CN104120088A - Method for generation of HPV58 L1 protein by Hansenula expression system - Google Patents
Method for generation of HPV58 L1 protein by Hansenula expression system Download PDFInfo
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Abstract
The invention relates to a method for generation of HPV58 L1 (human papillomavirus) protein by a Hansenula expression system. Specifically, the invention discloses a method for generation of a recombinant Hansenula cell for expressing the HPV58L1 protein and the recombinant Hansenula cell generated thereby. The invention also discloses a method for generating the HPV58L1 protein by the recombinant Hansenula cell and application of the generated HPV58L1 protein in preparation of preventive vaccines.
Description
Invention field
The invention belongs to medical bioengineering technical field, relate to the method that produces humanpapilloma virus 58 L1 protein, relate in particular to the method that produces humanpapilloma virus 58 L1 protein by 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 at present has identified out exceedes 200 kinds, HPV infects and has obvious tissue specificity, other HPV of different shaped for skin and mucous membrane to have a liking for tropism different, can bring out different papillary lesion, nearly more than 30 kinds of HPV types are relevant with genital tract infection, wherein have kind more than 20 and Tumor-assaciated.Bring out the good pernicious difference of pathology according to HPV, HPV can roughly be divided into two classes: 1) high-risk-type (as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68 etc.): high-risk HPV and mankind's Various Tissues malignant tumour are closely related, mainly cause severe atypical hyperplasia and infiltrating carcinoma, worldwide, recall rate is respectively HPV16 and HPV18 type at the HPV of front two type; In CONTINENTAL AREA OF CHINA, up-to-date epidemiology statistics data presentation, the infection rate of HPV52 and HPV58 has exceeded HPV18; 2) low risk (as HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV72, HPV81 etc.): low risk HPV can cause the optimum proliferative venereal disease of epidermic cell, as pointed condyloma and condyloma latum etc., the pointed condyloma wherein being brought out by HPV6 and HPV11 accounts for more than 90%.
HPV is mainly made up of virus coat and genomic dna.Genome is about 7900bp, has 8 encoding hiv protease genes.Wherein the albumen of 6 ORF codings, at the early expression of virus replication, is called early protein; 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.
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 by L1 albumen and L2 albumen coexpression in multiple expression system, VLP), wherein more to approach the structure of natural viral at the VLP of the generations such as Yeast system, baculovirus insect expression system and mammalian cell expression system.After the VLP immunity that utilizes heterogenous expression system to produce, can bring out in vivo generation neutralizing antibody, 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, as unicellular eukaryotic microorganisms, had both possessed prokaryotic organism and has grown fast, is easy to the features such as genetic manipulation, had again the functions such as eukaryotic cell translation post-treatment and modification.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 glycosylation side chain is long and pichia spp (Pichia Pastoris) problem that exogenous origin gene integrator copy number is lower.At present, application expressed by Hansenula yeast system is produced medicine (as Regular Insulin, trade(brand)name Wosulin) and all list marketings of HBV vaccine (trade(brand)name Hepavax-Gene).
The method of application expressed by Hansenula yeast system expression HPV16 and HPV18 type L1VLP is disclosed in the open CN102586287A of Chinese invention patent and CN102719453A.But whether can in debaryomyces hansenii system, realize high efficient expression and be assembled into VLP particle about the L1 albumen of HPV58 type, not open report at present.In above-mentioned 2 sections of published patent applications, do not provide the result of exogenous origin gene integrator copy number and the method for raising copy number of foreign gene of HPV16 and HPV18L1.Aspect the abduction delivering of foreign protein, HPV16 and the HPV18L1 induction time in fermentor tank all needs to exceed 20 hours, and the specifying information of protein expression level is not provided.In addition,, as an important protein purification index, while completing about purge process and purifying, the purity of foreign protein HPV16 and HPV18L1 is also open.
For realizing the high efficient expression of humanpapilloma virus 58 L1 protein in debaryomyces hansenii and can obtaining highly purified HPV58VLP albumen by purifying, in the present invention, applied the Double Selection strategy of Zeocin resistance screening in conjunction with G418 resistance screening, applied especially concentration go down to posterity up to the G418 resistant panel screening of 16mg/ml and stablize after restructuring expressed by Hansenula yeast bacterial strain.By detecting, the copy number of the HPV58 high expression level bacterial strain that screening obtains can exceed 60 copies.In fermentation inducement process, can significantly improve the expression level of humanpapilloma virus 58 L1 protein by improving abduction delivering temperature to 35 DEG C.In addition, be difficult for the feature of wash-out (still having 50% target protein hanging column when high density NaCl wash-out target protein) when the purifying HPV58VLP for chromatography media POROS50HS conventional in VLP purge process, the present invention uses POROS XS chromatography media, make target humanpapilloma virus 58 L1 protein can realize the wash-out of 80% above albumen in the time of high density NaCl wash-out, thereby significantly improved the output of humanpapilloma virus 58 L1 protein.
Summary of the invention
Aspect first, the invention provides a kind of method that produces the restructuring debaryomyces hansenii cell of expressing humanpapilloma virus 58 L1 protein, it comprises following steps:
A) by the exogenous polynucleotide insertion vector of the nucleotide sequence that comprises the humanpapilloma virus 58 L1 protein of encoding is carried out to construction expression construct;
B) transform debaryomyces hansenii cell by the expression construct obtaining in step a); With
C) the debaryomyces hansenii cell obtaining in step b) is screened, obtain the restructuring debaryomyces hansenii cell that contains described exogenous polynucleotide.
Aspect second, the present invention also provides the restructuring debaryomyces hansenii cell producing according to aforesaid method.
Aspect the 3rd, the present invention also provides a kind of method that produces humanpapilloma virus 58 L1 protein, comprises the following steps:
I) under the condition that is suitable for humanpapilloma virus 58 L1 protein expression, cultivate restructuring debaryomyces hansenii cell of the present invention; With
Ii) from culture, reclaim and purifying humanpapilloma virus 58 L1 protein.
An aspect in the end, the present invention also provides humanpapilloma virus 58 L1 protein that the method according to this invention the produces purposes in the vaccine infecting for the preparation of prevention HPV58.
Brief description of the drawings
Fig. 1 recombinate debaryomyces hansenii cellular expression levels SDS-PAGE detect.The abduction delivering of the different restructuring of 1-8:8 strain debaryomyces hansenii bacterial strain; 9: standard substance (10 μ g/mL); 10: protein molecular weight mark.
The Southern trace detected result of Fig. 2 taking MOX promoter fragment as probe.Taking ATCC26012 genomic dna as contrast.1: contrast (applied sample amount is 1000ng); 2: contrast (applied sample amount is 500ng); 3: contrast (applied sample amount is 250ng); 4: contrast (applied sample amount is 125ng); 5: the genomic dna (applied sample amount is 7.8ng, and its brightness is between 500ng and 1000ng contrast) of high expression level recombinant bacterium HP-1#/pRMHP2.1-58hp; 6: the genomic dna (applied sample amount is 7.8ng, and its brightness is between 500ng and 1000ng contrast) of high expression level recombinant bacterium HP-2#/pRMHP2.1-58hp.
Fig. 3 A: the Western Blot that in fermenting process, humanpapilloma virus 58 L1 protein is expressed detects (abduction delivering under 30 DEG C of conditions).1: pre-dsred protein molecular weight marker; 2: standard substance (10 μ g/mL); 3: induce 1 hour; 4: induce 3 hours; 5: induce 5 hours; 6: induce 7 hours; 7: induce 9 hours; 8: induce 10 hours.
B: the Western Blot that in fermenting process, humanpapilloma virus 58 L1 protein is expressed detects (abduction delivering under 35 DEG C of conditions).1: pre-dsred protein molecular weight marker; 2: standard substance (10 μ g/mL); 3: induce 1 hour; 4: induce 3 hours; 5: induce 5 hours; 6: induce 7 hours; 7: induce 8 hours; 8: induce 9 hours; 9: induce 10 hours.
The POROS50HS purifying electrophorogram of Fig. 4 A:HPV58L1 albumen.1: upper prop sample; 2: stream is worn liquid; 3-5: different concns NaCl elutriant; 6: scavenging solution.
The POROS XS purifying electrophorogram of B:HPV58L1 albumen.1: upper prop sample; 2: stream is worn liquid; 3-5: different concns NaCl elutriant; 6: scavenging solution; 7: protein molecular weight mark.
The CHT purifying electrophorogram of C:HPV58L1 albumen.1: upper prop sample; 2: stream is worn liquid; 3-6: different concns phosphoric acid salt elutriant; 7: scavenging solution; 8: protein molecular weight mark.
The transmission electron microscope observing result of the humanpapilloma virus 58 L1 protein of Fig. 5 purifying.
Detailed Description Of The Invention
The inventor has successfully set up the method for utilizing debaryomyces hansenii to produce humanpapilloma virus 58 L1 protein, and the humanpapilloma virus 58 L1 protein producing can be self-assembled into virus-like particle, can be used for the vaccine that preparation prevention HPV infects.
First the present invention provides a kind of method that produces the restructuring debaryomyces hansenii cell of expressing humanpapilloma virus 58 L1 protein, and it comprises following steps:
A) by the exogenous polynucleotide insertion vector of the nucleotide sequence that comprises the humanpapilloma virus 58 L1 protein of encoding is carried out to construction expression construct;
B) transform debaryomyces hansenii cell by the expression construct obtaining in step a); With
C) the debaryomyces hansenii cell obtaining in step b) is screened, obtain the restructuring debaryomyces hansenii cell that contains described exogenous polynucleotide.
The present invention also comprises the restructuring debaryomyces hansenii cell producing according to described method.
The aminoacid sequence that derives from the humanpapilloma virus 58 L1 protein of different HPV58 virus strain can there are differences.The inventor is by comparing to humanpapilloma virus 58 L1 protein sequences all in database, on the each amino acid position of humanpapilloma virus 58 L1 protein, choose the amino-acid residue that the frequency of occurrences is the highest, obtained the aminoacid sequence that is shown in SEQ ID NO:1, this sequence is the most representative consensus sequence of humanpapilloma virus 58 L1 protein.Therefore, the humanpapilloma virus 58 L1 protein in the present invention preferably has the aminoacid sequence shown in SEQ ID NO:1.
In order to utilize debaryomyces hansenii to express efficiently humanpapilloma virus 58 L1 protein, contriver, according to the aminoacid sequence shown in SEQ ID NO:1, carries out the codon optimized of nucleotide sequence for debaryomyces hansenii.Optimization principles comprises: a) select according to debaryomyces hansenii genetic code frequency of utilization table (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi species=4905) codon that frequency of utilization is high or the highest; B) avoid genetic transcription or protein translation to have the negative regulatory element of potential impact, as PolyAT district, PolyGC district, silencer (Sliencer) district and inner splice site etc.; C) comprehensively analyze in interior mRNA secondary structure comprising 5 ' end UTR, HPV58L1 coding region and 3 ' end UTR, 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 conventional restriction enzyme enzyme recognition site.Be shown in SEQ ID NO:2 through the nucleotides sequence of optimizing.The nucleotide sequence of the coding humanpapilloma virus 58 L1 protein using in the present invention is preferably the sequence shown in SEQ ID NO:2.
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:9) describing in the Chinese patent application of 201210021524.X.By the exogenous polynucleotide of the nucleotide sequence that comprises the humanpapilloma virus 58 L1 protein of encoding is cloned to 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, for example, carrier described in the Chinese patent CN100400665C having authorized.
In order to express in debaryomyces hansenii, the exogenous polynucleotide in described expression construct is operably connected with promotor and terminator.
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 using in the present invention is the MOX promotor from debaryomyces hansenii.Be applicable to terminator of the present invention and include but not limited to the MOX terminator from debaryomyces hansenii.
Expression construct is converted into debaryomyces hansenii cell can be undertaken by multiple methods known in the art, includes but not limited to the conversion of electroporation and PEG mediation.
In addition, in this area, develop multiplely for expressing foreign protein debaryomyces hansenii bacterial strain, 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 transforming the debaryomyces hansenii cell of expression construct of the present invention.
After conversion, can be according to the resistant gene carrying on the carrier debaryomyces hansenii cell of selecting to recombinate.Suitable resistant gene includes but not limited to Zeocin resistant gene and G418 resistant gene.According to the carrier using, also may screen restructuring debaryomyces hansenii cell with auxotrophy substratum.In one embodiment, with the Zeocin debaryomyces hansenii cell of selecting to recombinate.In another embodiment, with the G418 debaryomyces hansenii cell of selecting to recombinate.In a preferred embodiment, be used in combination Zeocin and the G418 debaryomyces hansenii cell of selecting to recombinate.The screening concentration of Zeocin can be 0.25,0.5,0.75,1.0 or 1.5mg/ml, preferably 0.5mg/ml.The screening concentration of G418 using can be 2,4,8,10,12,14,16,18 or 20mg/ml, preferably 16mg/ml.
Expression level and its copy number positive correlation in debaryomyces hansenii of exogenous polynucleotide in debaryomyces hansenii.Therefore, can also carry out the restructuring debaryomyces hansenii cell that further screening contains multiple copied exogenous polynucleotide by methods such as Southern trace or quantitative PCRs.The restructuring debaryomyces hansenii cell that preferably exogenous polynucleotide copy number is greater than 15, the restructuring debaryomyces hansenii cell that more preferably exogenous polynucleotide copy number is greater than 60.
The inventor is surprisingly found out that, be used in combination Zeocin and the G418 debaryomyces hansenii cell of selecting to recombinate, particularly use and select to obtain exogenous polynucleotide copy number up to the G418 of 16mg/ml and be greater than 15, be particularly greater than 60 stable restructuring debaryomyces hansenii cell.
On the other hand, the present invention also provides a kind of method that produces humanpapilloma virus 58 L1 protein, comprises the following steps:
I) under the condition that is suitable for humanpapilloma virus 58 L1 protein expression, cultivate restructuring debaryomyces hansenii cell of the present invention; With
Ii) from culture, reclaim and purifying humanpapilloma virus 58 L1 protein.
Various substratum and the basic culture condition that can be used for cultivating debaryomyces hansenii known in the art, 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 (as the fermentor tank of 30L) of different scales at flask according to desirable proteins amount.According to selected promotor, can in cultivation, add suitable inductor to induce the expression of described humanpapilloma virus 58 L1 protein.In the situation that using MOX or FMD promotor, can add methyl alcohol as inductor.Yeast fermentation is cultivated routine and is carried out at 30 DEG C, and the inventor is surprisingly found out that, restructuring debaryomyces hansenii cell of the present invention can make exogenous protein expression amount significantly improve 35 DEG C of inductions of carrying out protein expression.
The purifying of the humanpapilloma virus 58 L1 protein producing can use various protein purification mode known in the art, as saltout, the combination of ultrafiltration, precipitation, chromatography etc. or these modes.In the experimental program of an optimization, first carry out preliminary purification with chromatography media POROS XS, (Type II, 40 μ m) are further purified to utilize subsequently chromatography media Macro-Prep pottery hydroxyapatite.
Utilize the humanpapilloma virus 58 L1 protein of purifying prepared by method of the present invention can be self-assembled into virus-like particle (embodiment 9, Fig. 5), and in mouse, demonstrate good immunogenicity (embodiment 10), therefore the present invention also provides the purposes of described humanpapilloma virus 58 L1 protein in the vaccine infecting for the preparation of prevention HPV58.
Embodiment
Mode by embodiment is further illustrated to the present invention below, but therefore do not limit the present invention in described scope of embodiments.
The analysis of embodiment 1:HPV58L1 consensus amino acid sequences
The humanpapilloma virus 58 L1 protein of total length is made up of 498 amino acid, after GenBank retrieval, use Vector NTI software AlignX function to carry out aminoacid sequence compare of analysis, obtain the most representative HPV58L1 consensus amino acid sequences (consensus amino acid sequence, all adopt the sequence of the amino-acid residue that the frequency of occurrences is the highest at the each amino acid position of HPV58L1), its sequence is as shown in SEQ ID NO:1.
Optimization design and the synthetic of embodiment 2:HPV58L1 encoding gene
In order to utilize debaryomyces hansenii to express efficiently humanpapilloma virus 58 L1 protein, contriver, according to the aminoacid sequence shown in SEQ ID NO:1, carries out the codon optimized of nucleotide sequence for debaryomyces hansenii.Optimization principles comprises: a) select according to debaryomyces hansenii genetic code frequency of utilization table the codon that frequency of utilization is high or the highest; B) avoid genetic transcription or protein translation to have the negative regulatory element of potential impact, as PolyAT district, PolyGC district, silencer (Sliencer) district and inner splice site etc.; C) comprehensively analyze in interior mRNA secondary structure comprising 5 ' end UTR, HPV58L1 coding region and 3 ' end UTR, 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 conventional restriction enzyme enzyme recognition site.Be shown in SEQ ID NO:2 through the nucleotides sequence of optimizing.
According to above nucleotide sequence, the complete sequence synthetic of entrusting Sinogenomax Co., Ltd. to carry out, is cloned in (called after T-58hp) in T carrier, and it is carried out to sequence verification.
Embodiment 3: produce the expression construct of carrying HPV58L1 nucleotide sequence
The applied expressed by Hansenula yeast carrier of the present invention is that application number is the expressed by Hansenula yeast carrier pRMHP2.1 (SEQ ID NO:9) describing in the Chinese patent application of 201210021524.X.
(1) pcr amplification of MOX promotor and MOX terminator
Taking the mixed genomic DNA of debaryomyces hansenii strains A TCC26012 and ATCC34438 as template, obtaining size with following primer pair amplification is the MOX promotor of 1518bp, simultaneously at upstream introducing NotI restriction enzyme site;
MOX promotor upstream primer: 5 '-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3 ' (SEQ ID NO:3)
MOX promotor downstream primer: 5 '-TTTGTTTTTGTACTTTAGATTGATGTC-3 ' (SEQ ID NO:4)
Taking the mixed genomic DNA of debaryomyces hansenii strains A TCC26012 and ATCC34438 as template, obtaining size with following primer pair amplification is the MOX terminator of 311bp, simultaneously at downstream introducing BglII restriction enzyme site;
MOX terminator upstream primer: 5 '-GGAGACGTGGAAGGACATACCGC-3 ' (SEQ ID NO:5)
MOX terminator downstream primer: 5 '-GAAGATCTCAATCTCCGGAATGGTGATCTG-3 ' (SEQ ID NO:6)
(2) produce the expression construct of carrying HPV58L1 nucleotide sequence
Taking the recombinant plasmid T-58hp that carries 58hp as template, obtaining size with following primer pair amplification is the HPV58L1hp gene of 1545bp, introduces the overlap of MOX promoter region 3 ' end in upstream simultaneously, introduces the overlap that MOX terminator 5 ' is held in downstream;
58 upstream primers: 5 '-CATCAATCTAAAGTACAAAAACAAAATGTCGGTGTGGAGACCATCTGAAG-3 ' (SEQ ID NO:7)
58 downstream primers: 5 '-GCGGTATGTCCTTCCACGTCTCCTTATTTCTTGACTTTCTTCCTCTTAGTG-3 ' (SEQ ID NO:8)
Taking MOX promotor, 58hp gene, three fragments of MOX terminator as hybrid template, obtaining size with following primer pair amplification is the 58hp expression cassette of 3.4Kb, and amplified production carries NotI restriction enzyme site in upstream, carry BglII restriction enzyme site in downstream.
MOX promotor upstream primer: 5 '-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3 ' (SEQ ID NO:3)
MOX terminator downstream primer: 5 '-GAAGATCTCAATCTCCGGAATGGTGATCTG-3 ' (SEQ ID NO:6)
By NotI+BglII double digestion, 58hp expression cassette is cloned in pRMHP2.1 carrier, obtains expression construct pRMHP2.1-58hp.
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 obtaining in embodiment 3, after enlarged culturing, use E.Z.N.A Plasmid Mini Kit test kit (Omega Bio-Tek company) to extract plasmid, and carry out single endonuclease digestion with BglII, application E.Z.N.A Gel Extraction Kit test kit (Omega Bio-Tek company) reclaims, the sterilized water that is preheated to 55 DEG C with 50 μ L carries out wash-out, by measuring OD
260carry out DNA quantitative, and linearizing fragment is diluted to 100ng/ μ l, be stored in-20 DEG C of refrigerators, for subsequent use.
(2) processing of debaryomyces hansenii cell
The mono-bacterium colony of picking debaryomyces hansenii strains A TCC26012, in the small test tube of access containing the YPD liquid nutrient medium of 5ml, cultivates 12 hours for 30 DEG C; Get bacterium liquid 5ml and be forwarded in 200ml YPD substratum, cultivate 4-6 hour for 30 DEG C, to OD
600nmbe about 1.0-1.5, in the centrifugal 10min of 5000rpm; With the resuspended thalline of 200ml0.1mol/L phosphate buffered saline buffer (containing 25mmol/L DTT, pH7.5), fully mix, hatch 30min in 30 DEG C, the centrifugal 10min of 5000rpm, abandons supernatant, stays thalline.Wash thalline with the STM solution 200ml of precooling, thalline pressure-vaccum is even, in 4 DEG C of centrifugal 3min of 5000rpm, abandons supernatant, stays precipitation.With the ice-cold resuspended thalline of STM solution 100ml, in 4 DEG C of centrifugal 3min of 5000rpm, abandon supernatant, stay precipitation.According to the ice-cold resuspended thalline of STM solution of 50-200 μ l for biomass, and bacterium liquid is transferred in the centrifuge tube after high pressure, ice bath, prepares to transform.
(3) electricity of debaryomyces hansenii cell transforms
Add restructuring expressed by Hansenula yeast plasmid 15 μ l by the amount of plasmid: thalline=1:2, bacterium liquid 30 μ l, fully pressure-vaccum is even, is placed in ice bath to be transformed; To use in advance alcohol-pickledly, and after uv irradiating, take out in the electric revolving cup of-20 DEG C of refrigerations, add plasmid thalline mixed solution; Shock by electricity by the condition of voltage 2500V, resistance 150 Ω, electric capacity 50 μ F; After electric shock, add rapidly 1ml balance to the YPD solution of room temperature, after mixing gently, be transferred in EP pipe; Thalline after electricity is turned is placed 1h in 30 DEG C of water-baths, puts upside down gently 3 times at interval of 15min; By hatching the bacterium liquid of 2h, in the centrifugal 10min of 5000rpm, abandon supernatant; With the resuspended thalline of 200 μ l YPD solution, coat in the YPD flat board containing 0.5mg/mlZeocin with 100 μ l/ plates, be inverted and cultivate 3-7 days in 30 DEG C.
(4) the going down to posterity of restructuring expressed by Hansenula yeast bacterial strain, stable
The recombinant bacterial strain list bacterium colony growing in picking Zeocin resistant panel, be inoculated in 5ml containing in the YPD liquid nutrient medium of 0.5mg/ml Zeocin, in 30 DEG C, 200rpm shaking table cultivation 24-48 hour, after reaching 50 to OD value, transfer in 5ml containing in the YPD liquid nutrient medium of 0.5mg/ml Zeocin with the ratio of 1:1000, after being cultured to OD value and reaching 50, transfer in 5ml containing in the YPD liquid nutrient medium of 0.5mg/ml Zeocin with the ratio of 1:1000 again, by that analogy, pass continuously 10 times and carry out the preservation of bacterial classification.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 l60% 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 30 DEG C, 200rpm shaking table are cultured to OD value and reach 50, transfer in 5ml YPD liquid nutrient medium with the ratio of 1:1000, by that analogy, in not containing the YPD liquid nutrient medium of Zeocin resistance, pass 5 times continuously.
YPD flat board by the bacterium liquid coating after stable containing 16mg/ml G418, is inverted and cultivates 2-3 days in 30 DEG C.
Embodiment 5: the expression study of restructuring debaryomyces hansenii bacterial strain
From containing the multiple restructuring debaryomyces hansenii of picking list bacterium colony the YPD flat board of 16mg/ml G418, in the small test tube of access containing the YPG liquid nutrient medium of 5ml, cultivate 24 hours for 30 DEG C; Bacterium liquid is transferred in containing the 100ml triangular flask of 30ml YPM inducing culture, and initial density is OD
600=1, in 30 DEG C of shaking tables induction 72 hours, in bacterium liquid, adding final concentration every 12 hours was 0.5% methanol solution.
The bacterium liquid of getting after 10ml induction is transferred in the centrifuge tube of 50ml, in the centrifugal 10min of 10000rpm, abandons supernatant; With the resuspended bacterial sediment of lysis of 50ml, after fully mixing, in ultrasonic apparatus by " power 60%, time 20min, open 5s, close 5s " ultrasonication program carry out bacterial cell disruption, in shattering process, need to keep ice bath; By the bacterium liquid of ultrasonication, in the centrifugal 10min of 10000rpm, after collection supernatant, carry out SDS-PAGE detection, induction result (as shown in Figure 1) shows: more than the restructuring debaryomyces hansenii bacterial strain expression level of high expression level can reach 25 μ g/ml.
Embodiment 6: the exogenous polynucleotide copy number of restructuring debaryomyces hansenii cell detects
(1) extraction of pastoris genomic dna and quantitative
Inoculate high expression level yeast strain that 2 strain embodiment 5 obtain to substratum in the YPD liquid nutrient medium of 5ml, cultivate 16～24h for 30 DEG C; Get 2ml yeast culture liquid, the centrifugal 3min of room temperature 4500g collects thalline; Apply 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 add 50mg granulated glass sphere fully to shake 5min, add 50 μ l10mg/ml lywallzymes, 37 DEG C of temperature are bathed 1h; Add the 10%SDS of 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 the water-bath of 55 DEG C; Add the saturated phenol of 350 μ l and 350 μ l chloroforms, after fully mixing, in 13000rpm, centrifugal 10 minutes, collect the upper strata liquid after layering; (l) chloroform of approximately 700 μ, in 13000rpm, centrifugal 10 minutes, collects the upper strata liquid after layering to add equal-volume; Toward the 3mol/L sodium acetate solution that adds 140 μ l in the liquid of upper strata, after mixing gently, add again 700 μ l Virahols, mix rear room temperature and place 5min, in 13000rpm, centrifugal 10 minutes.Abandon supernatant, add 1ml70% ethanol to clean, in 13000rpm, centrifugal 10 minutes, remove supernatant, DNA precipitation is placed in room temperature and places after 30min, adds 100 μ l TE to dissolve.By measuring OD
260nmcarry out the quantitative of genomic dna, and linearizing fragment is diluted to 100ng/ μ l, be stored in-20 DEG C of refrigerators, for subsequent use.
(2) Southern trace method is carried out the quantitative of exogenous polynucleotide copy number
A: probe preparation
The MOX probe of describing in the Chinese patent application that the applied MOX probe of the present invention application number is 201210021524.X, the DIG DNA Labeling and Detection kit test kit (Cat No:11093657910) of application Roche company carries out probe preparation.Concrete steps are: to the PCR product that adds 10 μ l MOX promoter regions in EP tubule, (200ng/ μ l), and seal up with sealed membrane, boiling water boils 10 minutes.Put into immediately the dehydrated alcohol capsule (suddenly cooling) of precooling (20 DEG C).Dry pipe outer wall ethanol, centrifugal (about 10s), add successively 5 μ l intracellular toxins to check water, 2 μ l10x Hexanucleotide Mix, 2 μ l10x dTP Labeling Mixture, 1 μ l7Klenow Enzyme(labeling grade), mix sealed membrane sealing.37 DEG C of water-baths are spent the night ,-20 DEG C of preservations.
B:Southern trace
Application Beijing Mei Laibo medical science and technology company limited digoxin hybridization check test kit I(Cat No:DIGD-110) and the efficient hybridization solution of HyB (Cat No:Hyb-500) carry out the detection of Southern trace according to product description.
Southern trace detected result (as shown in Figure 2) shows: the high expression level restructuring debaryomyces hansenii HP-1#/pRMHP2.1-58hp bacterial strain and the HP-2#/pRMHP2.1-58hp bacterial strain exogenous polynucleotide copy number that obtain in the screening of the G418 of 16mg/ml resistant panel exceed 60.Show the Double Selection strategy of application Zeocin antibody screening in conjunction with G418 resistance screening, particularly adopted the recombinant bacterial strain that contributes to obtain high copy integration and can carry out humanpapilloma virus 58 L1 protein high expression level up to the G418 resistant panel screening of 16mg/ml concentration.
Zymotechnique and the optimization of embodiment 7:HPV58L1 restructuring expressed by Hansenula yeast bacterial strain
Fermentation seed liquid: get 1 frozen glycerol stock (HP-1#/pRMHP2.1-58hp), draw after melting in 50 μ l access 5ml YPD substratum, in 30 DEG C, 200rpm shaking table cultivation 20-24hr, A
600nmabout 2-5, each absorption in 2 bottles of 500ml YPD substratum of 1ml access after assay approval, in 30 DEG C, 200rpm shaking table cultivation 20-24hr to A
600nmabout 15-20, stand-by as fermentation seed liquid after assay approval.
Zymotechnique: 1) common process: the initial substratum that ferments contains yeast powder 300g, peptone 150g, glycerine 100g, basic salt (K
2sO
4273g, MgSO
4100g, 85%H
3pO
4400ml, KOH62g), 10L purified water is fully dissolved, and adds in 30L fermentor tank, and purified water is settled to 14L, and 121 DEG C, 30min sterilizing, adds 60ml PTM1 liquid microelement (CuSO after being cooled to 30 DEG C
45H
2o6.0g, KI0.088g, MnSO
4h
2o3.0g, Na
2moO
42H
2o0.2g, H
3bO
30.02g, CoCl
26H
2o0.5g, ZnCl
220.0g, FeSO
47H
2o65.0g, Biotin0.2g, dense H
2sO
45.0ml, purified water is settled to 1L, 0.22 μ m membrane filtration degerming), ammoniacal liquor regulates pH5.6, inoculates 1 bottle of 500ml fermentation seed liquid, and now fermentation volume is 15L.Initial mixing speed is 200rpm, air flow quantity 0.5Nm3/hr, tank pressure 0.5bar, and in fermentation, controlling oxygen dissolving value is 20-80%.The initial growth phase maintains after about 25hr, bacterium liquid A
600nmreach 20 left and right, dissolved oxygen starts fast rise, starts with 100ml/hr flow velocity flow feeding substratum (50% glycerine (W/V), 12ml PTM1), now enters stream and adds vegetative period.Cultivate after about 6-8hr bacterium liquid A
600nmreach 90 left and right, stop stream and add, ammoniacal liquor regulates pH value to 6.0.After dissolved oxygen bottom out, starting stream adds methyl alcohol (containing 12ml/L PTM1) and enters the abduction delivering phase, inducing temperature is set as 30 DEG C, methyl alcohol initial flow rate of acceleration is 50ml/hr, sampling per hour, methyl alcohol determination of electrode methanol concentration, by regulating methanol feeding speed to make methanol concentration be controlled at < 5g/L.2) Optimization Technology: Fermentation Process of Parameter is substantially with the description in " common process ", and difference is under inductive condition, inducing temperature to be increased to 35 DEG C.
Centrifugal and the expression amount of lower tank detects: lower tank after induction 10hr, under 4 DEG C of conditions, collect wet thallus with the centrifugal 30min of 5000rpm, and-20 DEG C are frozen for subsequent use.In addition, get in the centrifuge tube that 10ml fermented liquid is transferred to 50ml, in the centrifugal 10min of 10000rpm, abandon supernatant, with the resuspended bacterial sediment of cell pyrolysis liquid of 50ml, after fully mixing, carry out ultrasonication, ultrasonication liquid carries out the detection of Western trace, Western trace detected result is as shown in Fig. 3 A and 3B: 1) under common process condition, induce for 30 DEG C, the expression level of humanpapilloma virus 58 L1 protein is only about half of control sample (10 μ g/mL), due to diluted sample 5 times, therefore the fermented liquid expression amount of HPV58L1 is 25 μ g/mL.2) under Optimizing Technical, induce for 35 DEG C, the expression level of humanpapilloma virus 58 L1 protein is the more than 2 times of control sample (10 μ g/mL), due to diluted sample 5 times, therefore the fermented liquid expression amount of HPV58L1 is more than 100 μ g/mL.Therefore,, in fermentation inducement process, can significantly improve the output of humanpapilloma virus 58 L1 protein by improving abduction delivering temperature to 35 DEG C.
Purifying process research and the optimization of embodiment 8:HPV58L1 recombinant protein
Bacterial cell disruption: the HPV58 that gets-20 DEG C of preservations expresses wet thallus, adds 0.9% physiological saline to clean according to the ratio of 10ml/g wet thallus.After thalline washing, add broken damping fluid (containing 0.5mol/L NaCl by 20ml damping fluid/g wet thallus, 0.02%Tween-80,0.05mol/L MOPS) fully dissolve, the fragmentation of use high-pressure homogenization, cracking pressure 1500bar, the broken 5-8 time that circulates, microscopy percentage of damage > 90%.Bacterial cell disruption liquid with the centrifugal 30min of 10000rpm, is collected supernatant liquor, then is added 50% ammonium sulfate under 4 DEG C of conditions, and after precipitation 30min, the centrifugal 30min of 10000rpm under 4 DEG C of conditions, collects supernatant liquor.
The chromatography purification the first step: 1) common process: the bacterial cell disruption supernatant liquor filtering through 1 μ m, be splined on chromatography media POROS50HS, target protein is adsorbed onto on chromatography media, with 0.5M～1.5M NaCl gradient elution; 2) Optimization Technology: the bacterial cell disruption supernatant liquor filtering through 1 μ m, is splined on chromatography media POROS XS, with 0.5M～1.5M NaCl gradient elution.The above 2 kinds of different chromatography media absorption humanpapilloma virus 58 L1 proteins of application clean pillar with 0.5M NaOH again after different concns NaCl gradient elution.SDS-PAGE detects the purifying situation of humanpapilloma virus 58 L1 protein in chromatography process, and result is as shown in Fig. 4 A and Fig. 4 B: 1) application chromatography media POROS50HS, and after high density NaCl wash-out, the humanpapilloma virus 58 L1 protein still remaining more than 50% is not eluted; 2) application chromatography media POROS XS, after high density NaCl wash-out, only has less than 20% humanpapilloma virus 58 L1 protein and is not eluted.Show in the time carrying out the preliminary chromatography purification of humanpapilloma virus 58 L1 protein, application chromatography media POROS XS can be conducive to the wash-out of humanpapilloma virus 58 L1 protein, thereby significantly improves the yield of HPV58L1.
Chromatography purification second step: the humanpapilloma virus 58 L1 protein after preliminary purification is splined on to chromatography media Macro-Prep pottery hydroxyapatite (Type II, 40 μ m), target protein is incorporated on chromatography media, with 20～200mM phosphate concn gradient elution, target protein separates with impurity, collects the humanpapilloma virus 58 L1 protein (seeing Fig. 4 C) of wash-out.
Embodiment 9: the HPV58L1 recombinant protein of transmission electron microscope observing purifying
With the humanpapilloma virus 58 L1 protein sample after 3 times of dilution purifying of sterilized water, drip 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 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 filter paper bar, under incandescent light, dry.Application JEOL-1400 model transmission electron microscope observing VLP particle form take pictures (shown in result Fig. 5).
Embodiment 10: the immunogenicity research of the HPV58L1VLP preparing with debaryomyces hansenii restructuring
Humoral immunization effect ED is measured in application
50the immunogenicity of the method evaluation HPV58L1VLP of (median effective dose)
(1) immunity of mouse: 85 Balb/c female mices in 6 week age (purchased from Institute of Experimental Animals, Chinese Academy of Medical Sciences), clean level is raised.Be divided into 6 groups, comprise 5 experimental group and 1 control group, by required immunizing dose, humanpapilloma virus 58 L1 protein sample is diluted to (table 1).Immune programme for children is: 0th, once, mouse separation of serum are killed in last immunity for latter 14 days in each immunity in 3,6 weeks.
The grouping of table 1 mouse
(2) ELISA method is measured the serological conversion rate after HPV58L1VLP immune mouse, and concrete steps are: the intestinal bacteria humanpapilloma virus 58 L1 protein of recombinating is diluted to 0.5 μ g/ml with coated damping fluid, every hole adds 0.1ml, and 4 DEG C are spent the night.Next day, lavation buffer solution washing 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 DEG C of damp proof preservations.Each mice serum sample is diluted with 1:10000 with sample diluting liquid, get 0.1ml in above-mentioned coated reacting hole, put 37 DEG C and hatch 1 hour, wash 5 times.(doing blank, negative hole contrast) simultaneously.In reacting hole, add the sheep anti-mouse igg second antibody 0.1ml of the HRP mark of the fresh dilution of antibody diluent 1:10000, hatch 30 minutes for 37 DEG C, wash 5 times, last is all over washing with distilled water.In each reacting hole, add the tmb substrate solution 0.1ml of interim preparation, 37 DEG C are developed the color 10 minutes.In each reacting hole, add 50 μ l2M sulfuric acid 0.05ml with termination reaction.In microplate reader, in 450nm place (630nm is reference wavelength), to survey each hole OD value after the zeroing of blank hole.Cutoff value is calculated and positive findings is judged: Cutoff value=negative control value × 2.1; Sample OD value >Cutoff value is judged to the positive.
(3) humoral immunization effect ED
50calculating
According to the mouse positive rate calculation result of variant dosage level, the humoral immunization effect ED of HPV58L1VLP
50value is 0.134 μ g, shows that HPV58L1VLP possesses good immunogenicity.
Claims (18)
1. produce and express human papillomavirus type 58 L1(HPV58L1) method of the restructuring debaryomyces hansenii cell of albumen, it comprises following steps:
A) by the exogenous polynucleotide insertion vector of the nucleotide sequence that comprises the humanpapilloma virus 58 L1 protein of encoding is carried out to construction expression construct;
B) transform debaryomyces hansenii cell by the expression construct obtaining in step a); With
C) the debaryomyces hansenii cell obtaining in step b) is screened, obtain the restructuring debaryomyces hansenii cell that contains described exogenous polynucleotide.
2. the process of claim 1 wherein that the aminoacid sequence of described humanpapilloma virus 58 L1 protein is shown in SEQ ID NO:1.
3. the method for claim 2, the nucleotides sequence of wherein said coding humanpapilloma virus 58 L1 protein is shown in SEQ ID NO:2.
4. the method for any one in claim 1-3, is wherein operably connected in promotor and terminator in exogenous polynucleotide described in described expression construct.
5. the method for claim 4, wherein said promotor is MOX promotor.
6. the method for claim 4 or 5, wherein said terminator is MOX terminator.
7. the method for any one in claim 1-6, wherein said carrier comprises the nucleotide sequence shown in SEQ ID NO:9.
8. the method for claim 7, wherein c) middle Zeocin and the G418 screening restructuring debaryomyces hansenii cell of using of step.
9. the method for claim 8, wherein the concentration of Zeocin is 0.5mg/ml.
10. the method for claim 8, wherein the concentration of G418 is 16mg/ml.
In 11. claim 1-10, the method for any one, wherein transforms debaryomyces hansenii cell by electroporation in step b).
The method of any one in 12. claim 1-11, wherein the described debaryomyces hansenii cell in step b) is ATCC26012 debaryomyces hansenii cell.
The method of any one in 13. claim 1-12, the described exogenous polynucleotide that wherein said restructuring debaryomyces hansenii cell contains multiple copied.
14. 1 kinds of restructuring debaryomyces hansenii cells that the method according to claim 1-13 any one produces.
15. 1 kinds produce the method for humanpapilloma virus 58 L1 protein, comprise the following steps:
I) at the restructuring debaryomyces hansenii cell that is suitable for cultivating under the condition that described humanpapilloma virus 58 L1 protein expresses claim 14; With
Ii) from culture reclaim and purifying described in humanpapilloma virus 58 L1 protein.
The method of 16. claims 15, wherein step I) in be included in 35 DEG C of described humanpapilloma virus 58 L1 proteins of induction and express.
The method of 17. claims 15 or 16, wherein step I is used humanpapilloma virus 58 L1 protein described in POROS XS chromatography media purifying in i).
The purposes of the humanpapilloma virus 58 L1 protein that 18. methods according to any one in claim 15-17 produce in the vaccine infecting for the preparation of prevention HPV58.
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Address after: 100176 210, zone 3, block B, innovation building, 12 Hongda North Road, Beijing Economic and Technological Development Zone Patentee after: ABZYMO BIOSCIENCES Co.,Ltd. Patentee after: Jiangsu Ruike Biotechnology Co.,Ltd. Address before: 100176 210, zone 3, block B, innovation building, 12 Hongda North Road, Beijing Economic and Technological Development Zone Patentee before: ABZYMO BIOSCIENCES Co.,Ltd. Patentee before: JIANGSU RUIKE BIOTECHNOLOGY Co.,Ltd. |