CN104878022A - Nucleotide sequence for coding HPV58L1 and HPV52L1 proteins and application thereof - Google Patents

Abstract

The invention discloses a nucleotide sequence for coding HPV58L1 and HPV52L1 proteins and application thereof. In order to solve the problems of varieties and activities of proteins in HPV (human papilloma virus) vaccines, the codon is optimized, and thus, can implement high-level expression in yeast cells. The expression is utilized to generate HPV52 and HPV58 virus-like particles (VLPs) in the yeast cells, thereby being used for research and development of vaccines.

Description

The nucleotide sequence of coding HPV58L1, HPV52L1 albumen and application thereof

Technical field

The present invention relates to biological technical field, more particularly, relate to human papillomavirus coding and application thereof.

Background technology

Human papillomavirus (human papilkgna virus, HPV) is a kind of tumorigenesis DNA virus, and can cause human genital tract and skin of anus, mucous membrane that optimum and malignant tumour occurs, wherein closely-related with cervical cancer pathogenesis have HPV 16,18 etc.The new cases of annual whole world cervical cancer is about 500,000 people, death toll nearly 300,000, is the second largest cancer threatening women's health.

There is the human papillomavirus of more than 80 kinds, i.e. HPV.Wherein a lot of relevant to biological phenotype miscellaneous, described biological phenotype is from benign proliferative wart to pernicious cancer.HPV6 with HPV11 is and sexual organ or the optimum wart of respiratory mucosa, most general type that non-malignant pointed condyloma is relevant with mild dysplasia.HPV16 and HPV18 is the excessive risk type the most relevant with invasive carcinoma to the original position of uterine cervix, vagina, cysthus and anal tube.The cervical cancer of more than 90% and HPV16, HPV18 or less popular carcinogenic type HPV31 ,-33 ,-45, the infection of-52 and-58 is relevant.In to more than 90% cervical cancer, detect that the observations of HPV DNA provides the powerful Epidemiological Evidence that HPVs causes cervical cancer.Human papillomavirus is little (50-60nm), without coating, icosahedral DNA virus, maximum 8 early stage and 2 late genes of its coding.This virus genomic open reading-frame (ORF) called after E1 to E7, and L1 and L2, wherein E represents that early stage L represents late period.L1 and L2 encoding virus coat proteins, and E gene is relevant to the function of virus replication and cell transformation.L1 albumen is main capsid protein, and has the molecular weight of 55-60kDa.L2 albumen is secondary capsid protein.Immunologic data prompting is in viral capsid, and most of L2 albumen is positioned at L1 active site of protein.In different people papilloma virus, L1 and L2 albumen is high conservative.L1 albumen or the expression of L1 and L2 protein combination in yeast, insect cell, dried meat breast zooblast or bacterium cause the automatic assembling of virus-like particle (VLPs).VLPs form is similar to genuine virosome and can the neutralizing antibody of inducement efficient valency after being applied to animal or human.Because VLPs does not have oncovirus genome potentially, so they are the purposes of live virus in HPV vaccine development provide safe alternatives.For this reason, L1 and L2 gene has been confirmed as the immunology target of HPV infection and prevention and treatment of diseases vaccine development.

Epidemiology survey in Asia shows, and 52,58 types are just relevant to the cervical cancer of more than 30%.In normal population, the popularity degree of HPV52 is only second to HPV16, be about 2.42%, also certain ratio (a little less than HPV58) is occupied in patients of cervical ruin, but in invasive endometrial neck cancer, the popularity of HPV52 but reduces greatly, and this may be limited relevant with the conversion capability of HPV52E7 cancer protein.Cervical cancer be a progressive process caused by cervical erosion, and HPV52 to be cervical erosion occur and an important factor of development, the immunoprotection reaction therefore for HPV52 hypotype has great significance for the preventative vaccine of cervical cancer.

HPV vaccine development and commercialization has been hindered to the capsid protein obtaining high expression level in the host living beings of successful conversion, the difficulty that limits the production of purifying protein relevant.Therefore, although identify the wild-type nucleotide sequences of coding humanpapilloma virus 58 L1 protein, but that expects very much to develop humanpapilloma virus 58 L1 protein easily can upgrade source, and this albumen utilizes is formulating the nucleotide sequence of expressing the coding HPV58L1 be optimised in host cell.In addition, produce a large amount of HPV58L1VLPs for vaccine development with the matter that immunizes of native protein will be very useful.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, for the protein classes used in HPV vaccine and active problem, coding HPV58L1 or the HPV52L1 nucleotide sequence of albumen is provided, high level expression can be realized in yeast cell by codon optimized; In yeast cell, produce HPV52L1 or HPV58L1 virus-like particle (VLPs), for the research and development of vaccine by expressing simultaneously.

Object of the present invention is achieved by following technical proposals:

The nucleotide sequence of coding HPV52L1 albumen, as the nucleotide sequence in sequence table as described in SEQ ID No.1.

The nucleotide sequence of coding humanpapilloma virus 58 L1 protein, as the nucleotide sequence in sequence table as described in SEQ ID No.2.

A kind of carrier, the nucleotide sequence containing the above-mentioned HPV52L1 albumen of coding, or the nucleotide sequence of coding humanpapilloma virus 58 L1 protein.

A kind of eucaryon debaryomyces hansenii engineering bacteria, with the nucleotide sequence containing the above-mentioned HPV52L1 albumen of coding, or the carrier transfection eucaryon debaryomyces hansenii bacterial strain RB-11 of nucleotide sequence of coding humanpapilloma virus 58 L1 protein, obtains expression level through screening and the highest bacterial strain of plasmid copy number obtains.

RB-11 cell strain is that the improvement of LR9 cell strain derives strain (Roggenkamp.R etal., 1986Transformation ofthe methylotrophic yeast Hanseunla polymorpha by autonomous replication and integrationvectors, Mol.Gen Genet 202:302).RB-11 cell strain is 5 '-vitamin B13 glycosides phosphate decarboxylase gene defective mutant strain, has been widely used in the structure of industrial recombinant bacterium.

Methyl alcohol is utilized to carry out abduction delivering to the eucaryon debaryomyces hansenii bacterial strain RB-11 after transfection, in whole process, temperature is 28-32 DEG C, time is 72h, during beginning, methanol usage is 0.5% of cell suspending liquid quality, at interval of 24h, in cell suspending liquid, add methyl alcohol, the amount of adding methyl alcohol is 0.5% of cell suspending liquid quality.

Utilize eucaryon debaryomyces hansenii engineering bacteria to carry out the method for fermenting, during the fermentation, described eucaryon debaryomyces hansenii engineering bacteria is through outgrowth phase, the phase that derepresses and abduction delivering phase, and one of them mass parts is 1g:

(1) grow phase: fermentor tank pH value controls at 5.4-5.5, and leavening temperature controls at 28-32 DEG C, the fermention medium of use through sterilising treatment, by NH ₄h ₂pO ₄170-180 mass parts, MgSO ₄7H ₂o 40-50 mass parts, KCl 40-45 mass parts, NaCl 4-5 mass parts, glycerine 500-550 mass parts, fermentation defoaming agent 4-5 mass parts and deionized water 10000-15000 mass parts composition; In growth phase, in fermentor tank, dissolved oxygen can slowly decline, and when dropping to 60%, in fermentor tank, at the uniform velocity squeezes into supplemented medium, the supplemented medium of use through sterilising treatment, by NH ₄h ₂pO ₄80-90 mass parts, glycerine 250-300 mass parts and deionized water 500-550 mass parts form; When fermentor tank dissolved oxygen level is down to below 40%, air flow quantity is adjusted to 12-15L/min by 25-30L/min originally, mixing speed is adjusted to 700rpm by 1200-1500rpm originally; The whole growth phase time, cell growth state was good, and fermentor tank dissolved oxygen level can drop to less than 20%, cell OD at 20-25 hours ₆₀₀reach more than 80;

(2) derepress phase: when fermentor tank dissolved oxygen is gone back up to more than 60% by lower-most point, enter the phase that derepresses, maintain air flow quantity and agitation condition constant, add derepression culture base; Described derepression culture base through sterilising treatment, by NH ₄h ₂pO ₄25-30 mass parts, MgSO ₄7H ₂o 5-10 mass parts, KCl 5-8 mass parts, NaCl 0.5-1 mass parts, glycerine 1700-1900 mass parts and deionized water 1200-1350 mass parts form, and the phase that derepresses continues 20-25h;

(3) phase is induced: after the phase that derepresses, enter induction phase, inducing culture is added in fermentor tank, described inducing culture is through sterilising treatment, be made up of glycerine and methyl alcohol, the volume ratio of glycerine and methyl alcohol is (38-40): (60-65), and the consumption of regulation and control methyl alcohol, to make methyl alcohol in the liquid phase volume of whole fermentor tank, volume percent is 0.3%-0.5%, and induction phase continues 48-55h.

In growth phase, fermention medium is preferably by NH ₄h ₂pO ₄170-175 mass parts, MgSO ₄7H ₂o 40-45 mass parts, KCl 42-44 mass parts, NaCl 4.2-4.4 mass parts, glycerine 520-540 mass parts, fermentation defoaming agent 4-5 mass parts and deionized water 10000-12000 mass parts composition, described fermentation defoaming agent is THIF-298 type biological fermentation defoamers.

In growth phase, supplemented medium is preferably by NH ₄h ₂pO ₄85-87 mass parts, glycerine 260-280 mass parts and deionized water 520-540 mass parts form.

In growth phase, the volume ratio of supplemented medium and fermention medium is (0.8-1): (10-12).

Derepressing in phase, derepression culture base is preferably by NH ₄h ₂pO ₄26-28 mass parts, MgSO ₄7H ₂o 6-8 mass parts, KCl 7-8 mass parts, NaCl 0.6-0.8 mass parts, glycerine 1750-1850 mass parts and deionized water 1300-1350 mass parts form.Derepressing in phase, the volume ratio of fermention medium and derepression culture base is 4:(1-1.2).

In induction phase, the use volume of inducing culture be derepression culture matrix long-pending 30-40%.

The present invention utilizes wild type gene, on the basis of expression characteristic taking into full account eucaryon debaryomyces hansenii bacterial strain RB-11, provide two kinds of new nucleotide sequences, coding HPV52L1 albumen or humanpapilloma virus 58 L1 protein, after carrier construction and transfection RB-11, form respective VLPs in the product that induction fermentation obtains, and expression amount is apparently higher than the expression level of wild type gene, and can uses as vaccine.

Accompanying drawing explanation

Fig. 1 is the structure schematic diagram of expression vector pMPT-HPV52L1.

Fig. 2 is the structure schematic diagram of expression vector pMPT-HPV58L1.

Fig. 3 is the electrophoresis photographs of pcr amplification after expression vector pMPT-HPV58L1 transfectional cell.

Fig. 4 is transformant west-blot method the selection result figure of HPV58L1.

Fig. 5 is HPV58L1 VLPs image in buffer suspension liquid.

Fig. 6 is HPV52L1 VLPs image in buffer suspension liquid.

Fig. 7 is the contrast schematic diagram that the yeast expression VLPs of wild gene and optimized gene measures.

Embodiment

Technical scheme of the present invention is further illustrated below in conjunction with specific embodiment.

Embodiment 1 is encoded the synthesis of nucleotide sequence of HPV52L1 or humanpapilloma virus 58 L1 protein

In ncbi database, retrieval obtains wild-type DNA-sequence and the protein sequence of HPV52L1 and HPV58L1 respectively, wherein concerning HPV52L1, wild-type DNA-sequence HPV52L1-WT total length 1512 bases, protein sequence Genbank registration number is HQ537731.1, protein sequence total length 504 amino acid.

HPV52L1 aminoacid sequence is converted to the nucleotide sequence of eucaryon debaryomyces hansenii preference codon pattern.Rebuild HPV52L1 sequence engineered, to delete any possible premature transcription termination site to guarantee firm transcribing.In first-selected degenerate code, eucaryon debaryomyces hansenii preferences is in primary codon, in main use degenerate code, eucaryon debaryomyces hansenii preferences is in the first deputy codon, a few cases uses the codon of the 3rd to complete local directed complete set, DNAMAN software is utilized to avoid long complementary sequence, tumor-necrosis factor glycoproteins, by RNA structure prediction, avoid the hairpin structure of long complexity, get rid of strong secondary structure, increase specific intramolecule stability.The sequence occurring bringing disadvantageous effect to genetic transcription, translational efficiency is also avoided, as intron montage sequence, transcription termination sequence, internal promoter sequence (TATA) and ribosome bind site (GGAGG) etc. in the nucleotide sequence of gene.Transcription termination sequence mainly contains: ATTATTTTAT, TTTTTATA, TAG (being rich in T) TA (G) GT, (being rich in AT) TTT etc.Finally determine whole sequence, as shown in SEQ ID NO.1 in sequence table.Utilize the DNA sequence dna shown in SEQ ID NO.1 in full artificial synthesis composition sequence table.

Concerning HPV58L1, aminoacid sequence total length 498 amino acid, is converted to the nucleotide sequence of eucaryon debaryomyces hansenii preference codon pattern.Adopt the conversion thinking and countermeasure as above-mentioned HPV52L1, reconstruction HPV58L1 sequence is engineered, to delete any possible premature transcription termination site to guarantee firm transcribing.Final whole sequence, as shown in SEQ No.2 in sequence table.Utilize the DNA sequence dna shown in SEQ No.2 in full artificial synthesis composition sequence table.

The structure of embodiment 2 expression vector

(1) structure of HPV52L1 expression vector

Utilize the DNA sequence dna shown in SEQ ID NO.1 in full artificial synthesis composition sequence table, sequence two ends add the recognition site of restriction enzyme EcoR I and BamH I.With the DNA sequence dna that EcoR I suits with BamH I enzyme, electrophoresis reclaim enzyme cut after fragment, ﹣ 20 DEG C saves backup.

With debaryomyces hansenii CGMCC 22497 genomic dna for template, methanol oxidase gene (MOX) promotor, methanol oxidase gene (MOX) terminator of 350bp, the autonomously replicating sequence HARS of 1.0kb of clone 1.5kb; And from YIp5 (Genbank Acession NO.L09157) plasmid, clone the yeast saccharomyces cerevisiae urine uracil gene ScURA3 of 1.1kb; After above-mentioned 4 parts are connected, insert the multiple clone site of pBluescrip II plasmid, be built into shuttle vectors pMPT-02.

Carrier pMPT-02 is cut with EcoR I and BamH I enzyme, low melting-point agarose gel electrophoresis reclaim enzyme cut after large fragment, under the effect of T4-DNA ligase enzyme, connect with the DNA sequence dna synthesized shown in the SEQ ID NO.1 of same double digestion, ligation is overnight at 16 DEG C, reaction solution transforms TIANGEN Biotech (Beijing) Co., Ltd. TOP10 competent cell, with the plate screening transformant of band amicillin resistance, use primer primer fd15 '-ATGTCTGTTTGGAGACCGTC-' 3 and primer fd25 '-GGGATCCGTGTAATTATCTCTTGAC-' 3 by the transformant of pcr amplification screening containing carrier pMPT-HPV52L1 to the E. coli transformant that plate grows, screen the transformant enlarged culturing obtained, reclaim test kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.2.0) purification with the plasmid of precious biotechnology Dalian company limited and obtain expression vector pMPT-HPV52L1, accompanying drawing 1 is shown in by vector construction schematic diagram.

(2) build HPV58L1 expression vector with step in the same way, see accompanying drawing 2.

Utilize the DNA sequence dna shown in SEQ ID NO.2 in full artificial synthesis composition sequence table, sequence two ends add the recognition site of restriction enzyme EcoR I and BamH I.With the DNA sequence dna that EcoR I suits with BamH I enzyme, electrophoresis reclaim enzyme cut after fragment, ﹣ 20 DEG C saves backup.

With debaryomyces hansenii CGMCC 22497 genomic dna for template, methanol oxidase gene (MOX) promotor, methanol oxidase gene (MOX) terminator of 350bp, the autonomously replicating sequence HARS of 1.0kb of clone 1.5kb; And from YIp5 (Genbank Acession NO.L09157) plasmid, clone the yeast saccharomyces cerevisiae urine uracil gene ScURA3 of 1.1kb; After above-mentioned 4 parts are connected, insert the multiple clone site of pBluescrip II plasmid, be built into shuttle vectors pMPT-02.

Carrier pMPT-02 is cut with EcoR I and BamH I enzyme, low melting-point agarose gel electrophoresis reclaim enzyme cut after large fragment, under the effect of T4-DNA ligase enzyme, connect with the DNA sequence dna of the synthesis of same double digestion, ligation is overnight at 16 DEG C, reaction solution transforms TIANGEN Biotech (Beijing) Co., Ltd. TOP10 competent cell, with the plate screening transformant of band amicillin resistance, '-TTACTTCTTGACCTTC-' 3 is by the transformant of pcr amplification screening containing carrier pMPT-HPV58L1 to use primer primer fd35 '-ATGTCCGTGTGGCGTCCTTCGG-' 3 and primer fd4 5 to the E. coli transformant that plate grows, screen the transformant enlarged culturing obtained, reclaim test kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.2.0) purification with the plasmid of precious biotechnology Dalian company limited and obtain expression vector pMPT-HPV58L1, accompanying drawing 2 is shown in by vector construction schematic diagram.

Embodiment 3 expression vector electricity transforms eucaryon debaryomyces hansenii competence host cell

(1) pMPT-HPV52L1 experiences host cell

With Sac I respectively enzyme cut carrier pMPT-HPV52L1, make vector linearization.Linearizing carrier is by electrotransformation (the LN-101 type gene transfection instrument that Institutes Of Technology Of Tianjin produces, 1.5kV, 50 μ F, 200 Ω, 3-5mSec) transform eucaryon debaryomyces hansenii RB-11 (directly purchased from TUV biotech firm, RB-11 cell strain is that the improvement of LR9 cell strain derives strain Roggenkamp.R etal., 1986Transformation of the methylotrophic yeast Hanseunlapolymorpha by autonomous replication and integration vectors, Mol.Gen Genet 202:302, RB-11 cell strain is 5 '-vitamin B13 glycosides phosphate decarboxylase gene defective mutant strain, be widely used in the structure of industrial recombinant bacterium).Culture medium flat plate containing 1.34%YNB (yeast nitrogen basic medium) and 2% glucose screens transformant.The HPV 52L1 Insert Fragment of correct direction in the bacterium colony produced by PCR screening.Yeast conversion mixture is paved plate in MD solid medium, and be inverted cultivation 3-5 days at 30 DEG C.Picking colony also carries out clone and separate at flat lining out.The bacterium colony be separated grows in 37 DEG C to induce L1 to transcribe and protein expression subsequently in swivel pipe is cultivated.After 48 hours, precipitation is equivalent to 0D ₆₀₀the cell of=10 amounts, removing supernatant liquor and freezing precipitation be kept at-70 DEG C.

(2) pMPT-HPV58L1 experiences host cell: adopt and scheme substantially identical in (1), uses carrier pMPT-HPV58L1 to transform eucaryon debaryomyces hansenii RB-11.

The screening of embodiment 4 goal gene, order-checking and detection

(1) cytoclasis: get about 1mLOD ₆₀₀be the yeast thalline of 10 to 1.5mL centrifuge tube, remove supernatant after the centrifugal 5min of 6000rpm, add the pickling glass liquid identical with precipitation thalline volume, 100 μ L cytoclasis liquid, shake 5-10min as on vibrator.

(2) DNA extracting: after cytoclasis completes, adds 100 μ L sterilized waters in centrifuge tube, then adds 100 μ L phenol, chloroform liquid, and mix latter 4 DEG C and place ten minutes, the centrifugal 5min of 10000rpm, gets supernatant 1 μ L for amplified reaction.

(3) pcr amplification and agarose gel electrophoresis detect

Use primer primer fd3 5 '-ATGTCCGTGTGGCGTCCTTCGG-' 3 and primer fd45 '-TTACTTCTTGACCTTC-' 3 to the DNA profiling pcr amplification of above preparation, above primer can increase the MOX gene of eucaryon debaryomyces hansenii list copy and the HPV58L1 gene of integration simultaneously.The HPV58L1 fragment length that increases be 1500bp, MOX fragment length be 2060bp, loading 5 μ L in 1.0% sepharose well, the complete taking-up gel shooting of electrophoresis, electrophoresis photographs is shown in accompanying drawing 3, and wherein about 1500bp band is HPV58L1 gene, and about 2000bp band is MOX gene.Wherein 1,9 roads are high copy transformants, 5,7,8 roads are higher copy transformants, and 2,3,4,6 roads are low copy transformants, and 10 roads are Host Strains of negative control, 11 roads are DL2000Marker, are followed successively by 2000bp, 1000bp, 750bp and 500bp from top to bottom.Gene sequencing is carried out with the DNA fragmentation of DNA purification kit extracting 1500bp after the PCR primer agarose gel electrophoresis more than increased, consistent with design gene comparison result.

(4) multiple copied transformant real-time quantitative PCR detects copy number

Known MOX gene is the single copy gene of eucaryon debaryomyces hansenii, be interior mark with NOX gene, design primer pair same sample increases MOX gene fragment and HPV58L1 gene fragment simultaneously, and detect with quantitative real time PCR Instrument, because MOX gene is single copy in eucaryon debaryomyces hansenii genome, therefore the amount of the amount of HPV58L1 fragment/MOX fragment is the copy number of the HPV58L1 gene of each cell.Gene is transformed into recipient bacterium by plasmid vector and could high expression after stable integration, and its copy number reflects that the engineering strain of high expression expresses the characteristic of target protein to a certain extent.

Two pairs of primers all use software Primer5 to design, and by Dalian, synthesis is acted on behalf of by precious biotechnology company limited.The primer sequence of amplification HPV58L1 gene fragment is as shown in 5 '-TCTGAGCCCTATGGCGATT-' 3 and 5 '-AGCGGAAGACTGGATGACA-' 3, and fragment length is 156bp.The primer sequence of amplification MOX gene fragment is respectively MOX primer forward (Sp2): 5, and '-GCCACCTTCTACTCGTCCAG-' 3 and MOXprimer forward (As2): 5 '-ACTCCCAGTCGTCGTAGTCG-' 3, fragment length is 145bp.

Template DNA is with above-mentioned DNA method for extracting, and its dilution is as follows: standard substance dilute, and gets the DNA solution that a certain Sample extraction obtains and first dilutes 20 times, then carry out serial dilution, and each dilution 10 times, dilute 4 times, and this dilution requires accurate.Diluted sample, gets the DNA solution that other sample extractings obtain and first dilutes 20 times, then carry out serial dilution, and each dilution 10 times, dilute 2 times, and it is true that refinement is not wanted in this dilution.

Real-time quantitative PCR reaction system: cumulative volume 20 μ L, the wherein each 0.5 μ L of forward and reverse primer (10mM), EX PremierTaq is (containing 4mM Mg2 ⁺, 0.4mM dNTP, 0.5M ExTaq) 10 μ L, SYBR 0.5 μ L, template 5 μ L.Real-time quantitative PCR detects the quantitative real time PCR Instrument (model: Rotor-Gene3000) using Australian Corbett Research company.Real-time quantitative PCR reaction heat loop parameter: denaturation 94 DEG C of 5min, sex change 94 DEG C of 15secs, annealing and extension 60 DEG C of 50secs, 35 circulations.Melt inspection atopic: be slowly warmed up to 94 DEG C from 60 DEG C, within every 5 seconds, heat up once, each intensification 0.5 DEG C, often raises and once carries out signals collecting.Utilize Rotor-Gene5.0.28 (Corbett Research) software analysis samples copy number.

(5) adopt as above-mentioned step and method carries out the screening of HPV52L1 gene, order-checking and detection, the DNA fragmentation of test kit extracting is through gene sequencing, consistent with design gene comparison result.

Embodiment 5 cell cultures abduction delivering

Taken out from-70 DEG C of refrigerators by frozen bacterial classification and dissolve rapidly, after being blown and beaten with 0.2mL suction pipe/stir by bacteria suspension, connect 40 μ L bacteria suspensions in the triangular flask that 10mL MD substratum is housed, each sample connects the triangular flask of more than 2.After inoculation, shaking table cultivates 24 hours, culture temperature 31 ± 2 DEG C, shaking speed 160rpm.

Connect with suction pipe and cultivate the bacteria suspension 0.1mL that obtains, in the triangular flask that 10mL MD substratum is housed, cultivate 24 hours, culture temperature 31 ± 2 DEG C, shaking speed 160rpm.The bacteria suspension obtained is proceeded to sterilized 10mL centrifuge tube, centrifugal 10 minutes of 3000rpm on desk centrifuge, abandoning supernatant.Add sterilized water 10mL, fully after mixing with 3000rpm centrifugal 10 minutes, abandoning supernatant.Without carbon source substratum, the thalline concussion after washing is mixed with 10mL, proceed in triangular flask.

Add 0.5% (50 μ L) methyl alcohol cultivating above in the cell suspension obtained, be placed in by triangular flask and cultivate shaking table, temperature is 31 ± 2 DEG C, shaking speed 160rpm shaking culture.Rise next day and every morning, afternoon respectively add methyl alcohol once.Inducing culture, after 24 hours, adds 1mL without carbon source substratum, adds 0.5% (55 μ L) methyl alcohol, and be placed in by triangular flask and cultivate shaking table, temperature is 31 ± 2 DEG C, shaking speed 160rpm shaking culture.Inducing culture sampled detection after 72 hours, and bacteria suspension dilutes 30 times, measures OD with spectrophotometric ₆₀₀value, the OD of bacteria suspension after calculating inducing culture ₆₀₀value, gets the detection that suitable 1mL 200D cell carries out expression level.

Embodiment 6 eucaryon debaryomyces hansenii recombiant vaccine engineering bacteria 3 phase pilot scale fermentation technique

(1) bacterial classification preparation and seed amplify

The height screened in strain construction copy high expression level bacterial strain is prepared as primary seed, main generation seed, work seed three grades of seed banks, be placed in ﹣ 70 DEG C of refrigerator-freezers frozen, work seed is used for zymotechnique exploitation and vaccine fermentative production.The preparation of substratum: 2g/100ml glucose, 1.34g/100ml YNB; Primary-seed medium 200mL, secondary seed medium 1600mL, autoclaving.(1mL, an OD is got from ﹣ 70 DEG C of refrigerator-freezers ₆₀₀≈ 100) access 200mL primary-seed medium, cultivate 24 hours switching 1600mL secondary seed medium enlarged culturing, cultivate 22 hour cell OD for 31 DEG C for 31 DEG C ₆₀₀reach more than 10, as ferment-seeded.

(2) fermenting process, uses THIF-298 type biological fermentation defoamer as fermentation defoaming agent

Supplemented medium: NH ₄h ₂pO ₄87g is dissolved in 540mL water, autoclaving; Separately get 260g glycerine autoclaving.

Derepression culture base: amount to 3L, NH ₄h ₂pO ₄27g, MgSO ₄7H ₂o 6g, KCl 6.8g, NaCl 0.7g, above raw material is dissolved in 660mL water, autoclaving; Separately get 1.8L glycerine autoclaving.

Inducing culture: glycerine 400mL, adds 0.6L methyl alcohol after autoclaving.

Fermention medium: ferment initial volume 12L, comprises NH ₄h ₂pO ₄175g, MgSO ₄7H ₂o 40g, KCl 44g, NaCl 4.4g, glycerine 520g, separately add 4mL defoamer, 110-115 DEG C of autoclaving 30 minutes in tank.

Zymotechnique: carry out zymotechnique with the 30L fermentor tank (model: MSJ-J2) that Japanese ball water chestnut physics and chemistry device institute produces.The FC-280 type dissolved oxygen monitor carrying out online sterilizing East China Biological Engineering College of East China University of Science to fermentation cylinder for fermentation substratum carries out the monitor and forecast of dissolved oxygen.

The access of 1800mL secondary seed be equipped with in the fermentor tank of the 12L fermention medium of sterilizing, start fermentation, zymotechnique mainly comprises three phases, namely grows phase, the phase that derepresses and abduction delivering phase.

Growth phase: fermentor tank pH value controls at 5.4-5.5, and leavening temperature controls at 30 DEG C ± 2 DEG C, pipe pressure 0.5Kg/cm ².In fermentor tank, dissolved oxygen can slowly decline, and when dropping to 60% (volume ratio) left and right, connecting feed supplement pipeline, at the uniform velocity squeezing into about 800mL supplemented medium with peristaltic pump.When fermentor tank dissolved oxygen level is down to below 40%, air flow quantity is adjusted to 15L/min, and mixing speed is adjusted to 700rpm.Growth phase later stage (fermenting about 22 hours), if cell growth state is good, fermentor tank dissolved oxygen level can drop to less than 20%, cell OD ₆₀₀reach more than 80.

Derepress phase: peristaltic pump attaching plug is connected with dissolved oxygen monitor signal out-put supply before ging up by dissolved oxygen; Monitor dissolved oxygen controls 40, and dead band is 2, high-endly opens; Peristaltic pump power switch is closed.When observing fermentor tank dissolved oxygen and being gone back up to more than 60% by lower-most point, enter the phase that derepresses.Now open peristaltic pump power switch, setting pump speed 22rpm, starts stream and adds derepression culture base.Derepress the later stage, if Growth of Cells is good, OD ₆₀₀can more than 300 be reached.

Induction phase: the phase that derepresses continues 24 hours, enters induction phase, starts to add inducing culture, control methanol concentration 0.3% (V/V) by methyl alcohol electrode, whole fermentation time is approximately 96 hours.

Carrying out in fermenting process, the fermentation parameter can recorded according to summary of the invention adjusts, and all can realize ferment effect.

Embodiment 7 protein purification

Sample pretreatment: ﹣ 72 DEG C of freeze-stored cells 4 DEG C melt, and add 200mM PMSF to final concentration 4mM, suspension mixing after, with high pressure homogenizer under 1300BAR condition by cytoclasis twice.With the centrifugal 25min of Sigma 6K-15 whizzer 8000rpm, get supernatant liquor, with Sai Duolisi Sartocon 2Plus tangential flow systems 10KDa membrane ultrafiltration, concentrated 3 times, with 8 times of buffer A (25mM pH7.2 phosphate buffered saline buffer, 0.1M NaCl, 4mM EDTA, 0.03%Tween80) fully replace, remove small molecular weight impurity.

Strong cation exchange chromatography: application QXL strong anion displacement chromatography resin filling chromatography post (26mmID × 100mm), 0.5M NaOH cleaning-sterilizing used before use by this post.At room temperature use the average weighing apparatus chromatography column of buffer A.Under room temperature, ultrafiltration sample is with the speed upper prop of 15mL/min, wash with the speed stream of 15mL/min by the buffer A of 8 column volumes, 100% buffer A is to 100% buffer B (25mM pH7.2 phosphate buffered saline buffer, 2M NaCl, 4mMEDTA, 0.03%Tween80) linear gradient elution chromatography column, total linear gradient is 10 column volumes, collects the flow point of all absorption peaks.Rush post by the buffer B of 5 column volumes with the speed of 15mL/min after gradient elution, then rush post with the 1M NaOH of 10 column volumes, finally rush column equilibration by buffer A with the speed of 15mL/min.

Hydroxyapatite column: fully to replace the effective constituent under QXL strong anion displacement chromatography wash-out with 10KDa membrane ultrafiltration with balance liquid (50mM MOPS, 0.7M pH7.2NaCl), for hydroxyapatite column loading sample.

Abundant swelling rear filling (16mmID × 360mm) chromatography column of 40g hydroxyapatite 200mM pH7.0 phosphate buffered saline buffer.Flow velocity 3mL/min, the balance liquid of 6 column volumes.Sample is balance liquid ultrafiltration and concentration, permanent filter, gets 50mL balance liquid 3 times dilution, flow velocity 3mL/min loading.Respectively through the balance liquid of 4 column volumes, elutriant I (50mM MOPS, 0.7M pH7.2NaCl, the 5mM Na of 2 column volumes ₃pO ₄), flow velocity 3mL/min stream is washed.With 8 column volume 100% elutriant I to 100% elutriants II (50mM MOPS, 0.7M pH7.2NaCl, 200mMNa ₃pO ₄) gradient elution, collect the flow point of elution peak.With elutriant III (50mM MOPS, 0.7MpH7.2NaCl, the 500mM Na of 1.5 column volumes ₃pO ₄) wash-out, collect the flow point of elution peak.The content purity analyzing each elution peak is detected with Bradford method, SDS-PAGE electrophoresis, west-blot.

Adopt such scheme can according to cell fermentation and purifying obtains corresponding protein, i.e. humanpapilloma virus 58 L1 protein or HPV52L1 albumen.

1. target protein west-blot detects

(1) the yeast special protein extraction agent YeastBuster of Novagen is used to extract fermentation expression total protein of cell, centrifugally remove cell debris, supernatant liquor 2 × loading Buffer [100mM Tris-Cl, pH6.8,20% (w/v) glycerine, 2% (w/v) SDS, 0.2M DTT, a little tetrabromophenol sulfonphthalein] boiling water bath 10 minutes after 1: 1 mixing;

(2) through SDS-PAGE electrophoresis, application " molecular cloning " second edition protein polyacrylamide gel electrophoresis method, forwards to electrophoretic band on pvdf membrane, indicates transferring film effect by pre-dyed albumen Marker;

(3) BSA bag quilt is used, HPV58L1 (CAMVIR-1): sc-47699 monoclonal antibody is as primary antibodie, and Goatanti Mouse IgG-AP resists as two, detects target protein specificity, develops the color with BCIP/NBT colouring reagents box.Transformant west-blot method the selection result is shown in accompanying drawing 4.Wherein, 1,4 is albumen Marker, be followed successively by 170 from top to bottom, 130,100,70,55,40,35,25KDa.2 roads are that HPV58 total length L1 expresses transformant, and 3 roads are that HPV58 brachymemma L1 expresses transformant.

2. electron microscopic examination: form penton L1 capsomere for proving that 58L1 albumen and 52L1 albumen assemble really automatically, the latter is then fitted into virus-like particle automatically, makes partially purified HPV58L1 and HPV52L1 protein extract carry out transmission electron microscope art (EM).

Cell grows and precipitates under fermenting on a small scale, and the precipitation of generation carries out purification process.Precipitated by immunoblotting assay and clarify yeast extract to prove L1 protein expression and the reservation by purification step.Then make clarification yeast extract carry out on 45% sucrose cushion centrifugal, and the precipitation of generation is suspended in damping fluid to be analyzed by transmission EM.The representative HPV58L1 VLPs image produced shows in Figure 5, and HPV52L1 VLPs image shows in figure 6.The diameter range of the spherical particle in this crude samples is from 30 to 60nm, and some particles show regularly arranged capsomere.

3. use the wild-type DNA-sequence of HPV52L1 and HPV52L1 respectively, utilize the processing method of above-mentioned identical vector construction, cell transfecting and induction fermentation, to obtain total Yeast protein extract of wild type gene, contrast with total Yeast protein extract of the gene of corresponding design respectively

For proving that HPV52L1 VLP expresses, by elisa assay part HPV52L1WT (containing HPV52L1 wild gene) and the total Yeast protein extract of HPV52L1.Each personal same procedure makes the yeast cell growth of expression HPV52L1WT and HPV52L1, comprises swivel pipe cultivation, shaking flask and fermentor tank.Cracking yeast cell also prepares protein extract with the amount of the HPV52L1 virus-like particle (VLPs) determined every micrograms of total protein and produce.Sandwich ELISA is designed to prove that HPV52L1 VLP expresses.Complete HPV52L1 VLPS H582C3.F7 (F7) monoclonal antibody (mAb) of G-protein purifying is used in combining yeast protein extract.F7 specific recognition HPV52L1 VLP conformational epitope.Unconjugated albumen is washed off, and is used as to detect antibody by another HPV52L1 VLP conformation specific mAb H586E11.F4 (F4).The HPV52L1 VLPs that real conformation is correct combines, and by using the antibody puted together of anti-mouse IgG2bHRP and TMB (PierceBiotechnology, Inc., Rockford, IL) substrate to promote to detect.Flat board is read and concentration by measuring HPV 52L1 VLPs compared with reference standard thing with ng VLP/ μ g total protein at 450nm wavelength.

Specifically, F7 is used for spent the night in 4 DEG C of bags by the bottom of Immulon 4HBX 96 hole flat board.Wash dull and stereotyped three times with PBS and 0.05%Tween 20, use confining liquid (PBS+0.05%Tween 20+1%BSA) to close subsequently.Plate wash three times is by antigen (being diluted to total yeast cell lysate of 12.5 μ g/ml in confining liquid) duplicate, capable for A.The reference standard thing of the HPV52L1 VLPS of purifying is to arrange for 206ng/ml is applied to A capable 3 and 4 in the total Yeast protein of 12.5 μ g/ml.Then with reference to test sample along every row serial dilution twice.Room temperature, after lower three hours, removes excessive antigen by sucking-off and washs dull and stereotyped 3 times.In confining liquid, dilute F4 conformation specific mAb and be applied to each hole, under room temperature, carrying out one hour.Dull and stereotyped three times and dilute the antibody that anti-mouse IgG2bHRP-puts together in confining liquid of washing, and at room temperature apply 1 hour.TMB (PierceBiotechnology, Inc., Rockford, IL) is also applied 5 minutes to detect the antibody complex that HRP puts together by washing flat board.By adding 2M H ₂sO ₄termination detection reacts.Flat board is read and concentration by measuring HPV 52L1 VLPs compared with reference standard thing with ng VLP/ μ g total protein at 450nm wavelength.

Find out from Fig. 7 display data is known, two groups of amounts (52L1) using the amount (52L1WT) of the VLPs detected in the yeast of wild gene to be all significantly less than the VLPs detected in the yeast of the gene in the present invention after yeast codons optimization, ordinate zou is ng VLP/ μ g Total yeast protein.Yeast codons optimization makes HPV52L1VLPs expression level add 2-3 doubly.For wild type gene and the Optimization-type gene of HPV58L1, same procedure is adopted to carry out the comparison and detection of VLPs, wild-type and the Optimization-type of HPV58L1 show the character substantially identical with Optimization-type with the wild-type of HPV52L1, and namely yeast codons optimization makes HPV58L1 VLPs expression level add 2-3 doubly.

4. use VLPs univalent vaccine to carry out animal immune experiment

(1) mouse median effective dose (ED ₅₀): the SPF level NIH or the Balb/c mouse that use 60 6 ~ 8 week ages, be divided into 6 groups, often organizes 10 mouse.The VLPs univalent vaccine of HPV is prepared into concentration 160 μ g/mL, every each immune 1mL VLPs vaccine diluent of mouse, and concentration is respectively: former times, 1:5 times diluent, 1:20 times diluent, 1:80 times diluent, 1:160 times diluent, 1:320 times diluent.Adopt abdominal injection 1mL, within after immunity six weeks, put to death blood sampling, serum separation is no less than 0.5mL, deposit in-20 DEG C for subsequent use

(2) large ear rabbit immunity and sero-fastly to take: with the HPV58 VLP particulate antigen containing completely not formula adjuvant, immunity 2 large ear rabbits.Immunizing dose is 160 μ g, within after immunity 4 weeks and 10 weeks, respectively strengthens once.After immunity, the blood of collection once, is put 2h in 37 DEG C, then is moved into 4 DEG C and spend the night, the centrifugal 10min of 4000g by blood sampling in every two weeks, absorption supernatant, the polyvalent antibody of acquisition deposit in-20 DEG C for subsequent use.10th week put to death blood sampling, serum be separated be no less than 50mL, deposit in-20 DEG C for subsequent use.

(3) effect of above-mentioned pika experimental verification VLPs univalent vaccine and effect, can effectively excite pika to produce corresponding immune antibody.

Above to invention has been exemplary description; should be noted that; when not departing from core of the present invention, any simple distortion, amendment or other those skilled in the art can not spend the equivalent replacement of creative work all to fall into protection scope of the present invention.

Claims (10)

1. the nucleotide sequence of coding HPV52L1 albumen, as the nucleotide sequence in sequence table as described in SEQ ID No.1.

2. the nucleotide sequence of coding humanpapilloma virus 58 L1 protein, as the nucleotide sequence in sequence table as described in SEQ ID No.2.

3. a carrier, the nucleotide sequence SEQ ID No.1 containing coding HPV52L1 albumen, or the nucleotide sequence SEQ ID No.2 of coding humanpapilloma virus 58 L1 protein.

4. an eucaryon debaryomyces hansenii engineering bacteria, with the nucleotide sequence SEQ ID No.1 containing coding HPV52L1 albumen, or the carrier transfection eucaryon debaryomyces hansenii bacterial strain RB-11 of the nucleotide sequence SEQ ID No.2 of coding humanpapilloma virus 58 L1 protein, obtains the eucaryon debaryomyces hansenii bacterial strain RB-11 of transfection.

5. utilize the eucaryon debaryomyces hansenii bacterial strain RB-11 of methyl alcohol to transfection to carry out abduction delivering, it is characterized in that, in whole process, temperature is 28-32 DEG C, time is 72h, during beginning, methanol usage is 0.5% of cell suspending liquid quality, at interval of 24h, in cell suspending liquid, add methyl alcohol, the amount of adding methyl alcohol is 0.5% of cell suspending liquid quality.

6. utilize the eucaryon debaryomyces hansenii bacterial strain RB-11 of transfection to carry out the method for fermenting, it is characterized in that, during the fermentation, described eucaryon debaryomyces hansenii engineering bacteria is through outgrowth phase, the phase that derepresses and abduction delivering phase:

(1) grow phase: fermentor tank pH value controls at 5.4-5.5, and leavening temperature controls at 28-32 DEG C, the fermention medium of use through sterilising treatment, by NH ₄h ₂pO ₄170-180 mass parts, MgSO ₄7H ₂o 40-50 mass parts, KCl 40-45 mass parts, NaCl 4-5 mass parts, glycerine 500-550 mass parts, fermentation defoaming agent 4-5 mass parts and deionized water 10000-15000 mass parts composition; In growth phase, in fermentor tank, dissolved oxygen can slowly decline, and when dropping to 60%, in fermentor tank, at the uniform velocity squeezes into supplemented medium, the supplemented medium of use through sterilising treatment, by NH ₄h ₂pO ₄80-90 mass parts, glycerine 250-300 mass parts and deionized water 500-550 mass parts form; When fermentor tank dissolved oxygen level is down to below 40%, air flow quantity is adjusted to 12-15L/min by 25-30L/min originally, mixing speed is adjusted to 700rpm by 1200-1500rpm originally; The whole growth phase time, cell growth state was good, and fermentor tank dissolved oxygen level can drop to less than 20%, cell OD at 20-25 hours ₆₀₀reach more than 80;

(2) derepress phase: when fermentor tank dissolved oxygen is gone back up to more than 60% by lower-most point, enter the phase that derepresses, maintain air flow quantity and agitation condition constant, add derepression culture base; Described derepression culture base through sterilising treatment, by NH ₄h ₂pO ₄25-30 mass parts, MgSO ₄7H ₂o 5-10 mass parts, KCl 5-8 mass parts, NaCl 0.5-1 mass parts, glycerine 1700-1900 mass parts and deionized water 1200-1350 mass parts form, and the phase that derepresses continues 20-25h;

(3) phase is induced: after the phase that derepresses, enter induction phase, inducing culture is added in fermentor tank, described inducing culture is through sterilising treatment, be made up of glycerine and methyl alcohol, the volume ratio of glycerine and methyl alcohol is (38-40): (60-65), and the consumption of regulation and control methyl alcohol, to make methyl alcohol in the liquid phase volume of whole fermentor tank, volume percent is 0.3%-0.5%, and induction phase continues 48-55h.

7. fermentation process according to claim 6, is characterized in that, in growth phase, fermention medium is preferably by NH ₄h ₂pO ₄170-175 mass parts, MgSO ₄7H ₂o 40-45 mass parts, KCl 42-44 mass parts, NaCl 4.2-4.4 mass parts, glycerine 520-540 mass parts, fermentation defoaming agent 4-5 mass parts and deionized water 10000-12000 mass parts composition, described fermentation defoaming agent is THIF-298 type biological fermentation defoamers; Supplemented medium is preferably by NH ₄h ₂pO ₄85-87 mass parts, glycerine 260-280 mass parts and deionized water 520-540 mass parts form; The volume ratio of supplemented medium and fermention medium is (0.8-1): (10-12).

8. fermentation process according to claim 6, is characterized in that, derepression culture base is preferably by NH ₄h ₂pO ₄26-28 mass parts, MgSO ₄7H ₂o 6-8 mass parts, KCl 7-8 mass parts, NaCl 0.6-0.8 mass parts, glycerine 1750-1850 mass parts and deionized water 1300-1350 mass parts form.Derepressing in phase, the volume ratio of fermention medium and derepression culture base is 4:(1-1.2).

9. fermentation process according to claim 6, is characterized in that, the use volume of inducing culture be derepression culture matrix long-pending 30-40%.

10. the nucleotide sequence of coding HPV52L1 albumen, nucleotide sequence, as claimed in claim 3 carrier, as claimed in claim 4 the eucaryon debaryomyces hansenii engineering bacteria of coding humanpapilloma virus 58 L1 protein as claimed in claim 2 are preparing the application in virus-like particle as claimed in claim 1.