CN102178944A - Expression and application of human papilloma viruses type 16 and 18 L1 proteins in inset cells - Google Patents

Abstract

The invention provides a method for preparing a bivalent vaccine for human papilloma viruses HPV16 and HPV18, which comprises the following steps of: optimizing gene sequences of L1 proteins of the HPV16 and HPV18; cloning the optimized gene sequences into a baculovirus expression vector and transfecting the inset cells to obtain a recombinant baculovirus; infecting the insect cells by using the baculovirus; and recovering and purifying the L1 proteins expressing the HPV16 and HPV18, and mixing with an adjuvant to prepare the bivalent vaccine. The invention also provides the optimized gene sequences of the L1 proteins of the HPV16 and HPV18, vectors containing the gene sequences, and cells containing the vectors.

Description

Human papilloma virus 16 and 18 expression and the application of type L1 albumen in insect cell

Technical field

The invention belongs to biological technical field, and relate to human papilloma virus 16 and expression and the vaccine production method thereof of 18 type L1 albumen in insect cell.

Background technology

The statistics of The World Health Organization (WHO) shows that cervical cancer is the infection disease of serious harm WomanHealth, and its sickness rate is only second to breast carcinoma, occupies second of whole world women's malignant tumor, in some developing countries even occupy the first.Cervical cancer is one of modal gynecologic malignant tumor, and the whole world has 500,000 women to suffer from cervical cancer the every year approximately, and nearly has that 300,000 women die from cervical cancer, and wherein, 80% death occurs in developing country.According to incompletely statistics, the existing cervical cancer patient of China is about 13.8 ten thousand, has every year 50000 people to die from cervical cancer approximately, and sickness rate be year by year rise, the trend of rejuvenation.In nineteen ninety-five, (human papillomavirus, infection HPV) is defined as the pathogenic cause of disease of cervical cancer to WHO with the human papillomavirus.Nearly all cervical cancer all causes by HPV, wherein all exists HPV to infect up to 99.7% cervical cancer patient, and surpasses 2/3 cases of cervical cancer and caused by HPV 16 types and 18 types.

(Human papillomavirus HPV) is a kind of epithelium sexually transmitted disease (STD) poison of having a liking for to the human papillomavirus, and it has the specificity of height.HPV is the general name of a papova, and it forms a section, and wherein each viral form is similar, but DNA restriction map spectrum is different, the antigenicity difference of nucleocapsid protein matter.Present kind surplus the HPV type of having determined nearly 160 is divided into skin-type HPV and reproductive tract epithelium HPV according to the position at the epithelium place of its infection, and wherein about 35 kinds of types can infect female genital, about 20 kinds relevant with tumor.Cause the height of the danger of tumor according to HPV, HPV is divided into low dangerous type HPV and high-risk type HPV.Low dangerous type HPV comprises HPV6,11,42,43, types such as 44, and it causes benign lesions such as external genitalia condyloma latum usually, comprises low pathological changes (CINI) in the epithelium of cervix uteri.High-risk type HPV comprises HPV16,18,31,33,35,39,45,51,52,56,58,59, types such as 68.High-risk type HPV, particularly HPV16 and 18 types, the generation that becomes (CIN II/III) with cervical cancer and Cervical intraepithelial neoplasia is relevant.

In recent ten years, the HPV vaccine research can be divided into two classes, i.e. preventative vaccine research and therapeutic vaccine research.Preventative vaccine is a target antigen with main capsid protein L 1 of HPV16 and less important capsid protein L2 generally, its role is to bring out body and produces specific neutralizing antibody and effective local immune response, with long-term infection and the infection again that stops HPV.When the capsid protein of HPV is expressed in eucaryon and prokaryotic expression system, can oneself assembling or form virus-like particle (VLP), its structure and epitope and natural virion are quite similar.VLP can combine and enter cell with cell receptor, presents and brings out stronger cellular immunization thereby help antigenic processing.Normally (its activity of conversion is removed therapeutic vaccine with modified HPV16 early protein, but still keep antigenicity) as target antigen, but the cellullar immunologic response of its inducing specific, thereby can be used for controlling or eliminate the optimum and pernicious focus that infects HPV, and can be used as the postoperative auxiliary treatment of this class disease.In great majority cervical cancers and precancerous lesion thereof relevant with HPV16, the E6 of HPV16 and E7 albumen continuous expression, and this continuous expression is tumour cell transformation and to keep malignant characteristics necessary, and do not have this two kinds of albumen in the normal structure.Therefore, E6 is the desirable target antigen that is used for the therapeutic vaccine of relevant cervical cancer of HPV16 and precancerous lesion with E7 albumen.At the middle and advanced stage cervical cancer patient residual tumor cell in back of performing the operation, can use this therapeutic vaccine, with the epithelial cell that kills and wounds, removes these tumor cells and infected by the cellular immunization that excites patient, thereby prevent or limit the recurrence and the diffusion of tumor.Preventative and effect therapeutic HPV vaccine also has intersection.For example, have the expression of HPV late protein in optimum wart and slight CIN pathological changes, therefore, preventative vaccine also has certain therapeutical effect to these diseases.Yan Zhi some vaccines as mosaic vaccine, HPV pseudovirus vaccine etc., possess prevention and treatment dual function simultaneously in recent years.

The HPV vaccine causes in prevention aspect the persistent infection of cervical cancer, and effective percentage reaches 100%, and it can reduce by 75% with the cervical cancer incidence rate, and safety is good.Existing in the world at present a plurality of HPV16 candidate vaccines enter the I/II clinical trial phase, and this vaccine will be saved thousands of women's life, and therefore the health to the women produces significant impact.

Merck ﹠ Co., Inc. in 1991 from the mandate that patents of Australian CSL company, express by using saccharomyces cerevisiae, prepared tetravalence HPV vaccine at HPV 6,11,16 and 18 types.And the HPV vaccine technologies of GlaxoSmithKline PLC company derives from MedImmune company, and this vaccine is the divalent vaccine, and commodity are called " Cervarix " (prevention 16,18 types), and it uses insect cell to express.These two kinds of vaccines all can make animal subject produce neutralizing antibody to HPV virus rapidly, and to the person poultry safety, effectively the various diseases, particularly syphilis and the cervical cancer that are caused of prophylactic treatment HPV virus.A plurality of national patent protections such as these technology are published on the internal authority technical magazine, partial content has obtained the U.S..

Lausanne, SUI university research personnel work out a kind of spray vaccine for the treatment of cervical cancer recently, and its effect is identical with vaccinate.By sucking this spray vaccine, can stimulate and produce antibody at this virus, its effect is the same with vaccinate.This spray is easy to use, short treating period.Patient only need use twice, its 2 weeks of interbody spacer, and vaccinate need be injected 3 times, and 6 months consuming time.

Reported also that a kind of effective vaccine based on 7 kinds of human papillomavirus (HPV) can prevent most cervical cancers in the whole world in 2004.Yet its expense is very high, and at some not too common HPV hypotypes, its protective effect that provides also is difficult to clearly confirm.Consider the cost problem, the vaccine of common hypotype may be more cheap at 2 kinds (HPV16,18) or 3 kinds (HPV16,18 and 45), and is also more real.Especially, the vaccine of anti-HPV16 and HPV18 is very meaningful, because it can prevent 70% cervical cancer.

For this reason, the present invention is based on the L1 Argine Monohydrochloride sequence of the popular highly pathogenic HPV16/18 type strain of China, developed the preparation method of new HPV16/18 bivalent vaccine, it prevents to have important practical significance for large-scale production HPV16/18 bivalent vaccine and for China women's cervical cancer.

Summary of the invention

Human papillomavirus's main capsid protein L 1 gene can the oneself assemble or formation virus-like particle (VLP) behind protokaryon and eukaryotic expression, brings out cellular immunization thereby can combine with cell receptor.Therefore, L1 albumen is the target antigen of the most worthy of prevention cervical cancer.The present invention as object of study, has developed the preparation method of new HPV16/18 bivalent vaccine with the main capsid protein L 1 gene of the popular highly pathogenic HPV 16 of China and 18 two kind of hypotype.Utilize this method, the present invention has realized the high expressed of the main capsid protein L 1 of HPV 16 and HPV 18, and obtained biological character VLP preferably, it has good antigenicity and immunogenicity, prevents to have important practical sense for large-scale production HPV16/18 bivalent vaccine and for China women's cervical cancer.

Therefore, in one aspect, the invention provides the method for preparation at the bivalent vaccine of human papillomavirus HPV16 and HPV18, it may further comprise the steps:

1) optimizes the proteic gene order of L1 of HPV16 and HPV18 respectively;

2) gene order after optimizing in the step 1) is cloned into rhabdovirus expression vector respectively, to obtain recombiant plasmid;

3) use described recombiant plasmid, with corresponding baculovirus genome, the difference transfection insect cell is to obtain recombinant baculovirus;

4) infect the insect cell of fresh cultured respectively with described recombinant baculovirus;

5) be mixed with into bivalent vaccine with adjuvant from the insect cell difference separation and purification HPV16 of step 4) and the L1 albumen of HPV18, and with it.

In a preferred embodiment, the gene order after the proteic optimization of the L1 of HPV16 and HPV18 is respectively shown in SEQ ID NO.1 and 2.

In a preferred embodiment, the employed rhabdovirus expression vector of method of the present invention is pAcSG2, and employed baculovirus genome is baculovirus genome AcNPV.

In a preferred embodiment, the employed insect cell of method of the present invention is selected from Sf-9 cell, Sf-21 cell and High Five cell.

In a preferred embodiment, method of the present invention with the L1 protein adsorption of the HPV16 of separation and purification and HPV18 to adjuvant, with the preparation bivalent vaccine.In a preferred embodiment, the employed adjuvant of method of the present invention is preferably selected from aluminum hydroxide adjuvant and aluminum phosphate adjuvant.

In one aspect, the invention provides isolated nucleic acid molecule, it comprises the gene order after the proteic optimization of L1 of HPV16 and HPV18.Preferably, isolated nucleic acid molecule of the present invention comprises the sequence shown in SEQ ID NO.1 or 2.

In one aspect, the invention provides the carrier that comprises isolated nucleic acid molecule of the present invention.Preferably, carrier of the present invention is an expression vector.In a preferred embodiment, carrier of the present invention is suitable for for example expressing in Sf-9 cell, Sf-21 cell or the High Five cell etc. at insect cell, and it is rhabdovirus expression vector preferably, is more preferably pAcSG2.

In one aspect, the invention provides the cell that comprises isolated nucleic acid molecule of the present invention or carrier.In a preferred embodiment, cell of the present invention is an insect cell, preferred Sf-9 cell, Sf-21 cell or High Five cell.

In one aspect, the purposes that also provides isolated nucleic acid molecule of the present invention to be used to prepare bivalent vaccine, described bivalent vaccine is used to prevent the infection of HPV16 and HPV18.

In one aspect, also provide isolated nucleic acid molecule of the present invention to be used to prepare the proteic purposes of L1 of HPV16 and HPV18.

The beneficial effect of the invention

Compared with prior art, technical scheme of the present invention has following beneficial effect:

(1) prepared vaccine helps the cervical cancer prevention to China women at the L1 Argine Monohydrochloride sequence of the popular highly pathogenic HPV16/18 type strain of China;

(2) in the preparation process of vaccine, the proteic gene order of HPV16/18L1 is optimized, help to improve the expression of L1 albumen in insect cell, thereby have important practical significance for large-scale production HPV16/18 bivalent vaccine and for the clinical practice of HPV vaccine.

Below in conjunction with drawings and Examples embodiment of the present invention are described in detail, but it will be understood by those skilled in the art that following drawings and Examples only are used to illustrate the present invention, rather than to the qualification of scope of the present invention.With the following detailed description of preferred embodiment, it is obvious that various purposes of the present invention and favourable aspect will become to those skilled in the art with reference to the accompanying drawings.

Summary of drawings

Fig. 1 shows the double digestion evaluation of pVL16-L1 and pVL18-L1.Wherein, swimming lane 1 is a dna molecular amount standard; Swimming lane 2 is Xho I and the Nco I double digestion product of pVL16-L1; Swimming lane 3 is Xho I and the Nco I double digestion product of pVL18-L1.

Fig. 2 shows the enzyme action evaluation of the pcr amplification product of recombinant virus.Wherein, swimming lane 1 is a dna molecular amount standard; Swimming lane 2 is the genome pcr amplification product of rBV16; Swimming lane 3 is the genome pcr amplification product of rBV18.

Fig. 3 shows that the proteic SDS-PAGE of HPV16 L1 that rBV16 expresses identifies.Wherein, swimming lane 1 is the contrast of Sf-9 cell pyrolysis liquid; Swimming lane 2 and 3 is the lysate of the Sf-9 cell of rBV16 infection; Swimming lane 4 is the molecular weight of albumen standard.

Fig. 4 shows that the proteic SDS-PAGE of HPV18 L1 that rBV18 expresses identifies.Wherein, swimming lane 1 is the contrast of Sf-9 cell pyrolysis liquid; Swimming lane 2,3,4 and 5 is the lysate of the Sf-9 cell of rBV18 infection; Swimming lane 6 is the molecular weight of albumen standard.

Fig. 5 shows that the proteic SDS-PAGE of the HPV16/18 L1 of purification identifies.Wherein, swimming lane 1 is the molecular weight of albumen standard; Swimming lane 2 is the HPV16 L1 albumen of purification; Swimming lane 3 is the HPV18 L1 albumen of purification.

Fig. 6 is the transmission electron microscope photo of HPV16/18 L1.Wherein, Fig. 6 A shows the VLP of HPV16 L1; Figure B shows the VLP of HPV18 L1.

Fig. 7 shows the proteic Western trace evaluation of HPV16/18 L1.Wherein, swimming lane 1 is the molecular weight of albumen standard; Swimming lane 2 is a HPV16 L1 albumen; Swimming lane 3 is the contrast of Sf-9 cell; Swimming lane 4 is a HPV18 L1 albumen; Swimming lane 5 is the contrast of Sf-9 cell.

Fig. 8 shows the interior fluorescence of cell after the pseudovirus of preparation infects the 293FT cell.

The specific embodiment

Referring now to following be intended to illustrate but do not limit embodiments of the invention the present invention is described.

The optimization of the proteic gene order of L1 of embodiment 1.HPV16/18

The proteic aminoacid sequence of L1 of popular highly pathogenic HPV16 of China and HPV18 type strain is respectively shown in SEQ ID NO.7 and 8.

Use the commercially available software OptimumGene that gets ^TML1 gene order to HPV16 and HPV18 type is optimized, to improve the expression of L1 gene in insect cell.Wherein, the HPV16 L1 gene order after the optimization is shown in SEQ ID NO.1; HPV18 L1 gene order after the optimization is shown in SEQ ID NO.2.

The structure of embodiment 2.pVL16-L1 and pVL18-L1 transfection plasmid

Utilize primer to 5 '-CTC GAG GAA TTC AGG ATG CAA GTG ACG TTTATT-3 ' (SEQ ID NO.3) and 5 '-A GAG CTC CCA TGG AGG TTA CAA CTTGCG TTT CTT-3 ' (SEQ ID NO.4), and primer to 5 '-CTC GAG GAA TTCAGG ATG TGC TTG TAC ACG CGC-3 ' (SEQ ID NO.5) and 5 '-A GAG CTCCCA TGG AGG TTA TTT GCG AGC TCT CAC G-3 ' (SEQ ID NO.6), by PCR increase respectively the HPV16 type and the HPV18 type L1 gene through optimizing of synthetic.The PCR reaction condition is 95 ℃ of pre-degeneration 5 minutes; 95 ℃ of degeneration of 30 circulation 30 seconds were annealed 45 seconds for 55 ℃, and 72 ℃ were extended 2 minutes; Last 72 ℃ were extended 10 minutes.Behind the pcr amplification, obtain the L1 genetic fragment that two ends have Xho I and Nco I restriction enzyme digestion sites respectively.

Xho I that use is obtained commercially and Nco I (for example available from precious biological engineering (Dalian) company limited) (derive from the test kit of U.S. company BD, BD BaculoGold to L1 genetic fragment and baculovirus vector pAcSG2 ^TM, 560129) and carry out double digestion respectively, carry out 3 hours (the L1 genetic fragment of 1 μ g or pAcSG2 carrier add 10U Xho I and 10UNco I) in 37 ℃.L1 genetic fragment behind the enzyme action is inserted into respectively among the same baculovirus vector pAcSG2 through Xho I and NcoI enzyme action.The reaction system that connects is: L1 genetic fragment 100 μ g, pAcSG2 carrier 150 μ g, T4DNA ligase 1U; Reaction condition is that 16 ℃ of connections are spent the night.With the transformed into escherichia coli (E.coli) respectively of the carrier after connecting, then through colony screening, increase that bacterium is cultivated and plasmid extraction obtains to comprise the transfection plasmid of target gene fragment.The transfection plasmid that is obtained is identified by Xho I and Nco I double digestion.

As shown in Figure 1, the recombiant plasmid HPV16L1-pAcSG2 (being called for short pVL16-L1) and the HPV18L1-pAcSG2 (being called for short pVL18-L1) of target gene fragment have been obtained to comprise.

The preparation of embodiment 3. recombinant baculovirus

PVL16-L1 and pVL18-L1 recombiant plasmid (are derived from the test kit of U.S. company BD, BD BaculoGold with the AcNPV genomic DNA respectively ^TM, 560129) and cotransfection Sf-9 cell (buy from American I nvitrogen company) together.27 ℃ down cultivate 4 days after, the cell volume observe transfection under mirror after increases.Cultivate after 5 days collecting cell culture supernatant (wherein containing recombinant virus rBV16 and rBV18 respectively).Cell conditioned medium liquid is inoculated into the Sf-9 cell of fresh cultured respectively, cultivated 3 days down at 27 ℃.Get culture supernatant repeated transmission generation once.Afterwards, get culture supernatant, add isopyknic 2 * precipitated liquid (containing 20%PEG6000,1M NaCl), spend the night 4 ℃ of mixing.Then under 4 ℃, with 13000rpm with centrifugal 30 minutes of mixture, and abandoning supernatant.The precipitation of gained is with 100 μ l sterilized water resuspensions, and adds the 5U E.C. 3.4.21.64, spends the night 50 ℃ of digestion.Add isopyknic phenol/chloroform in digest, vibration is mixed, and with 13000rpm centrifugal 10 minutes; Get supernatant, add isopyknic chloroform, vibration is mixed, with 13000rpm centrifugal 10 minutes once more; Get supernatant, add the 3M NaAc and 0.6 of 1/10 volume * isopropyl alcohol, and placed 15 minutes in-20 ℃.Under 4 ℃ centrifugal 10 minutes then with 13000rpm, and abandoning supernatant.70% washing with alcohol of the precipitation usefulness pre-cooling of gained 2 times, at room temperature dry 10 minutes then.After the drying, precipitation suspends with 50 μ l sterilized water, thereby obtains the genome of recombinant virus rBV16 and rBV18.Get 5 μ l recombinant virus genomes, and respectively with Auele Specific Primer to SEQ ID NO.3 and SEQ ID NO.4, and SEQ ID NO.5 and SEQ ID NO.6 carry out PCR and identify.The PCR reaction condition is as follows: 95 ℃ of pre-degeneration 5 minutes; 95 ℃ of degeneration of 30 circulation 30 seconds were annealed 45 seconds for 55 ℃, and 72 ℃ were extended 2 minutes; Last 72 ℃ were extended 10 minutes.The result that PCR identifies shows, contains the HPV16 L1 and size purpose fragment (referring to Fig. 2) for the HPV18 L1 of 1700bp of size for 1600bp in the genome of recombinant virus rBV16 and rBV18 respectively.

Collect infected Sf-9 cell, add cell pyrolysis liquid (50mmol/L Tris-HCl (pH8.0); 15mmol/L NaCl; 0.1mmol/L PMSF; 0.1%NP40; 5mmol/LEDTA) carry out cracking.With 100 μ l cell lysates and 100 μ l, 2 * albumen sample-loading buffer (100mmol/L Tris HCl (pH 6.8); 200mmol/L DTT; 8%SDS; 0.02% Coomassie brilliant blue R-250; 24% glycerol) mixing, and boiling water bath 10 minutes.Get 10 μ l samples then and on 10% polyacrylamide gel, carry out the SDS-PAGE electrophoresis, and carry out Coomassie brilliant blue dyeing, whether express with testing goal albumen.The result shows, recombinant virus rBV16 and rBV18 express the HPV18 L1 albumen (referring to Fig. 3 and 4) that HPV16 L1 albumen that molecular weight is 59.4KD and molecular weight are 63.7KD respectively.Further analyze by software Quatity One (Bio-Rad company), the result shows, the HPV16 L1 albumen of expressing accounts for 9.8% (referring to Fig. 3) of whole Sf-9 total protein of cell, and the HPV18 L1 albumen of expression accounts for 6.5% (referring to Fig. 4) of whole Sf-9 total protein of cell.

The proteic separation and purification of embodiment 4.HPV16/18 L1

In the insect cell of 96 hours infection recombinant baculovirus of cultivation, add cell pyrolysis liquid (50mmol/L Tris-HCl (pH8.0); 15mmol/L NaCl; 0.1mmol/L PMSF; 0.1%NP40; 5mmol/L EDTA), the ultrasonic disruption cell, and in 4 ℃ with 10000g centrifugal 15 minutes, remove cell debris.With 4 ℃ of centrifuged supernatant of 100000g 90 minutes.With the precipitation of gained be resuspended in buffer A (50mM Tris, pH 8.0; 1M NaCl; 10mMMgCl ₂10mM CaCl ₂2mM PMSF; 10ug/ml Leupeptin) in, adds the solid CsCl of 5.2 grams then, and volume is adjusted to 13ml (the CsCl final concentration is 0.4g/ml), further, obtain purified HPV16 and HPV18 L1 albumen with centrifugal 22 hours of 4 ℃ of 100000g with buffer A.The albumen of gained is identified by SDS-PAGE, the results are shown among Fig. 5.

Also can pass through sucrose density gradient centrifugation purification HPV16 and HPV18L1 albumen.Recombiant protein is taken out of on the sucrose density gradient of present 50-60%.Concrete purification process is as follows: supernatant is laid on 30% the sucrose solution (being dissolved among the TBS), with 150000g, 4 ℃ centrifugal 6 hours; The precipitation that obtains 50mM Tris-100mM NaCl resuspension, and be laid on the sucrose density gradient solution (being dissolved among the 50mM Tris-100mM NaCl) of 20%-60%, with 150000g, 4 ℃ are centrifugal 22 hours; Sucking-off purpose ash white area band, and with the buffer A dilution, then by 100000g, 4 ℃ of centrifugal HPV16/18 L1 albumen that came deposition and purification in 90 minutes.

The proteic morphologic observation of embodiment 5.HPV16/18 L1

The learn from else's experience HPV16/18 L1 protein liquid of purification drips copper mesh, dyes with 2% phosphotungstic acid, and observes by transmission electron microscope.The result shows that HPV16/18 L1 albumen is rendered as virus-like particle, and (Virus-like particles, VLP), its diameter is about 52nm (referring to Fig. 6).

Embodiment 6. detects reorganization HPV16/18 L1 albumen by the Western trace

Use anti-HPV16 L1 antibody (to derive from SANTA CRUZ BIOTECHNOLOGY company respectively, sc-18052) and anti-HPV18L1 antibody (derive from abcam company, ab31492), detect HPV16 and HPV18 L1 albumen behind the purification by the Western trace.Western trace result shows that molecular weight is the HPV16 L1 of 59.4KD and the proteic specific protein leucorrhea of HPV18 L1 (referring to Fig. 7) that molecular weight is 63.7KD, and this shows that expressed HPV16 and HPV18 L1 albumen have good antigenicity.

Embodiment 7. usefulness reorganization HPV16/18 L1 protein immune animal

HPV16 and HPV18 L1 albumen with the quantitative purification of Bio-Rad Protein Assay test kit.Respectively with the HPV16 L1 albumen of 0.1 μ g, 2 μ g or 20 μ g and HPV18 L1 protein adsorption to 0.5mg Al (OH) ₃, to be prepared into the HPV bivalent vaccine of three kinds of dosage.Use the HPV bivalent vaccine of preparation, respectively at 0,2,4 weeks coming immune Balb/c mice three times by intramuscular injection.Mice is put to death in back three days of last immunity, and collects serum.

The preparation of embodiment 8.HPV16/18 pseudovirion

Respectively with L1 and L2 gene (the GeneBank accession number AF084952 of HPV16 L2 of HPV16 and HPV18, the GeneBank accession number DQ003068 of HPV18 L2) is cloned into pcDNA3.1 carrier (available from Invitrogen company), to make up pcDNA3.1-16L1, pcDNA3.1-16L2, pcDNA3.1-18L1 and pcDNA3.1-18L2 recombinant vector.With pcDNA3.1-16L1 and pcDNA3.1-16L2 cotransfection 293FT cell (buying ATCC) in the U.S.; With pcDNA3.1-18L1 and pcDNA3.1-18L2 cotransfection 293FT cell.The cell of gained was cultivated 3-5 days at 37 ℃.Harvesting and multigelation three times carry out the chloroform extracting, and collect supernatant.The supernatant of gained is the HPV16 pseudovirus, and (pseudovirus 16, and psV16) (pseudovirus 18, and psV18) granule is frozen standby with the HPV18 pseudovirus.Pseudovirus psV16 and psV18 to preparation on the 293FT cell that 96 orifice plates are cultivated carry out titer determination.In brief, get the pseudovirus seed liquor and carry out 10 times of serial dilutions, 8 culture hole of each dilution factor inoculation; Postvaccinal cell was cultivated 5 days at 37 ℃, observed the intracellular fluorescence (referring to Fig. 8) of each culture hole down in fluorescence microscope then, calculated the titre of pseudovirus.

The proteic immunogenicity analysis of embodiment 9.HPV16/18 L1

HPV16/18 L1 albumen (in carbonate buffer solution) with purification spends the night 4 ℃ of bags by 96 orifice plates.(the solution washing plate of PBS solution+0.5%Tween-20) three times adds the mice serum of PBS dilution, hatches 1 hour for 37 ℃ to use PBST then.After hatching, use PBST solution washing plate three times, add the anti-mouse IgG antibody (buying company) of HRP labelling, hatched 1 hour for 37 ℃ in KPL.After hatching, use PBST solution washing plate three times, add OPD substrate colour developing liquid (citric acid 0.51%, Na ₂HPO ₄12H ₂O 1.84%, o-phenylenediamine (OPD) 0.05%, H ₂O ₂50 μ l), the lucifuge colour developing is 10 minutes.Use 2N H afterwards ₂SO ₄Cessation reaction, and measure the OD value at 492nm place with microplate reader (BIO-RAD company).The result shows that HPV16/18 L1 albumen can induce body to produce specific antibody, and this shows that reorganization HPV16/18 L1 albumen has good immunogenicity (referring to table 1).

Mice serum is by dilution in 1: 500, mixed with the pseudovirus psV16 of 100PFU and psV18 respectively with diluent, and 37 ℃ of incubations 1 hour.Behind the incubation, pseudovirus is joined on the 293FT cell of monolayer, and 37 ℃ of absorption 1 hour.After the absorption, continued cultured cell 7 days in 37 ℃.At last, by the intracellular fluorescence of fluorescence microscope 293FT.The result shows; the proteic mice serum of anti-HPV16/18 L1 can combine with pseudovirus psV16 and psV18; and the inhibition pseudovirus enters generation fluorescence in the 293FT cell; this shows and contains the proteic neutralizing antibody of anti-HPV16/18 L1 in the mice serum that it can protect body to avoid the infection of HPV16/18 type virus (referring to table 2).

The immunogenicity determining of table 1.HPV16 L1 vaccine

The immunogenicity determining of table 2.HPV18 L1 vaccine

Claims (8)

1. preparation is at the method for the bivalent vaccine of human papillomavirus HPV16 and HPV18, and it may further comprise the steps:

1) optimize the proteic gene order of L1 of HPV16 and HPV18 respectively, the gene order after the proteic optimization of L1 of preferred HPV16 and HPV18 is respectively shown in SEQ ID NO.1 and 2;

2) gene order after optimizing in the step 1) is cloned into rhabdovirus expression vector respectively, to obtain recombiant plasmid, preferred described rhabdovirus expression vector is pAcSG2;

3) use described recombiant plasmid, with corresponding baculovirus genome, the difference transfection insect cell, to obtain recombinant baculovirus, preferred described baculovirus genome is baculovirus genome AcNPV, and described insect cell is preferably selected from Sf-9 cell, Sf-21 cell and High Five cell;

4) infect the insect cell of fresh cultured respectively with described recombinant baculovirus;

5) be mixed with into bivalent vaccine from the insect cell difference separation and purification HPV16 of step 4) and the L1 albumen of HPV18, and with it with adjuvant, described adjuvant is preferably selected from aluminum hydroxide adjuvant and aluminum phosphate adjuvant.

2. isolated nucleic acid molecule, its sequence is shown in SEQ ID NO.1.

3. isolated nucleic acid molecule, its sequence is shown in SEQ ID NO.2.

4. the carrier that contains the nucleic acid molecules of claim 2 or 3, described carrier is expression vector preferably, is more preferably rhabdovirus expression vector, is more preferably pAcSG2.

5. the cell that contains the carrier of claim 4, described cell is insect cell preferably, is more preferably Sf-9 cell, Sf-21 cell or High Five cell.

6. claim 2 and/or 3 nucleic acid molecules are used to prepare the purposes of bivalent vaccine, and described bivalent vaccine is used to prevent the infection of HPV16 and HPV18.

7. the nucleic acid molecules of claim 2 is used to prepare the proteic purposes of L1 of HPV16.

8. the nucleic acid molecules of claim 3 is used to prepare the proteic purposes of L1 of HPV18.

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