer) district and inner splice site etc.; C) carry out analysis-by-synthesis to comprising 5 ' end UTR, HPV6L1 coding region and 3 ' end UTR in interior mRNA secondary structure, avoid the formation of complicated RNA secondary structure, the free energy of mRNA secondary structure is reduced; D) adopt as far as possible and on all four 5 ' the UTR district of debaryomyces hansenii promotor downstream native sequences at upstream of coding region; E) eliminate restriction enzyme enzyme recognition site commonly used.Be shown in SEQ ID NO:4 through the nucleotides sequence of optimizing.The nucleotide sequence of employed coding HPV6L1 albumen is preferably the sequence shown in the SEQ ID NO:4 among the present invention.
The adaptable expressed by Hansenula yeast carrier of the present invention is that application number is the expressed by Hansenula yeast carrier pRMHP2.1 (comprising the sequence that is shown in SEQ ID NO:15) that describes in the Chinese patent application of 201210021524.X.The exogenous polynucleotide of the nucleotide sequence by will comprising coding HPV6L1 albumen is cloned the carrier into pRMHP2.1, can obtain expression construct of the present invention.It will be understood by those skilled in the art that expression construct of the present invention can also use other vector construction, the carrier described in the Chinese patent CN100400665C that has for example authorized.
In order to express in debaryomyces hansenii, the exogenous polynucleotide in the described expression construct operably is connected with terminator with promotor.
As used herein, " operably connecting " refers to that the function of at least two polynucleotide connects.For example, operably connect and comprise being connected between promotor and another polynucleotide, wherein said promoter sequence is initial and mediate transcribing of these another polynucleotide.Operably connect and comprise being connected between terminator and another polynucleotide, wherein said terminator stops transcribing of these another polynucleotide.
Be applicable to promotor of the present invention and include but not limited to MOX, FMD, AOX1 and DHAS promotor.In some embodiments, the promotor of using among the present invention is the MOX promotor from debaryomyces hansenii.In other embodiments, the promotor of using among the present invention is the FMD promotor from debaryomyces hansenii.Be applicable to terminator of the present invention and include but not limited to MOX terminator from debaryomyces hansenii.
Expression construct is converted into the debaryomyces hansenii cell can carry out with multiple methods known in the art, includes but not limited to the conversion of electroporation and PEG mediation.
In addition, developed multiplely for expressing foreign protein debaryomyces hansenii bacterial strain in this area, included but not limited to CGMCC2.2498 debaryomyces hansenii, ATCC34438 debaryomyces hansenii and ATCC26012 debaryomyces hansenii cell.These debaryomyces hansenii bacterial strains also can be applied to the present invention.In some embodiments, be ATCC26012 debaryomyces hansenii cell for the debaryomyces hansenii cell that transforms expression construct of the present invention.
After conversion, can be according to the resistant gene that carries on the carrier debaryomyces hansenii cell of selecting to recombinate.Suitable resistant gene includes but not limited to Kan resistant gene and Zeocin resistant gene.According to employed carrier, also may screen restructuring debaryomyces hansenii cell with the auxotrophy substratum.
Expression level and its copy number positive correlation in debaryomyces hansenii of exogenous polynucleotide in debaryomyces hansenii.Therefore, can also further screen the restructuring debaryomyces hansenii cell that contains the multiple copied exogenous polynucleotide by methods such as Southern trace or quantitative PCRs.Preferred exogenous polynucleotide copy number is greater than 5 restructuring debaryomyces hansenii cell, and more preferably the exogenous polynucleotide copy number is greater than 30 restructuring debaryomyces hansenii cell.
On the other hand, the present invention also provides a kind of method of the HPV6L1 of generation albumen, may further comprise the steps:
I) under the condition that is suitable for the HPV6L1 protein expression, cultivate restructuring debaryomyces hansenii cell of the present invention; With
Ii) from culture, reclaim and purifying HPV6L1 albumen.
Various substratum and the basic culture condition that can be used for cultivating debaryomyces hansenii known in the art, those skilled in the art can select or revise as required.The cultivation of restructuring expressed by Hansenula yeast bacterial strain of the present invention can be carried out or carry out at the bio-reactor (such as the fermentor tank of 30L) of different scales at flask according to the desirable proteins amount.According to selected promotor, can in cultivation, add suitable inductor and induce described HPV6L1 protein expression.In the situation of using MOX or FMD promotor, add methyl alcohol as inductor.
The purifying of the HPV6L1 albumen that produces can use various protein purification mode known in the art, as saltout, the combination of ultrafiltration, precipitation, chromatography etc. or these modes.In one embodiment, at first use chromatography media POROS 50HS (Applied Biosystems) to carry out preliminary purification, utilize subsequently chromatography media Macro-Prep pottery hydroxyapatite (Type II, 40 μ m) to be further purified.
(embodiment 9 to utilize the HPV6L1 albumen of the purifying of method of the present invention preparation can be self-assembled into virus-like particle, Fig. 6), and demonstrate good immunogenicity (embodiment 10) in mouse, so the present invention also provides the purposes of described HPV6L1 albumen in the vaccine that infects for the preparation of prevention HPV6.
Embodiment
The below will further specify the present invention by the mode of embodiment, but therefore not limit the present invention in the described scope of embodiments.
The analysis of embodiment 1:HPV6L1 consensus amino acid sequences
The HPV6L1 albumen of total length is comprised of 500 amino acid, through the GenBank retrieval, obtains to contain altogether 117 of 500 amino acid whose total length HPV6L1 protein sequences.Use VectorNTI software AlignX function to carry out the aminoacid sequence compare of analysis, obtain the most representative HPV6L1 consensus amino acid sequences (consensus amino acid sequence, namely all adopt the sequence of the highest amino-acid residue of probability of occurrence at each amino acid position of HPV6L1), its sequence is shown in SEQ ID NO:1.
The synthetic of embodiment 2:HPV6L1 encoding gene
The present invention has synthesized 3 kinds of different HPV6L1 nucleotide sequences altogether, is called 6wt, 6sc and 6hp:
1) the 6wt-nucleotide sequence identical with the natural HPV6L1 gene order shown in the GenBank accession number AF335604.1 is shown in SEQ ID NO:2;
2) the 6sc-inventor is according to the aminoacid sequence shown in the SEQ ID NO:1, and for the nucleotide sequence of yeast saccharomyces cerevisiae genetic code preferences brand-new design, sequence is shown in SEQ ID NO:3;
3) the 6hp-inventor is according to the aminoacid sequence shown in the SEQ ID NO:1, and for the nucleotide sequence of debaryomyces hansenii genetic code preferences brand-new design, sequence is shown in SEQ ID NO:4.
According to above nucleotide sequence, entrust Sinogenomax Co., Ltd. to carry out respectively the complete sequence synthetic of 6wt, 6sc and 6hp, be cloned in (respectively called after T-6wt, T-6sc and T-6hp) in the T carrier, and it is carried out sequence verification.
Embodiment 3: produce the expression construct of carrying different HPV6L1 nucleotide sequences
The applied expressed by Hansenula yeast carrier of the present invention is that application number is the expressed by Hansenula yeast carrier pRMHP2.1 (it comprises sequence shown in the SEQ ID NO:15) that describes in the Chinese patent application of 201210021524.X.
(1) pcr amplification of MOX promotor and MOX terminator
Take the mixed genomic DNA of debaryomyces hansenii strains A TCC26012 and ATCC34438 as template, to obtaining the MOX promotor that size is 1518bp, introduce simultaneously the NotI restriction enzyme site with following primer in the upstream;
MOX promoter primer: 5 '-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3 ' (SEQ ID NO:16)
MOX promoter primer: 5 '-TTTGTTTTTGTACTTTAGATTGATGTC-3 ' (SEQ ID NO:17)
Take the mixed genomic DNA of debaryomyces hansenii strains A TCC26012 and ATCC34438 as template, to obtaining the MOX terminator that size is 311bp, introduce simultaneously the BglII restriction enzyme site with following primer in the downstream;
MOX terminator primer: 5 '-GGAGACGTGGAAGGACATACCGC-3 ' (SEQ ID NO:18)
MOX terminator primer: 5 '-GAagatctCAATCTCCGGAATGGTGATCTG-3 ' (SEQ ID NO:19)
(2) produce the expression construct of carrying different HPV6L1 nucleotide sequences
Take the recombinant plasmid T-6wt that carries 6wt as template, obtain size as the HPV6L1wt gene of 1551bp take primer 1 and primer 2 amplification, introduce simultaneously the overlap of MOX promoter region 3 ' end in the upstream, introduce the overlap of MOX terminator 5 ' end in the downstream;
Primer 1:5 '-CATCAATCTAAAGTACAAAAACAAAATGTGGCGGCCTAGCGACAGCACAG-3 ' (SEQ ID NO:5)
Primer 2: 5 '-GCGGTATGTCCTTCCACGTCTCCTTACCTTTTAGTTTTGGCGCGCTT-3 ' (SEQ ID NO:6)
Take the recombinant plasmid T-6sc that carries 6sc as template, obtain size as the HPV6L1sc gene of 1551bp take primer 3 and primer 4 amplifications, introduce simultaneously the overlap of MOX promoter region 3 ' end in the upstream, introduce the overlap of MOX terminator 5 ' end in the downstream;
Primer 3:5 '-CATCAATCTAAAGTACAAAAACAAAATGTGGAGACCATCTGATTCTACTG-3 ' (SEQ ID NO:7)
Primer 4:5 '-GCGGTATGTCCTTCCACGTCTCCTTATCTTTTCGTCTTGGCTCTTTTC-3 ' (SEQ ID NO:8)
Take the recombinant plasmid T-6hp that carries 6hp as template, obtain size as the HPV6L1hp gene of 1551bp take primer 5 and primer 6 amplifications, introduce simultaneously the overlap of MOX promoter region 3 ' end in the upstream, introduce the overlap of MOX terminator 5 ' end in the downstream;
Primer 5:5 '-CATCAATCTAAAGTACAAAAACAAAATGTGGAGACCATCGGACTCTACTG-3 ' (SEQ ID NO:9)
Primer 6:5 '-GCGGTATGTCCTTCCACGTCTCCTTAGCGCTTCGTCTTCGCTCTTTTC-3 ' (SEQ ID NO:10)
Respectively take MOX promotor, 6wt gene/6sc gene/6hp gene, three fragments of MOX terminator as template, obtain big or small 6wt expression cassette as 3.4Kb/6sc expression cassette/6hp expression cassette take primer 7 and primer 8 amplifications, simultaneously carry the NotI restriction enzyme site in the upstream, carry the BglII restriction enzyme site in the downstream.
Primer 7:5 '-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3 ' (SEQ ID NO:11)
Primer 8:5 '-GAagatctCAATCTCCGGAATGGTGATCTG-3 ' (SEQ ID NO:12)
Respectively 6wt expression cassette, 6sc expression cassette and 6hp expression cassette are cloned in the pRMHP2.1 carrier by the NotI+BglII double digestion, obtain expression construct pRMHP2.1-6wt, pRMHP2.1-6sc and pRMHP2.1-6hp.
Embodiment 4: the generation of restructuring debaryomyces hansenii cell
(1) extraction of recombinant expression construct physique grain and enzyme are cut
Picking is transformed into the intestinal bacteria bacterium colony of the recombinant expression construct physique grain that obtains among the embodiment 3, use E.Z.N.A Plasmid Mini Kit test kit (Omega Bio-Tek company) to extract plasmid after the enlarged culturing, and carry out single endonuclease digestion with Bgl II, using E.Z.N.A Gel Extraction Kit test kit (Omega Bio-Tek company) reclaims, the sterilized water that is preheated to 55 ℃ with 60 μ L carries out wash-out, by measuring OD
260It is quantitative to carry out DNA, and linearizing fragment is diluted to 100ng/ μ l, is stored in-20 ℃ of refrigerators, for subsequent use.
(2) processing of debaryomyces hansenii cell
The single bacterium colony of picking debaryomyces hansenii strains A TCC26012 accesses in the small test tube of the YPD liquid nutrient medium that contains 5ml, cultivates 12 hours for 37 ℃; Get bacterium liquid 5ml and be forwarded in the 200ml YPD substratum, cultivated 4-6 hour for 37 ℃, be about 1.0-1.5 to OD600, in the centrifugal 10min of 6000rpm; With the resuspended thalline of 200ml0.1mol/L phosphate buffered saline buffer (containing 25mmol/L DTT, pH7.5), abundant mixing is hatched 30min in 37 ℃, and the centrifugal 10min of 6000rpm abandons supernatant, stays thalline.STM solution 200ml with precooling washes thalline, and the thalline pressure-vaccum is even, in 4 ℃ of centrifugal 3min of 6000rpm, abandons supernatant, stays precipitation.With the ice-cold resuspended thalline of STM solution 100ml, in 4 ℃ of centrifugal 3min of 6000rpm, abandon supernatant, stay precipitation., and bacterium liquid is transferred in the centrifuge tube behind the high pressure with the 100-200 μ l ice-cold resuspended thalline of STM solution according to biomass, ice bath is prepared to transform.
(3) electricity of debaryomyces hansenii cell transforms
Press plasmid: the amount of thalline=1: 2 adds restructuring expressed by Hansenula yeast plasmid 15 μ l, bacterium liquid 30 μ l, and fully pressure-vaccum is even, places ice bath to be transformed; To use in advance alcohol-pickledly, and behind the uv irradiating, take out in the electric revolving cup of-20 ℃ of refrigerations, add plasmid thalline mixed solution; Condition by voltage 2500V, resistance 150 Ω, electric capacity 50 μ F shocks by electricity; The rear rapidly adding of electric shock 1ml balance is transferred in the EP pipe to the YPD solution of room temperature gently behind the mixing; Thalline after electricity turned is placed 1h in 37 ℃ of water-baths, every interval 15min puts upside down 3 times gently; With hatching the bacterium liquid of 1h, in the centrifugal 10min of 6000rpm, abandon supernatant; With the resuspended thalline of 200 μ l YPD solution, coat in the YPD flat board that contains 0.25mg/mlZeocin with 100 μ l/ plates, be inverted in 37 ℃ and cultivated 3-7 days.
(4) restructuring expressed by Hansenula yeast bacterial strain goes down to posterity, stablizes
The recombinant bacterial strain list bacterium colony that grows on the picking Zeocin resistant panel, be inoculated in the YPD liquid nutrient medium that 5ml contains 0.25mg/ml Zeocin, cultivated 24-36 hour in 37 ℃, 200rpm shaking table, after reaching 50 to the OD value, transfer with 1: 1000 ratio and to contain in the YPD liquid nutrient medium of 0.25mg/ml Zeocin in 5ml, after being cultured to the OD value and reaching 50, transfer with 1: 1000 ratio again and contain in the YPD liquid nutrient medium of 0.25mg/ml Zeocin in 5ml, by that analogy, pass continuously 10 times and carry out the preservation of bacterial classification.The preservation system is bacterium liquid: 60% glycerine=1: 1, and amount is preserved bacterial classification as required, is generally 500 μ l bacterium liquid+500 μ l, 60% glycerine;
The restructuring expressed by Hansenula yeast bacterial strain access 5ml that reaches 10 times is not contained in the YPD liquid nutrient medium of Zeocin resistance, after 37 ℃, 200rpm shaking table are cultured to the OD value and reach 50, transfer in 5ml YPD liquid nutrient medium with 1: 1000 ratio, by that analogy, in not containing the YPD liquid nutrient medium of Zeocin resistance, pass 5 times continuously.
The YPD that bacterium liquid coating after stablizing is contained 10mg/ml G418 is dull and stereotyped, is inverted in 32 ℃ and cultivates 2-4 days, and the eugonic single bacterium colony of picking carries out the copy number detection from each flat board.
Embodiment 5: the exogenous polynucleotide copy number of restructuring debaryomyces hansenii cell detects
(1) extraction of pastoris genomic dna and quantitative
The single bacterium colony of the eugonic yeast strain that inoculation embodiment 4 obtains substratum in the YPD liquid nutrient medium of 5ml is cultivated 16～24h for 37 ℃; Get 2ml yeast culture liquid, the centrifugal 3min of room temperature 4500g collects thalline; Use the resuspended thalline of 500 μ l SCED solution (1mol/L sorbyl alcohol, 10mmol/L Trisodium Citrate, 10mmol/L EDTA, 10mmol/L DTT dithiothreitol (DTT)), and adding 50mg granulated glass sphere fully shakes 5min, add 50 μ l 10mg/ml lywallzymes, 37 ℃ of temperature are bathed 1h; The 10%SDS that adds 60 μ l, the Proteinase K of 30 μ l, the RNaseA enzyme of 10 μ l, room temperature is placed after 10 minutes and cultivate 2h in 55 ℃ water-bath; Add the saturated phenol of 350 μ l and 350 μ l chloroforms, fully mixing is rear in 13000rpm, and centrifugal 10 minutes, the upper strata liquid after the collection layering; Add equal-volume (about 700 μ l) chloroform, in 13000rpm, centrifugal 10 minutes, the upper strata liquid after the collection layering; Add the 3mol/L sodium acetate solution of 140 μ l in the liquid of upper strata, add gently 700 μ l Virahols behind the mixing again, room temperature is placed 5min behind the mixing, in 13000rpm, and centrifugal 10 minutes.Abandon supernatant, add 1ml 70% ethanol and clean, in 13000rpm, centrifugal 10 minutes, remove supernatant, after the DNA precipitation places room temperature to place 30min, add 100 μ l TE dissolving.Carry out the quantitative of genomic dna by measuring OD260, and linearizing fragment is diluted to 100ng/ μ l, be stored in-20 ℃ of refrigerators, for subsequent use.(2) Southern trace method is carried out the quantitative of exogenous polynucleotide copy number
A: probe preparation
The applied MOX probe of the present invention application number is the MOX probe of describing in the Chinese patent application of 201210021524.X, and the DIG DNALabeling and Detection kit test kit (Cat No:11093657910) of using Roche company carries out the probe preparation.Concrete steps are: add the PCR product (200ng/ μ l) of 10 μ l MOX promoter regions in the EP tubule, and seal up with sealed membrane, boiling water boiled 10 minutes.Put into immediately the dehydrated alcohol capsule (suddenly cooling) of precooling (20 ℃).Dry pipe outer wall ethanol, centrifugal (about 10s), add successively 5 μ l intracellular toxins and check water, 2 μ l 10xHexanucleotide Mix, 2 μ l 10x dTP Labeling Mixture, 1 μ l, 7 Klenow Enzyme (labeling grade), mixing, sealed membrane sealing.37 ℃ of water-baths are spent the night ,-20 ℃ of preservations.
The b:Southern trace
Digoxin hybridization check test kit I (Cat No:DIGD-110) and the efficient hybridization solution of HyB (Cat No:Hyb-500) of using Beijing Mei Laibo medical science and technology company limited carry out the detection of Southern trace according to product description.
(3) copy number of different restructuring debaryomyces hansenii bacterial strains relatively
Southern trace detected result (seeing Fig. 1) shows: the copy number of HP/pRMHP2.1-6wt bacterial strain, HP/pRMHP2.1-6sc bacterial strain and HP/pRMHP2.1-6hp bacterial strain that screening obtains approaches, and the copy number average is greater than 31 but less than 63.
The prokaryotic expression of embodiment 6:HPV6L1 albumen and the preparation of rabbit polyclonal antibody
(1) pcr amplification of HPV6L1 gene and expression plasmid clone makes up
Take pRMHP2.1-6hp as template, obtain size as the HPV6L1hp gene fragment (not being with terminator codon) of 1519bp take primer 9 and primer 10 amplifications, introduce simultaneously the NdeI restriction enzyme site in the upstream, introduce the SalI restriction enzyme site in the downstream;
Primer 9:5 '-ggaattccatATGTGGAGACCATCGGACTCTACTG-3 ' (SEQ ID NO:13)
Primer 10:5 '-CCGGTCGACGCGCTTCGTCTTCGCTCTTTTCC-3 ' (SEQ ID NO:14)
The 6hp gene fragment clone is entered in the pET43.1a carrier (Novagen company) of NdeI+XhoI double digestion the recombinant plasmid called after pET43.1a-6hp that sequence verification is correct by the NdeI+SalI double digestion.
(2) abduction delivering of HPV6L1 albumen in intestinal bacteria
Recombinant plasmid pET43.1a-6hp transforms escherichia coli expression bacterial strain BL21-CodonPlus (DE3)-RIPL, obtains to express engineering bacteria BL21-CodonPlus (DE3)-RIPL/pET43.1a-6hp.Picking list bacterium colony access contains in the 5ml LB liquid nutrient medium test tube of (containing Amp 50 μ g/ml), and 37 ℃ add IPTG to final concentration 0.5mmol/L when being cultured to OD600 and being 0.6-0.9, and induction time is 5h, and the control tube of not inducing is set simultaneously.Collect bacterium liquid, SDS-PAGE detects the expression (Fig. 2 A) of recombinant protein.
(3) purifying of HPV6L1 albumen
Preparation of samples: picking is expressed the single bacterium colony access of engineering bacteria BL21-CodonPlus (DE3)-RIPL/pET43.1a-6hp and is contained in the 5ml LB liquid nutrient medium test tube of (containing Amp 50 μ g/ml), 37 ℃ of overnight incubation, next day, the inoculum size access by 1% contained in the 400ml LB liquid nutrient medium 1L triangular flask of (containing Amp 50 μ g/ml), to OD
600Be about at 0.8 o'clock, add IPTG to final concentration 0.5mmol/L, collect bacterium liquid and centrifugal after inducing 5h, bacterial sediment is resuspended in PBS solution in the ratio of 1: 10 (g/ml), carrying out ultrasonic bacteria breaking under ice bath (5s, 5s, 400W, 60cycles), the centrifugal 10min centrifugal collecting precipitation of 10000r/min.
Purge process (Fig. 2 B): with ratio adding solution (100mM phosphoric acid salt, 500mM NaCl, the 20mM imidazoles of ultrasound precipitation in 1: 10 (g/ml), the 8M urea, pH8.0) abundant stirring and dissolving in, the centrifugal 10min of 10000r/min collects supernatant 0.45 μ m membrane filtration.Be splined on Chelating Sepharose FF chromatography media, balance liquid A is 100mM phosphoric acid salt, 500mM NaCl, the 20mM imidazoles, the 8M urea, pH8.0, elutriant B are 100mM phosphoric acid salt, 150mM NaCl, the 500mM imidazoles, 8M urea, pH8.0, elution step: wash foreign protein with 20%B stream first, again with 100%B wash-out target protein.Collect the target protein elution peak.Imidazoles is removed in dialysis.
(4) rabbit polyclonal antibody preparation
Rabbit immunity and serum are collected: 2 new zealand rabbits of HPV6L1 protein immunization of using the prokaryotic expression of purifying, immunization method adopts back multi-point injection method, immune programme for children be the 0th, 3,6,8 the week each immunity once, immunizing dose is 250 μ g/ time, antigen needs to inject after Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FIA) are fully emulsified again, first immunisation is used FCA, follow-up immunization is used FIA, take a blood sample and separation of serum by carotid artery in the 9th week, every rabbit can separation of serum 40-60ml.
Antibody purification (Fig. 2 C): antiserum(antisera) is after 0.45 μ m filters, and upper prop carries out affinitive layer purification in chromatography media rProtein A Sepharose FF.Under the pH neutrallty condition, antibodies with citric acid-sodium citrate damping fluid (pH4.0) wash-out, is collected the antibody elution peak on chromatography media, use immediately 1M Tris.Cl, and the pH9.0 pH that neutralizes is neutral.
Embodiment 7: the expression study of different restructuring debaryomyces hansenii bacterial strains
(1) abduction delivering of restructuring debaryomyces hansenii bacterial strain
Picking restructuring debaryomyces hansenii list bacterium colony accesses in the small test tube of the YPG liquid nutrient medium that contains 5ml from flat board, cultivates 24 hours for 37 ℃; Bacterium liquid is transferred in the 100ml triangular flask that contains 30ml YPM inducing culture, and initial density is OD
600=1, induced 72 hours in 37 ℃ of shaking tables, in bacterium liquid, added final concentration every 12 hours and be 0.5% methanol solution (being the 0.15ml/ bottle).
Bacterium liquid after getting 25ml and inducing is transferred in the centrifuge tube of 50ml, in the centrifugal 10min of 10000rpm, abandons supernatant; With the resuspended bacterial sediment of the lysis of 50ml, fully behind the mixing, the ultrasonication program by " power 60%, time 20min, open 5s, close 5s " in ultrasonic apparatus is carried out bacterial cell disruption, needs to keep ice bath in the shattering process; With the bacterium liquid of ultrasonication, in the centrifugal 10min of 10000rpm, it is frozen for subsequent use in-20 ℃ to collect supernatant.
(2) restructuring debaryomyces hansenii bacterial strain HPV6L1 protein expression detects
Use Western trace semi-quantitative method and carry out the detection of HPV6L1 protein expression level
Glue and electrophoresis: the polyacrylamide gel of preparation 12%, add sample to be checked and standard substance, upper strata glue is with the voltage of 8V/cm, and separation gel carries out electrophoresis with the voltage of 15V/cm, and when arriving the separation gel bottom to tetrabromophenol sulfonphthalein, stop electrophoresis downcuts separation gel;
The semidrying transferring film: pvdf membrane was processed 15 seconds with methyl alcohol first, was soaked at least 5min of anodal liquid with after the rinsed with deionized water 3 times again; Running gel is soaked in the negative pole liquid, and the electrotransfer device is installed in order, and the electric current of 150mA carries out electricity and turns 35-45min; After transferring film is finished, check whether dye in advance the protein marker band is all shifted.
Sealing: with tweezers the film adding is had in the valve bag of 20ml 5% milk, extrude bubble, seal with plastic film sealing machine.Shaking table shook slowly sealing 1 hour or spent the night (spend the night and should put into the refrigerator cold-storage preservation).
Primary antibodie is hatched: how anti-ratio adding with 1/200 fills in the valve bag of 5% milk with the anti-HPV6L1 albumen of the rabbit for preparing, and behind the mixing film is put into, and extrudes bubble, seals with plastic film sealing machine.Shaking table shakes slowly hatches 1.5 hours.
Wash film: film is taken out wash 5 times with TBST, each about 100ml, the time is 5 minutes.Be 10 minutes for the last time.
Two anti-hatching: the goat-anti rabbit two anti-ratios addings with 1/1000 of HRP mark are filled in the valve bag of 5% milk, behind the mixing film is put into, extrude bubble, seal with plastic film sealing machine.Shaking table shakes slowly hatches 2 hours.
Wash film: film is taken out wash 5 times with TBST, each about 100ml, the time is 5 minutes.Be 10 minutes for the last time.
Colour developing: film is put into nitrite ion, and shaking table shakes slowly to the band appearance, washes color development stopping with flowing water, takes pictures after drying.
(3) result
By the impact of more different HPV6L1 nucleotide sequences for protein expression, the result shows (Fig. 3): HPV natural gene 6wt and the expression level of 6sc gene in debaryomyces hansenii optimized according to the yeast saccharomyces cerevisiae codon-bias are far below the gene 6hp according to debaryomyces hansenii codon-bias optimization design, therefore, in the debaryomyces hansenii bacterial strain, realize that for HPV6L1 albumen high level expression is vital for debaryomyces hansenii host genetic codon bias complex optimum.
Zymotechnique and the purifying process research of embodiment 8:HPV6L1 restructuring expressed by Hansenula yeast bacterial strain
Fermentation seed liquid: get 1 frozen glycerol stock (HP/pRMHP2.1-6hp).Draw after melting in the 80 μ l access 5ml YPD substratum, in 37 ℃, 200rpm shaking table cultivation 18-24hr, A
600nmAbout 3-5 respectively draws behind the assay approval in 2 bottles of 500ml YPD substratum of 1ml access, in 37 ℃, 200rpm shaking table cultivation 18-24hr to A
600nmAbout 15-20, stand-by as fermentation seed liquid behind the assay approval.
Fermentation control: the initial substratum that ferments contains yeast powder 300g, peptone 150g, glycerine 100g, basic salt (K
2SO
4273g, MgSO
4100g, 85%H
3PO
4400ml, KOH 62g), the 10L purified water is fully dissolved, and adds in the 30L fermentor tank, and purified water is settled to 14L, and 121 ℃, the 30min sterilization adds 60ml PTM1 liquid microelement (CuSO after being cooled to 30 ℃
45H
2O 6.0g, KI0.088g, MnSO
4H
2O 3.0g, Na
2MoO
42H
2O 0.2g, H
3BO
30.02g, CoCl
26H
2O0.5g, ZnCl
220.0g, FeSO
47H
2O 65.0g, Biotin 0.2g, dense H
2SO
45.0ml purified water is settled to 1L, 0.22 μ m membrane filtration degerming), ammoniacal liquor is regulated pH5.6, inoculates 2 bottles of 500ml fermentation seed liquids, and this moment, fermentation volume was 15L.Initial mixing speed is 200rpm, air flow quantity 0.5Nm3/hr, tank pressure 0.5bar, and the control oxygen dissolving value is 20-80% in the fermentation.After the initial growth phase keeps about 22hr, bacterium liquid A
600nmReach about 20, dissolved oxygen begins fast rise, and beginning is with 100ml/hr flow velocity flow feeding substratum (50% glycerine (W/V), 12ml PTM1), and enter stream and add vegetative period this moment.After cultivating about 6-8hr, bacterium liquid A
600nmReach about 90, stop stream and add, ammoniacal liquor is regulated pH value to 6.0.Beginning stream after the dissolved oxygen bottom out adds methyl alcohol (containing 12ml/L PTM1) and enters the abduction delivering phase, methyl alcohol initial flow rate of acceleration is 50ml/hr, per hour sampling, methyl alcohol determination of electrode methanol concentration, by regulating methanol feeding rate-controlling methanol concentration＜5g/L, and being used for Western trace detection (5 times of dilutions of sample) after collecting the wet thallus ultrasonication, Western trace detected result is as shown in Figure 4.
Lower tank is centrifugal: carry out lower tank after inducing 10hr, collect wet thallus with the centrifugal 30min of 5000rpm under 4 ℃ of conditions.The wet thallus-20 of results is ℃ frozen.
Bacterial cell disruption: the HPV6 that gets-20 ℃ of preservations expresses wet thallus, adds 0.9% physiological saline according to the ratio of 10ml/g wet thallus and cleans.After the thalline washing, add broken damping fluid by 20ml damping fluid/g wet thallus and (contain 0.5mol/L NaCl, 0.02%Tween-80,0.05mol/L MOPS) fully dissolving uses high-pressure homogenization broken, cracking pressure 1500bar, circulate microscopy percentage of damage＞90% broken 6-8 time.Bacterial cell disruption liquid with the centrifugal 30min of 9000rpm, is collected supernatant liquor under 4 ℃ of conditions, add 45% ammonium sulfate again, and behind the precipitation 30min, the centrifugal 30min of 9000rpm under 4 ℃ of conditions collects supernatant liquor.
Chromatography purification: through the bacterial cell disruption supernatant liquor of 1 μ m filtration, be splined on chromatography media POROS50HS (Applied Biosystems), target protein is adsorbed onto on the chromatography media, with 0.5M～1.5MNaCl gradient elution, target protein and impurity obtain initial gross separation, collect the HPV6L1 albumen (Fig. 5 A) of wash-out.First pure HPV6L1 albumen is splined on chromatography media Macro-Prep pottery hydroxyapatite (Type II, 40 μ m), target protein is incorporated on the chromatography media, with 20～200mM phosphate concn gradient elution, target protein separates with impurity, collects the HPV6L1 albumen (Fig. 5 B) of wash-out.
The depolymerization of HPV6L1VLP and reunion: the HPV6L1 albumen behind the purifying adds 25mM DTT, and 0.05% Polysorbate80 is under the pH8.2 condition, room temperature depolymerization 2 hours.DTT is removed in dialysis, the dialysis system: 1.0M NaCl, and 20mM phosphoric acid salt, 0.05%Polysorbate80, pH 7.2.In ratio dialysis in 1: 100, change each 30min 3 times.Carry out dialysis again in the reunion damping fluid dialysis system: 1M NaCl, 5mM Ca
2+, 60mM Trisodium Citrate, pH6.2,0.05%Polysorbate80.In 4 ℃, dialyse and obtain the HPV6L1VLP of reunion after 24 hours.
The stability of HPV6L1 albumen relatively before and after meeting again: 1) THERMAL STABILITY: under the controlled rate condition, temperature is increased to 70 ℃ gradually by 25 ℃, because scattering of light changes in the solution that protein aggregation causes, and can detect by the absorbance value at 350nm place.Table 1 shows: the HPV6L1 albumen of not meeting again begins to occur obvious gathering at 50 ℃, and obvious gathering does not occur in the time of 50 ℃ the HPV6L1 albumen after meeting again, and heats just to begin to assemble to 60 ℃.2) acceleration for stabilization Journal of Sex Research: under 37 ℃ of conditions, after relatively placing certain hour, the aggregation extent of the HPV6L1 albumen of not meeting again and the HPV6L1 albumen of reunion, by as seen from Table 2, the accelerated stability of HPV6L1 albumen is significantly better than the HPV6L1 albumen of reunion not after meeting again.Thermal Synthetic stability and accelerated stability result of study are met again and have been improved the intrinsic stability of HPV6L1 albumen for temperature-induced gathering.
Table 1: the thermal stability analysis of HPV6L1 albumen before and after meeting again
Table 2: the accelerated stability analysis of HPV6L1 albumen before and after meeting again
Embodiment 9: the HPV6L1 recombinant protein of transmission electron microscope observing purifying
HPV6L1 protein sample with behind 2 times of dilutions of sterilized water purifying drips a droplet on cured dish.Get surface and sample liquid Surface Contact that copper mesh makes supporting film, leave standstill 1min and take out, take out copper mesh, absorb unnecessary drop with the filter paper bar, slightly dry.Get 2% acetic acid uranium solution, drip a droplet on cured dish.Absorption has the copper mesh of sample to be positioned over dye liquor surface (sample contacts with dye liquor), leaves standstill 2min.Take out copper mesh, absorb unnecessary drop with the filter paper bar, dry under the incandescent light.Use JEOL-1400 model transmission electron microscope observing VLP particle form and take pictures (result is shown in Figure 6).
Embodiment 10: with the immunogenicity research of the HPV6L1VLP of debaryomyces hansenii restructuring preparation
Use and measure humoral immunization effectiveness ED
50The immunogenicity of the method evaluation HPV6L1VLP of (median effective dose)
(1) immunity of mouse: 70 6 all Balb/c female mices in age (available from Institute of Experimental Animals, Chinese Academy of Medical Sciences), the cleaning level is raised.Being divided into is 6 groups, comprises 5 experimental group and 1 control group, by required immunizing dose the HPV6L1 protein sample is diluted (table 3).Immune programme for children is: 0th, 3,6 each immunity of week once, mouse and separation of serum were killed in last immunity in rear 14 days.
Table 3: the grouping of mouse
(2) the ELISA method is measured the serological conversion rate behind the HPV6L1VLP immune mouse, and concrete steps are: with coated damping fluid the intestinal bacteria HPV6L1 albumen of recombinating is diluted to 0.5 μ g/ml, every hole adds 0.1ml, and 4 ℃ are spent the night.Next day, the lavation buffer solution washing was 3 times, got rid of most residual liquid.With antibody diluent sealing 30 minutes, lavation buffer solution washing 3 times detected after drying, or dries rear 4 ℃ of damp proof preservations.With sample diluting liquid each mice serum sample was diluted with 1: 10000, get 0.1ml in above-mentioned coated reacting hole, put 37 ℃ and hatched 1 hour, wash 5 times.(doing simultaneously blank, negative hole contrast).Add the sheep anti-mouse igg second antibody 0.1ml of the HRP mark of antibody diluent fresh dilution in 1: 5000 in reacting hole, hatched 30 minutes for 37 ℃, wash 5 times, last is all over washing with distilled water.The tmb substrate solution 0.1ml that adds interim preparation in each reacting hole, 37 ℃ were developed the color 10 minutes.In each reacting hole, add 50 μ l 2M sulfuric acid 0.05ml with termination reaction.On microplate reader, in the 450nm place (630nm is reference wavelength), to survey each hole OD value after the zeroing of blank hole.The Cutoff value is calculated and positive findings is judged: Cutoff value=negative control value * 2.1; Sample OD value＞Cutoff value then is judged to the positive.
(3) humoral immunization is renderd a service ED
50Calculating
According to table 4 calculation result, the humoral immunization of HPV6L1VLP is renderd a service ED
50Value is 0.315 μ g, has shown that HPV6L1VLP possesses good immunogenicity.
Table 4: the humoral immunization of mouse is renderd a service detection case
Wherein: being higher than 50% sun, to turn extent of dilution be 2, and being lower than 50% sun, to turn extent of dilution be 4.By calculate obtaining: distance is than being that to be higher than extent of dilution that 50% sun turns be that the logarithm of 0.301, ED50 is that to turn extent of dilution be 3.17 to 0.5017,50% sun to 0.667, log10.So, ED
50Value is 0.315 μ g.
Claims (14)
1. the method for the restructuring debaryomyces hansenii cell of human papillomavirus 6 type L1 (HPV6L1) albumen is expressed in a generation, and it comprises following steps:
A) the exogenous polynucleotide insertion vector of the nucleotide sequence by will comprising coding HPV6L1 albumen comes the construction expression construct;
B) use a) the middle expression construct conversion debaryomyces hansenii cell that obtains of step; With
C) to step b) in the debaryomyces hansenii cell that obtains screen, obtain to contain the restructuring debaryomyces hansenii cell of described exogenous polynucleotide.
2. the process of claim 1 wherein that the aminoacid sequence of described HPV6L1 albumen is shown in SEQ ID NO:1.
3. the method for claim 2, the nucleotides sequence of wherein said coding HPV6L1 albumen is shown in SEQ ID NO:4.
4. each method among the claim 1-3 wherein operably is connected in promotor and terminator in exogenous polynucleotide described in the described expression construct.
5. the method for claim 4, wherein said promotor is MOX promotor or FMD promotor.
6. claim 4 or 5 method, wherein said terminator is the MOX terminator.
7. each method among the claim 1-6, wherein said carrier comprises the nucleotide sequence shown in the SEQ ID NO:15.
8. each method, wherein step b among the claim 1-7) in transform the debaryomyces hansenii cell by electroporation.
9. each method, wherein step b among the claim 1-8) in described debaryomyces hansenii cell be ATCC26012 debaryomyces hansenii cell.
10. each method among the claim 1-9, wherein said restructuring debaryomyces hansenii cell contains the described exogenous polynucleotide of multiple copied.
11. restructuring debaryomyces hansenii cell of producing of each method according to claim 1-10.
12. a method that produces HPV6L1 albumen may further comprise the steps:
I) the restructuring debaryomyces hansenii cell of cultivation claim 11 under the condition that is suitable for described HPV6L1 protein expression; With
Ii) from culture, reclaim and the described HPV6L1 albumen of purifying.
13. the method for claim 12, it also comprises the step of purified HPV6L1 albumen being carried out depolymerization and repolymerization.
14. according to claim 12 or the purposes of HPV6L1 albumen in the vaccine that infects for the preparation of prevention HPV6 that produce of 13 method.
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| CN104845985A (en) * | 2014-02-18 | 2015-08-19 | 上海泽润生物科技有限公司 | Recombinant human papilloma virus protein expression |
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| US7276243B2 (en) * | 2003-03-24 | 2007-10-02 | Merck & Co., Inc. | Optimized expression of HPV31 L1 in yeast |
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| CN104745605A (en) * | 2013-12-26 | 2015-07-01 | 上海泽润生物科技有限公司 | Expression of 6 and 11 subtype proteins of recombinant human papilloma virus by pichia yeast |
| CN104745605B (en) * | 2013-12-26 | 2020-02-07 | 上海泽润生物科技有限公司 | Expression of recombinant human papilloma virus 6 and 11 subtype protein pichia pastoris |
| CN104845985A (en) * | 2014-02-18 | 2015-08-19 | 上海泽润生物科技有限公司 | Recombinant human papilloma virus protein expression |
| CN111154777A (en) * | 2014-02-18 | 2020-05-15 | 上海泽润生物科技有限公司 | Recombinant human papilloma virus protein expression |
| CN111154777B (en) * | 2014-02-18 | 2023-08-15 | 上海泽润生物科技有限公司 | Recombinant human papilloma virus protein expression |
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