CN101280316B

CN101280316B - Therapeutic vaccine for human cervical carcinoma

Info

Publication number: CN101280316B
Application number: CN2008101127659A
Authority: CN
Inventors: 黄来强; 郑义; 杨峥嵘; 万骏; 马原栋
Original assignee: Shenzhen Graduate School Tsinghua University
Current assignee: Shenzhen Graduate School Tsinghua University
Priority date: 2008-05-26
Filing date: 2008-05-26
Publication date: 2010-06-09
Anticipated expiration: 2028-05-26
Also published as: CN101280316A

Abstract

The invention discloses therapeutic vaccine of human cervical carcinoma. The active component of the vaccine is recombinant adenovirus; the recombinant adenovirus is prepared according to the following methods: recombinant adenovirus comprises HPV16mE67, HPV18mE67 and HPV58mE67, the transcriptional fusion three-gene fragment of the short nucleotide fragment connected with the three components is inserted into the recombinant adenovirus carrier obtained in the viral DNA of the Adeno-X expression system, and then the recombinant adenovirus carrier is inducted in the cell to obtain the recombinant adenovirus. The animal test result indicates that the vaccine can be expressed in the cell, the animal is directly immunized through the purified adenovirus pellet, the formed tumor has the obvious treatment effect, and the functional mechanism is mainly performed, because the strong CTL response in the animal vivo can be generated. The vaccine in the invention can be applied for treating the tumor caused by the HPV infection.

Description

A kind of therapeutic vaccine of human cervical carcinoma

Technical field

The present invention relates to a kind of therapeutic vaccine of human cervical carcinoma.

Background technology

(human papilloma-virus, persistent infection HPV) often can cause generations such as Genital warts, cervical cancer, penile cancer, cancer of anal margin and incidence cancer to human papillomavirus, and is wherein outstanding with cervical cancer harm especially.Present global cervical cancer patient surpasses 1,400 ten thousand, mainly concentrate on developing country, and surplus the annual newly-increased case 470,000, wherein half people's death at least, cervical cancer is in second in women's tumor mortality rate, become great public health problem (the Zur Hausen H.Papillomavirus in human cancers.Pro Assoc AmPhysicians of puzzlement society, 1999,111 (6): 581-587.Koutsky LA, Galloway DA, Holmes KK.Epidemiology of genital human papillomavirus infection.Epidemiol.Rev.1988,10:122-163.Munger K, Baldwin A, Edwards KM.Mechanisms of HumanPapillomavirus-induced Oncogenesis.J Virol, 2004,78 (21): 11451-11460.GarlandSM.Human papillomavirus update with a particular focus on cervical disease.Pathology, 2002,34 (3): 213-224.).Cervical cancer traditional therapy unsatisfactory curative effect clinically, mainly show as: the middle and advanced stage chemotherapy of patients is efficient to be generally less than 20%, radiotherapy or the operation nearly 4 one-tenth recurrences in back, and prognosis extreme difference.Studies show that, utilize up-to-date Recent advances in molecular biology, the research biological engineering vaccine is to improve curative effect, be one of the present direction that in the cervical cancer control, is hopeful to make a breakthrough most (Jansen KUand Shaw AR.Human papillomavirusvaccines and prevention of cervical cancer.Annu.Rev.Med.2004,55:319-331.AmandaPsyrri* and Daniel DiMaio Human papillomavirus in cervical and head-and-neck cancer.Nature clinical practice ONCOLOGY 2008,5 (1): 24-31.).

HPV is a kind of double-stranded DNA virus, and energy oneself assembling or formation virus-like particle (VLP) after its capsid protein L 1 and L2 express can bring out body and produce specificity neutralizing antibody and effectively local immunity reaction; HPV has 8 early proteins, wherein E6 and E7 can make cancer suppressor protein p53 and Rb1 inactivation respectively, cause the cell cycle out of control, Telomerase activates and cell immortalityization, its continuous expression is tumour cell transformation and to keep malignant characteristics necessary, with be that target position can cause the reaction of stronger specific T-cells, E6 and E7 albumen do not exist in the normal human, and antigenicity, specificity is all stronger, belong to vaccine target position (Roden RB preferably, Grrenstone HL, KimbauerR, et al.Invitro generation and type-specific neutralization of a human papillomavirus type16 virion pseudotype.virology, 1996,70 (9): 5875-5883.Porgador A.Induction of antitumorimmunity using bone marrow-generated dendritic cells.JImmunol, 1996,156 (8): 2918-1926.Evans M, Boryiewicz LK, Evans AS, et al.Antigenprocessing defects in cervical carcinomas limit the presentation of a CTL epitope fromhuman papillomavirus 16E6.J Immunol, 2001,167 (9): 5420-5428.Berezutskaya E, BagchiS.The human papillomavirus E7 oncoprotein functionally interacts with the S4 subunit ofthe 26S proteasome.J Biol Chem, 1997,277 (48): 30135-30140.).

The HPV vaccine research is very active over nearly 10 years, existing in the world several HPV candidate vaccines enter clinical II or III phase experimental stage, the Gardasil (Merck company) that but comprises FDA official approval in 2006 listing, mostly be preventative VLP vaccine, therapeutic vaccine research still has (No authors listed.Newvaccine prevents cervical cancer.FDA Consum.2006 to be strengthened; 40 (5): 37.).Especially it should be noted that in addition, large epidemiology survey shows: the virus that HPV is made up of a plurality of types, lack the group antigen determinant, and different areas HPV infection type constituent ratio there are differences, therefore developing polyvalent vaccine could really play prevention effect.With the cervical cancer is example, external many based on 16 and 18 types, 58 types rare (＜2%), China's 58 types infect then very general (about 10-30%), recall rate is only second to 16 types (about 50%) among the cervical cancer patient, in south many areas in addition with 16 types (the Shi W that almost maintains an equal level, Bu P, Liu J, et al.Human papillomavirus type16 E7 DNA vaccine:mutation in the open reading frame of E7 enhances specificcytotoxic T-lymphocyte induction and antitumor activity.JVirology.1999,73 (9): 7877-7881. woods jade is pure, Liu Baoyin, Li Jie etc. the discovery of human papillomavirus 58 types and various molecule epidemic disease-ology research in Chinese uterine neck diseases. medical research communication, 1996; 25 (11): 21.DaiM, Bao YP, Li N, et al.Human papillomavirus infection in Shanxi Province, People ' sRepublic of China:a population-based study.Br J Cancer.2006,95 (1): 96-101.Chan PK, Li WH, Chan MY, et al.High prevalence of human papillomavirus type 58in Chinesewomen with cervical cancer and precancerous lesions.J Med Virol, 1999,59 (2): 232-238.).The polyvalent vaccine exploitation is subjected to restrictions such as carrier capacity and production cost; and the protection effect of common hypotype is not difficult to confirm yet; at present external still based on the research of monovalence or divalence most commonly encountered diseases poison type vaccine; domestic 58 types are infected also do not cause enough attention; enter in the HPV candidate vaccine of clinical experimental stage none so far in the world at 58 hypotypes; this causes interior import vaccine of long duration inapplicable in China most probably; therefore, independent development is extremely urgent at the HPV vaccine of China's national situation.

At present, the development of therapeutic HPV vaccine mainly contains following method (Shreya Kanodia, Diane M.DaSilva, et al.Recent advances in strategies for immunotherapy of humanpapillomavirus-induced lesions Int.J.Cancer:2008; 122:247-259.).

1, with virus is the vaccine of carrier.Mainly be to be carrier with vaccinia virus, adenovirus, adeno-associated virus, major advantage: can cause that the host produces intensive cell and humoral immune reaction, but former infection may have a negative impact to the effect of vaccine.

2, with the bacterium be the vaccine of carrier.Such vaccine can produce high titer antibody, but cell immune response is good less than the vaccine effect that with virus is not carrier.As with listeria bacteria, Salmonellas, Shigellae or intestinal bacteria vaccine as carrier.

3, polypeptide vaccine.Major advantage: synthetic convenient, but cell and humoral immune reaction all a little less than, and such vaccine is subjected to the restriction of HLA.

4, protein vaccine.Such vaccine is not subjected to the restriction of HLA, can produce antibody, in order to produce the intensive ctl response, often need carry out the coupling connection with other molecule, as heat shock protein(HSP) or interpolation adjuvant.

5, dna vaccination.Major advantage is easy to prepare, good stability, but its security merits attention.

6, based on the vaccine of dendritic cell.Its immunogenicity is strong, but often need feed back in the body in vitro culture again, begins to be much accounted of in tumor treatment.

7, tumour-cell vaccine or derive from tumour cell composition or tumor related antigen, or tumour cell is modified.

The subject matter of HPV vaccine research existence at present is: 1. how to form immunological memory; 2. how to induce effective mucosal immunity; 3. how to control production cost.

Present VLP vaccine prevention is effective but cost is too high, can not be widely used in developing country; Synthetic polypeptide vaccine is the same with dna vaccination, and immune effect is relatively poor, even be aided with various adjuvants, still can not prevent homotype HPV to infect or make fully and generate tumor regression, and its application is limited by the MHC-1 quasi-molecule also, and difficulty has big progress in a short time.By contrast, have long-term, the extensive human vector-viral vaccine of inoculating history and be only the strongest HPV vaccine strategy of present developing country feasibility.Adenovirus carrier exogenous gene expression level height not only wherein, cause that immune response is strong and lasting, preparation method's maturation, immunization route is convenient, or known unique virus vector that can bring out the potent mucosal immunity, having replaced retrovirus becomes current application carrier the most widely.The replication defective adenoviral improvement of constantly recombinating in recent years in addition, its safety in utilization strengthens greatly, and is further ripe in order to the condition of exploitation HPV vaccine.

Therapeutic vaccine mainly with E6 and E7 as target protein, its objective is and induce body to produce specific cell immunoreaction, remove the HPV that has infected, precancerous lesion and the tumour that treatment is caused by infection.But the E6 of high-risk HPV and E7 albumen are the carcinogens of generally acknowledging, play an important role in relevant immortalization of HPV and tumorigenicity.E7 albumen energy inactivation tumor suppressor protein Rb, and effect (the Michel N of plain E of activated end granzyme, reactivation cycle and A arranged, Osen W.Gissmann L, et al.Enhanced immunogenicity of HPV 16E7 fusion proteins in DNAvaccination.Virology, 2002,294:47259.).E6 can make the G of P53 albumen control by E6AP-ubiquitin approach degraded P53 albumen ₁/ S node is out of hand, cause the unstable and transgenation increase of cell chromosome, make foreign DNA be incorporated into probability in the karyomit(e) (the Zheng ZM and.Baker CC.papillomavirus genomestructure that raises greatly, expression, and post-transcriptional regulaion.Front Biosci.2006,11:2286-2302.); Also can act on apoptosis-related protein simultaneously, suppress apoptosis, activated end granzyme, keep telomere length and make cell immortalityization.So the activity of conversion that must remove E6 and E7 just can be used for the structure of vaccine.Studies show that, the N end of E6 is main relevant with proteic stability, deletion mutantion meeting remarkably influenced albumen takes place at (Grossman SR of external transformation period in the N end, Mora R, Laimins LA.Intracellular localization andDNA-Ifinding properties of human papillomavirus type 18 E6 protein expressed with abaculovirus vectorl, J virol, 1989,63 (1): 366-374.).Two zinc of E6 refer to that the district changes viral activity of conversion and causes that the albumen trans-activation is necessary, all relevant (KandaT with interaction between protein, WatanabeS, Zanma S, et al.Human papillomavirus type 16 E6 proteins with glycine substitution forcysteine in the metal-binding motif.Virolog, 1991,185 (2): 536-543.).Many data confirm that the C end of E6 contains most epitope.Nakagawa etc. have studied the relation of HPV18 type E6 each site of albumen and HEK293 cell transformation and p53 inactivation in great detail, find to dezincify and refer to outside the structure, the L-52 sudden change influences also highly significant (Nakagawa S to it, Watanabe S, Yoshikawa H, et al.Mutaional analysis of humanpapillomavirus type 16 E6 protein:transforming function for human cells and degradationof p53 in vitro.Virolog, 1995,212 (2): 535-542.).

E7 albumen is the acidic protein of 105 amino-acid residues of total length, is the main transforming protein of HPV18.E7 albumen is divided into 3 functional zone: the 1st, the 2 conserved regions height homologies of 1 district and 2 districts and adenovirus ElA; 3 districts refer to the district for zinc." L-X-E-X-C " in 2 districts is considered to the critical sites in conjunction with Rb.Studies show that this site mutation can obviously reduce the activity of conversion of E7 albumen to monkey CA-1 clone, but do not influence human keratinocyte's immortalization, but zinc refers to the sudden change distinguished then proteic conversion of completely destroy E7 and immortalization ability.E7 albumen may be to exist with dimeric form, and this dimer combines with Zine with " Cys-X-X-Cys " motif, the change of 1 zinc phalangeal process can weaken itself and Zine binding ability, shorten the transformation period, reduce stability, lose its convertibility; 2 zinc phalangeal processes become the serious shortening that then thoroughly loses itself and Zine bonded ability, cause the transformation period, completely lose its convertibility, and might lose antigenicity.

In addition, there is the research report that HPV16 type E7 albumen is carried out the T20V sudden change, can improve its inductive ctl response, but do not influence the proteic functionally active of E6 and E7 (Schreurs MWJ, Kueter EWM, ScholtenKBJ, et al.A single amino acid substitution improves the in vivo immunogenicity of theHPV16 oncoprotein E7 (11-20) cytotoxic T lymphocyte epitope.Vaccine, 2005,23:4005.).

Summary of the invention

An object of the present invention is to provide a kind of transcribing and merge three gene fragments.

Provided by the present invention transcribing merged three gene fragments, comprises HPV16mE67, HPV18mE67 and HPV58mE67 and the short nucleotide fragments that is connected the three; Described HPV16mE67 amino acid sequence coded is a sequence 6 in the sequence table; Described HPV18mE67 amino acid sequence coded is a sequence 7 in the sequence table; Described HPV58mE67 amino acid sequence coded is a sequence 8 in the sequence table; The described short nucleotide fragments that connects HPV16mE67, HPV18mE67 and HPV58mE67 be the ribosome internal entry site (internal ribosome entry site, IRES).

The deoxyribonucleotide sequence of described HPV16mE67 is a sequence 1 in the sequence table; The deoxyribonucleotide sequence of described HPV18mE67 is a sequence 2 in the sequence table; The deoxyribonucleotide sequence of described HPV58mE67 is a sequence 3 in the sequence table, and the deoxyribonucleotide sequence of described ribosome internal entry site is a sequence 4 in the sequence table.

Above-mentioned deoxyribonucleotide sequence of transcribing fusion three gene fragments is a sequence 5 in the sequence table, and wherein HPV16mE67, HPV18mE67 and HPV58mE67 all have initiator codon and terminator codon.

Contain above-mentioned recombinant expression vector, transgenic cell line and reorganization bacterium of transcribing fusion three gene fragments and also belong to protection scope of the present invention.

Described recombinant expression vector is the duplicate deficit type recombinant adenovirus expression vector.

Above-mentioned duplicate deficit type recombinant adenovirus expression vector can be used for preparing the vaccine that prevents and/or treats the human cervical carcinoma.

Contain the above-mentioned duplicate deficit type recombinant adenovirus that merges three gene fragments of transcribing and also belong to protection scope of the present invention.

Described duplicate deficit type recombinant adenovirus prepares with the Adeno-X expression system.

Another object of the present invention provides a kind of therapeutic vaccine of human cervical carcinoma, and its activeconstituents is above-mentioned duplicate deficit type recombinant adenovirus.

According to other characteristic distributions of China HPV infection type, select HPV16,18 and 58 these three modal hypotypes (to contain domestic cervical cancer HPV cases of infection more than 80%, southeast ratio is higher), the site that influences its activity of conversion and raising ctl response is suddenlyd change, to the E6 gene, select its P53 binding site of sudden change and one of them zinc fingers; To the E7 gene, select its Rb binding site of sudden change and one of them zinc to refer to, not influencing under protein stability and the immunogenic prerequisite, thoroughly remove its activity of conversion.Make up E6 and the proteic fusion gene of E7 of each hypotype HPV, and changed it over to adenovirus carrier, detected the removal situation of E6 and E7 albumen activity of conversion.Animal test results shows that this vaccine can be expressed in cell, the adenovirus particles direct immunization animal with purifying has the obvious treatment effect to established tumour, and its mechanism of action can produce the intensive ctl response in animal body mainly due to this vaccine.

Description of drawings

Fig. 1 is the agarose gel electrophoresis result of HPV16,18 and 58 type E6 and E7 gene PCR amplified production

Fig. 2 is the agarose gel electrophoresis detected result of fusion gene HPV16mE67, HPV18mE67 and HPV58mE67

Fig. 3 is the agarose gel electrophoresis result of recombinant plasmid pShuttle2-16mE67-IRES2-18mE67-IRES2-58mE67 after by Nhe I and NotI double digestion

Fig. 4 is for changing recombinant adenoviral vector Adeno-X-HPV16 over to, and 18, the colibacillus PCR qualification result of 58mE67

Fig. 5 is recombinant plasmid pAdeno-X-HPV16,18, and the form of 58mE67 transfection HEK293 cell cell after 8 days

Fig. 6 is the titer determination result of recombinant adenovirus

Fig. 7 is recombinant plasmid pAdeno-X-HPV16, behind the 18mE67-EGFP transfection HEK293 cell 48h, and the fluorescence microscope result of transgenic cell

Fig. 8 is the Western Blot detected result behind the reorganization Adenovirus Transfection HEK293 cell

Fig. 9 is the one-tenth knurl situation of mouse

Figure 10 is the measuring result of mouse tumor volume

Figure 11 is the measuring result of mouse survival rate

Figure 12 is the CTL detected result

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).It is synthetic that the primer and dna sequence dna are given birth to the worker by Shanghai if no special instructions.

The source and the sudden change thereof of embodiment 1, HPV E6 and E7 gene

The HPV genomic dna is from the outpatient's of Shenzhen institute of traditional Chinese medicine (young woman) cervical smear sample, and pcr amplification turns out to be the genomic dna of HPV after the order-checking.With this HPV genomic dna is template, design HPV16 respectively, the Auele Specific Primer of 18 and 58 type E6 and E7 gene, the Auele Specific Primer of amplification HPV16 type E6 gene is P1 and P2, the Auele Specific Primer of amplification HPV16 type E7 gene is P3 and P4, the Auele Specific Primer of amplification HPV18 type E6 gene is P5 and P6, the Auele Specific Primer of amplification HPV18 type E7 gene is P7 and P8, the Auele Specific Primer of amplification HPV58 type E6 gene is P9 and P10, the Auele Specific Primer of amplification HPV58 type E7 gene is P11 and P12 (concrete primer sequence is as shown in table 1), with the archaeal dna polymerase HPV16 that increases respectively, 18 and 58 type E6 and E7 gene, and on primer, add corresponding restriction enzyme site, the PCR reaction system is as follows:

DNTP Mixture 10 * Pyrobest Buffer II Pyrobest DNA polymerase dna profiling upstream primer (20 μ m) downstream primer (20 μ m) dH2O	10μl 10μl 0.5μl 1μl(10ng) 2.5μl 2.5μl 73.5μl
	10μl 10μl 0.5μl 1μl(10ng) 2.5μl 2.5μl 73.5μl	Cumulative volume	100μl

PCR reaction conditions: 95 ℃ of pre-sex change 5min of elder generation; 95 ℃ of 1min then, 56 ℃ of 1min, 72 ℃ of 1min, totally 32 circulations, last 72 ℃ are extended 10min.

Pcr amplification product is carried out agarose gel electrophoresis detect, concrete detected result as shown in Figure 1.Wherein, M is DL2000Marker, and swimming lane 1-3 is respectively the sepharose detected result of HPV16,18 and 58 type E6 gene PCR amplified productions; Swimming lane 4-6 is respectively the sepharose detected result of HPV16,18 and 58 type E7 gene PCR amplified productions.The result shows that the acquisition size is that HPV16,18 and 58 type E6 genes and the size of 500bp is the HPV16 of 300bp, 18 and 58 type E7 genes.

Link to each other with pMD18T simple (available from Takara company) respectively after pcr amplification product is purified and check order, sequencing result shows, the HPV16 that amplification obtains, 18 consistent with the sequence of GenBank Accenssion Number EF029179, EF202153 and D90400 with the deoxyribonucleotide sequence of 58 type E6 genes, the HPV16 that obtains of increasing, 18 consistent with the sequence of GenBank AccenssionNumber EU430687, EF202153 and D90400 with the deoxyribonucleotide sequence of 58 type E7 genes.

The HPV16 that above-mentioned amplification is obtained, 18 suddenlys change with the site relevant with activity of conversion of 58 type E6 and E7 gene and the position that can strengthen cellular immune function respectively, and the sequence after the sudden change confirms to suddenly change successfully through sequencing analysis, and concrete mutation method is as follows:

Pcr amplification product with above-mentioned HPV16 type E6 gene is a template, is that primer carries out pcr amplification with PM1 and PM2, pcr amplification product is linked to each other with pMD18T simple and checks order the recombinant plasmid called after pMD18T-16E6m1 that sequencing result is correct; Being template again with pMD18T-16E6m1, is that primer carries out pcr amplification with PM3 and PM4, pcr amplification product is linked to each other with pMD18T simple and checks order the PCR product called after HPV16mE6 that sequencing result is correct.Pcr amplification product with above-mentioned HPV16 type E7 gene is a template, is that primer carries out pcr amplification with PM5 and PM6, pcr amplification product is linked to each other with pMD18T simple and checks order the recombinant plasmid called after pMD18T-16E7ml that sequencing result is correct; Being template again with pMD18T-16E7m1, is that primer carries out pcr amplification with PM7 and PM8, pcr amplification product is linked to each other with pMD18T simple and checks order the PCR product called after HPV16mE7 that sequencing result is correct.Pcr amplification product with above-mentioned HPV18 type E6 gene is a template, is that primer carries out pcr amplification with PM9 and PM10, pcr amplification product is linked to each other with pMD18T simple and checks order the recombinant plasmid called after pMD18T-18E6m1 that sequencing result is correct; Being template again with pMD18T-18E6m1, is that primer carries out pcr amplification with PM11 and PM12, pcr amplification product is linked to each other with pMD18T simple and checks order the PCR product called after HPV18mE6 that sequencing result is correct.Pcr amplification product with above-mentioned HPV18 type E7 gene is a template, is that primer carries out pcr amplification with PM13 and PM14, pcr amplification product is linked to each other with pMD18T simple and checks order the recombinant plasmid called after pMD18T-18E7m1 that sequencing result is correct; Being template again with pMD18T-18E7m1, is that primer carries out pcr amplification with PM15 and PM16, pcr amplification product is linked to each other with pMD18T simple and checks order the PCR product called after HPV18mE7 that sequencing result is correct.Pcr amplification product with above-mentioned HPV58 type E6 gene is a template, is that primer carries out pcr amplification with PM17 and PM18, pcr amplification product is linked to each other with pMD18T simple and checks order the recombinant plasmid called after pMD18T-58E6m1 that sequencing result is correct; Being template again with pMD18T-58E6m1, is that primer carries out pcr amplification with PM19 and PM20, pcr amplification product is linked to each other with pMD18T simple and checks order the PCR product called after HPV58mE6 that sequencing result is correct.Pcr amplification product with above-mentioned HPV58 type E7 gene is a template, is that primer carries out pcr amplification with PM21 and PM22, the PGR amplified production is linked to each other with pMD18T simple and checks order the recombinant plasmid called after pMD18T-58E7m1 that sequencing result is correct; Being template again with pMD18T-58E7m1, is that primer carries out pcr amplification with PM23 and PM24, pcr amplification product is linked to each other with pMD18T simple and checks order the PCR product called after HPV58mE7 that sequencing result is correct.

Wherein, HPV16mE6 is that the leucine (L) from the 57th of N-terminal with its amino acid sequence coded sports glycine (G), and the 110th halfcystine (C) sports glycine (G); HPV16mE7 is that the leucine (L) from the 22nd of N-terminal with its amino acid sequence coded sports glycine (G), and the 91st halfcystine (C) sports glycine (G); HPV18mE6 is that the leucine (L) from the 52nd of N-terminal the with its amino acid sequence coded sports glycine (G), the 68th halfcystine (C) sports glycine (G), HPV18mE7 is that the halfcystine (C) from the 27th of N-terminal the with its amino acid sequence coded sports glycine (G), and the 98th halfcystine (C) sports glycine (G); HPV58mE6 is that the leucine (L) from the 50th of N-terminal the with its amino acid sequence coded sports glycine (G), and the 66th halfcystine (C) sports glycine (G); HPV58mE7 is that the glutamine (E) from the 26th of N-terminal the with its amino acid sequence coded sports glycine (G), and the 92nd halfcystine (C) sports glycine (G).

Table 1 design of primers

Annotate: italics partly is a restriction enzyme site in the table, and the band underscore partly is the mutational site.

The active removal situation of the acquisition of embodiment 2, fusion gene and mE67 gene transformation detects

One, the acquisition of fusion gene

E6 and the E7 gene of the wild-type HPV16 that respectively the foregoing description 1 is obtained with T-Vector carrier (available from Takara company), the E6 of HPV18 and E7 gene, mE6 and the mE7 gene of HPV16 after the E6 of HPV58 and E7 gene and the sudden change, the mE6 of HPV18 and mE7 gene, the mE6 of HPV58 is connected with the mE7 gene, produce fusion gene HPV16E67, HPV18E67, HPV58E67, HPV16mE67, HPV18mE67 and HPV58mE67, concrete method of attachment is as follows: with HPV16E6, HPV16mE6 primer P1, P2 carries out pcr amplification, HPV16E7, HPV16mE7 primer P3, P4 carries out pcr amplification, the electrophoresis product carries out enzyme with HindIII respectively and cuts, reclaim enzyme and cut product, with HPV16E6 and HPV16E7, HPV16mE6 is connected with the T4 ligase enzyme with the product of HPV16mE7 after the HindIII enzyme is cut, be template to connect product then, with P1 and P4 is that primer carries out pcr amplification, pcr amplification product is linked to each other with pMD18T simple, with the carrier difference called after T-HPV16E67 and the T-HPV16mE67 that contain fusion gene that obtains; Same method, with HPV18E6, HPV18mE6 primer P5, P6 carries out pcr amplification, HPV18E7, HPV18mE7 primer P7, P8 carries out pcr amplification, the electrophoresis product carries out enzyme with SalI respectively and cuts, reclaim enzyme and cut product, with HPV18E6 and HPV18E7, HPV18mE6 is connected with the T4 ligase enzyme with the product of HPV18mE7 after the SalI enzyme is cut, be template to connect product then, with primer P5 and P8 is that primer carries out pcr amplification, pcr amplification product is linked to each other with pMD18T simple, with the carrier difference called after T-HPV18E67 and the T-HPV18mE67 that contain fusion gene that obtains; With HPV58E6, HPV58mE6 primer P9, P10 carries out pcr amplification, HPV16E7, HPV16mE7 primer P11, P12 carries out pcr amplification, the electrophoresis product carries out enzyme with EcoRI respectively and cuts, reclaim enzyme and cut product, with HPV58E6 and HPV58E7, HPV58mE6 is connected with the T4 ligase enzyme with the product of HPV58mE7 after the EcoRI enzyme is cut, be template to connect product then, with primer P9 and P12 is that primer carries out pcr amplification, pcr amplification product is linked to each other with pMD18T simple, with the carrier difference called after T-HPV58E67 and the T-HPV58mE67 that contain fusion gene that obtains.

The above-mentioned carrier T-HPV16mE67, the T-HPVi8mE67 that contain fusion gene that obtains and T-HPV58mE67 are carried out PCR, electrophoresis detection respectively, and the result as shown in Figure 2.Wherein, 1 is the electrophoresis detection result of HPV16mE67PCR product, and 2 is the electrophoresis detection result of HPV18mE67PCR product, and 3 is the electrophoresis detection result of HPV58mE67PCR product.T-HPV16mE67, T-HPV18mE67 and T-HPV58mE67 plasmid are checked order, and the result shows the deoxyribonucleotide sequence of HPV16mE67 shown in sequence in the sequence table 1, and its amino acid sequence coded is shown in sequence in the sequence table 6; The deoxyribonucleotide sequence of HPV18mE67 is shown in sequence in the sequence table 2, and its amino acid sequence coded is shown in sequence in the sequence table 7; The deoxyribonucleotide sequence of HPV58mE67 is shown in sequence in the sequence table 3, and its amino acid sequence coded is shown in sequence in the sequence table 8.

Two, the active removal situation of HPV16mE67, HPV18mE67 and HPV58mE67 gene transformation detects

1, the acquisition of transgenic cell

Fusion gene HPV16E67 with above-mentioned steps one acquisition, HPV18E67, HPV58E67, HPV16mE67, after HPV18mE67 and HPV58mE67 cut with NheI and BamHI enzyme, be connected into respectively between the NheI and BamHI restriction enzyme site of eukaryon expression plasmid pIRES2-EGFP (available from BD company), obtain containing the recombinant expression vector pIRES2-HPV16E67 of fusion gene HPV16E67, the recombinant expression vector pIRES2-HPV18E67 that contains fusion gene HPV18E67, the recombinant expression vector pIRES2-HPV58E67 that contains fusion gene HPV58E67, the recombinant expression vector pIRES2-HPV16mE67 that contains fusion gene HPV16mE67 contains the recombinant expression vector pIRES2-HPV18mE67 of fusion gene HPV18mE67 and contains the recombinant expression vector pIRES2-HPV58mE67 of fusion gene HPV58mE67.(see the recombinant expression vector of above-mentioned structure for details Lipofectamin with liposome method ^TMThe explanation of 2000 test kits) difference transfection Balb/c 3T3 cell through the G418 screening, obtains containing the cell clone of corresponding gene.Simultaneously with the Balb/c 3T3 cell that do not change any eukaryon expression plasmid in contrast, go the activity of conversion situation in order to observation.

2, conventional cell in vitro is cultivated, and observes its contact inhibition situation

With plasmid pIRES2-HPV16E67, pIRES2-HPV18E67, pIRES2-HPV58E67, pIRES2-HPV16mE67, pIRES2-HPV18mE67 and pIRES2-HPV58mE67 difference transfection Balb/c 3T3 cell, through the G418 screening, obtain containing the cell clone of corresponding gene.Cultivate above-mentioned transgenic cell after 3 days, carry out om observation, found that, the cell that changes plasmid pIRES2-HPV16E67, pIRES2-HPV18E67, pIRES2-HPV58E67 over to is the plocoid storied length of laying equal stress on, prompting loses the growth contact inhibition, and there is the contact inhibition phenomenon all the time in the cell that changes plasmid pIRES2-HPV16mE67, pIRES2-HPV18mE67, pIRES2-HPV58mE67 over to.

3, soft agar is cultivated

At diameter is the agar of plastic culture dish upper berth one deck 0.35% of 35mm, inoculates 5 * 10 respectively in each culture dish ⁴Genetically modified Balb/c 3T3 cell that the above-mentioned steps 1 of individual logarithmic phase obtains and the Balb/c 3T3 cell that does not change any carrier for expression of eukaryon over to are handled 5 repetitions, 37 ℃ of 5%CO for every kind ₂The conventional cultivation for 3 weeks, inverted microscope is observed colony down and is formed situation.The result shows, cultivating 2-3 on the soft agar medium after week, the cell that changes plasmid pIRES2-HPV16E67, pIRES2-HPV18E67, pIRES2-HPV58E67 over to is the colony growth, and it is the same with not genetically modified Balb/c 3T3 cell to change the cell of plasmid pIRES2-HPV16mE67, pIRES2-HPV18mE67, pIRES2-HPV58mE67 over to, there is no the above colony of 50 cells and forms.

4, tumorigenesis experiment in the nude mouse

Get Balb/c mouse (available from Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center), every mouse hypodermic inoculation 5 * 10 ⁶The transgenosis of the acquisition of individual above-mentioned steps 1 and not genetically modified Balb/c 3T3 cell, 5 mouse of every kind of cell inoculation are observed the subcutaneous one-tenth knurl of mouse situation.The result shows, inoculate after 40 days, none becomes knurl with the mouse of the Balb/c 3T3 cell inoculation that changes plasmid pIRES2-HPV16mE67, pIRES2-HPV18mE67, pIRES2-HPV58mE67 over to with the mouse of not genetically modified Balb/c 3T3 cell inoculation, and 80% subcutaneous one-tenth knurl being arranged with the mouse of the Balb/c 3T3 cell inoculation that changes plasmid pIRES2-HPV16E67, pIRES2-HPV18E67, pIRES2-HPV58E67 over to, the mean diameter of knurl is 2.5mm.

Soft agar is cultivated and the interior tumorigenesis of nude mouse experiment showed, that the mE67 gene of three hypotypes after the sudden change has lost the activity of conversion of E6 and E7 gene, for the safe handling of vaccine provides guarantee.

The structure of embodiment 3, recombinant adenovirus plasmid

The back links to each other with the carrier pCDNA 3.1 (+) that cuts with same enzyme the HPV16mE67 gene that the foregoing description 2 is made up with the BamHI enzyme is cut through NheI, generation recombinant plasmid pCDNA3.1 (+) 16mE67; With plasmid pIRES2-EGFP (available from BD company) is template, with P13, P14 is that primer carries out pcr amplification, obtain the IRES2 fragment of 663bp after the product separation and purification, the IRES2 fragment is checked order, its deoxyribonucleotide sequence is shown in sequence in the sequence table 4.This IRES2 fragment is connected between the BamHI and EcoRI restriction enzyme site of plasmid pCDNA3.1 (+) 16mE67 after with BamHI and EcoRI double digestion, obtains recombinant expression vector pCDNA3.1 (+) 16mE67-IRES2.The back links to each other with the carrier pCDNA 3.1 (+) that cuts with same enzyme the HPV18mE67 gene that the foregoing description 2 is made up with the XhoI enzyme is cut through EcoRI, generation recombinant plasmid pCDNA3.1 (+) 18mE67; With plasmid pIRES2-EGFP (available from BD company) is masterplate, carry out pcr amplification with P13, P14 ' for primer, pcr amplification product is connected into BamHI and the XhoI restriction enzyme site of plasmid pCDNA3.1 (+) 18mE67 after with BamHI and XhoI double digestion, obtains recombinant expression vector pCDNA3.1 (+) 18mE67-IRES2.Use this pCDNA3.1 (+) 18mE67-IRES2 plasmid of EcoRI and XhoI double digestion then, reclaim the small segment of 1471bp, and it is connected between the EcoRI and XhoI site of above-mentioned pCDNA3.1 (+) 16mE67-IRES2, with recombinant expression vector called after pCDNA 3.1 (+)-Kozak 16mE67-IRES2-18mE67-IRES2 that produces.HPV58mE67 gene P9 with the foregoing description 2 structures, P12 ' carries out pcr amplification for primer, pcr amplification product is behind XhoI and NotI double digestion, link to each other with the carrier pCDNA3.1 (-) that cuts with same enzyme (Invitrogen company), produce recombinant plasmid pCDNA3.1 (-) 58mE67, use NheI and the above-mentioned pCDNA 3.1 of XhoI double digestion (+)-Kozak16mE67-IRES2-18mE67-IRES2 plasmid then, reclaim the small segment of 2915bp, and it is connected between the NheI and XhoI site of above-mentioned pCDNA3.1 (-) 58mE67, with recombinant expression vector called after pCDNA3.1 (-) 16mE67-IRES2-18mE67-IRES2-58mE67 that produces.

With NheI and NotI double digestion recombinant expression vector pCDNA3.1 (-) 16mE67-IRES2-18mE67-IRES2-58mE67, obtain the fusion gene 16mE67-IRES2-18mE67-IRES2-58mE67 of 3.7kb.This fusion gene is checked order, and sequencing result shows that the deoxyribonucleotide sequence of 16mE67-IRES2-18mE67-IRES2-58mE67 is shown in sequence in the sequence table 5.

All remove the E6 terminator codon of HPV16mE6, HPV18mE6 and HPV58mE6 and the E7 initiator codon of HPV16mE7, HPV18mE7 and HPV58mE7 among the fusion gene 16mE67-IRES2-18mE67-IRES2-58mE67, and kept E6 initiator codon and E7 terminator codon.More than all molecular biology working specification and Takara MutanBEST Kit (Takara company) illustrate and carry out routinely in operation.

Recombinant expression vector pCDNA3.1 (-) 16mE67-IRES2-18mE67-IRES2-58mE67 through inserting between the NheI and NotI restriction enzyme site of shuttle plasmid pShuttle2 (available from BD company) behind NheI and the NotI double digestion, is obtained recombinant expression vector pShuttle-16mE67-IRES2-18mE67-IRES2-58mE67.With PI-Sce I and I-Ceu I double digestion (NewEngland Lab company), it is as follows that enzyme is cut system with recombinant expression vector pShuttle2-16mE67-IRES2-18mE67-IRES2-58mE67:

Reagent	Volume
Reagent	Volume	PI-Sce I (1units/ μ l) I-Ceu I (5units/ μ l) recombinant expression vector pShuttle-16mE67-IRES2-18mE67-IRES2-58mE67 (500ng/ μ l) 10 * Double Digest Buffer 10 * BSA dH2O	2μl 0.5μl 2μl 3μl 3μl 5μl
Cumulative volume	30μl		2μl 0.5μl 2μl 3μl 3μl 5μl

Enzyme is cut in the rearmounted 37 ℃ of water-baths of system mixing accurately enzyme cut 3h, reclaim and purifying enzyme is cut product.Enzyme is cut product and is carried out the agarose gel electrophoresis detection, and detected result as shown in Figure 3.Wherein, M1 is DL2000marker, swimming lane 1 is cut the agarose gel electrophoresis result of product for recombinant expression vector pShuttle2-16mE67-IRES2-18mE67-IRES2-58mE67 enzyme, swimming lane 4 is the agarose gel electrophoresis result after empty carrier pShuttle2 enzyme is cut, and M2 is DL15000marker.

The enzyme that contains 16mE67-IRES2-18mE67-IRES2-58mE67 that reclaims is cut product be connected in the Adeno-X viral DNA in the Adeno-X expression system (available from BD company), put in the PCR instrument 16 ℃ of connections and spend the night with Takaraligation Mixture.To connect product and cut digestion (Swa I restriction enzyme site is between PI-Sce I and I-Ceu I), fail the gene fragment that is connected with the Adeno-X viral DNA with removal with Swa I enzyme.Reclaim enzyme once more and cut product, the recombinant adenoviral vector called after Adeno-X-HPV16 that fusion gene 16mE67-IRES2-18mE67-IRES2-58mE67 and Adeno-X viral DNA correctly are connected, 18,58mE67.

Use recombinant adenoviral vector Adeno-X-HPV16,18,58mE67 transformed into escherichia coli DH5 α, the Amp resistant panel is selected positive colony, is that primer carries out pcr amplification with the P3 in the table 1 and P4, P7 and P8, P11 and P12 respectively, and the PCR detected result is as shown in Figure 4.Wherein, M is the dna molecular amount standard of DL2000bp, 1 is the agarose gel electrophoresis detected result of the pcr amplification product of HPV16E7, and 2 is the agarose gel electrophoresis detected result of the pcr amplification product of HPV18E7, and 3 is the agarose gel electrophoresis detected result of the pcr amplification product of HPV58E7.

Extract recombinant adenoviral vector Adeno-X-HPV16,18, the DNA of 58mE67 is with the recombinant plasmid called after pAdeno-X-HPV16 of gained, 18,58mE67.To recombinant plasmid pAdeno-X-HPV16,18,58mE67 carries out pcr amplification, enzyme is cut evaluation, enzyme is cut the correct recombinant plasmid of evaluation serve the Hai Boya order-checking.Pac I enzyme is cut pAdeno-X-HPV16, and 18,58mE67 is used for transfection HEK293 cell.

The preparation of embodiment 4, defective recombinant sexual gland virus and the mensuration of titre

One, defective recombinant sexual gland virus rAd-HPV16,18, the preparation of 58mE67

With the recombinant plasmid pAdeno-X-HPV16 after the linearizing, 18,58mE67 transfection HEK293 cell, cultivate cell to cell after the transfection occur tangible ill effect (cytopathic effect, CPE).After the transfection 8 days, the concrete form of cell as shown in Figure 5.Wherein, A is transfection recombinant plasmid pAdeno-X-HPV16,18, and the form of the HEK293 cell of 58mE67, B is the form of normal HEK293 cell.

Recombinant adenovirus rAd-HPV16,18, the preparation of 58mE67 is according to Adeno-X ^TMExpression SystemUser Manual carries out, and detailed process is as follows:

(1) wash the HEK293 cell 2 times of the obvious ill effect of above-mentioned appearance with PBS, directly the HEK293 cell is blown and beaten (without pancreatin), 1, centrifugal 5min under the 500rpm room temperature; 500 μ l PBS re-suspended cells;

(2)-20 ℃ and 37 ℃ of water-bath multigelation cells 3 times, each back short period of time mixing cell suspension that melts;

(3) the centrifugal 10min of 12000rpm room temperature collects the supernatant contain virus, adds 10% glycerine, after the filtration sterilization as recombinant virus stoste put-70 ℃ frozen standby;

(4) cultivate the HEK293 cell, treat cell be paved with bottle at the bottom of during 50-70%, add the virus stock solution used 250 μ l of step (3) preparation, continue to cultivate;

(5) the ill effect of observation of cell when treating that cell more than 50% floats at the bottom of the culture plate, repeats above-mentioned (1)-(3) step and prepares recombinant adenovirus rAd-HPV16,18,58mE67 deposits for-20 ℃, uses in order to measuring virus titer or further preparing infectious titer.

Two, the preparation of defective recombinant sexual gland virus rAd-LacZ

With plasmid pShuttle2-LacZ (available from BD company) PI-Sce I and I-Ceu I double digestion, enzyme is cut product and is linked to each other with Adeno-X viral DNA in the Adeno-X expression system of cutting through same enzyme, with the recombinant adenoviral vector called after Adeno-X-LacZ that obtains.Extract the DNA of recombinant adenoviral vector Adeno-X-LacZ, with the recombinant plasmid name pAdeno-X-LacZ of gained.With recombinant plasmid pAdeno-X-LacZ transfection HEK293 cell, preparation defective recombinant sexual gland virus rAd-LacZ.The same step 1 of detailed process of defective recombinant sexual gland virus rAd-LacZ preparation.

Three, recombinant adenovirus rAd-HPV16,18, the titer determination of 58mE67

Because of the HEK293 cell can be expressed adenovirus activatory early protein E1 and E3, recombinant plasmid pAdeno-X-HPV16,18,58mE67 can pack and be discharged in the substratum in the HEK293 cell, reclaim the recombinant adenovirus rAd-HPV16 of above-mentioned steps one preparation, 18,58mE67 with the ultra-high speed whizzer, use the titre of adenovirus titration kit measurement recombinant adenovirus then, the titer determination result as shown in Figure 6.Wherein, 1-10 classifies as and adds different concns recombinant adenovirus rAd-HPV16, and 18, the HEK293 cell of 58mE67, the 11st and 12 classify normal HEK293 cell as, and the cell of CPE effect is represented not produce in the grey hole, and CPE represents in the hole to produce the cell of CPE effect.The result shows that virus titer is 3.2 * 10 ⁷Pfu/ml.

Four, fluorescence microscope

Recombinant plasmid pAdeno-X-HPV16,18mE67-EGFP (the same pAdeno-X-HPV16 of its building process, 18,58mE67, replace recombinant plasmid pAdeno-X-HPV16,18, the HPV58mE67 gene among the 58mE67 with EGFP) begin behind the transfection HEK293 cell 48h, the situation that HEK293 cell expressing green fluorescence is observed in timing, the expression intensity of estimation HPV58mE67.400X microscopically fluoroscopic examination result as shown in Figure 7, the result shows that HPV58mE67 can express in the HEK293 cell.

Five, Western blot detects

With the infection multiplicity (MOI) of above-mentioned step 3 purifying 5: 1 recombinant adenovirus rAd-HPV16,18,58mE67 infects the COS-7 cell, collecting cell after one day, the RIPA lysing cell extracts albumen, respectively with mouse anti human HPV16,18E6 monoclonal antibody (available from Chemicon company), HPV16E7 monoclonal antibody (available from Neomarker company), HPV18E7 monoclonal antibody (available from GeneTex company) and HPV58 monoclonal antibody (available from Santa Cruz company) are one anti-, and Western blot detects the expression of HPV16mE67, HPV18mE67 and HPV58mE67.

Western blot detected result as shown in Figure 8.Wherein, the Western blot detected result of the COS-7 cell of

swimming lane

1,3,5 and 7 expression untransfected adenovirus;

Swimming lane

2,4,6 and 8 is represented transfection recombinant adenovirus rAd-HPV16 respectively, 18, and the Western blot detected result that the COS-7 cell of 58mE67 detects with different antibodies.The result shows that HPV16mE67 and HPV18mE67 have higher expression, but the expression amount of HPV58mE67 is few than the expression amount of HPV16mE67 and HPV18mE67, may have the polarity problems of expression.

Embodiment 6, animal experiment

One, suppresses tumor growth

(agree thing Instr Ltd. of Johnson ﹠ Johnson) because the TC-1 cell and can express HPV16E6 and HPV16E7 albumen, get mouse subcutaneous injection 5*10 above shank thereafter available from Shanghai ⁵Individual TC-1 cell formed tumour after 4 days.The one-tenth knurl situation of mouse as shown in Figure 9.Wherein, A is the model group mouse of injection TC-1 cell, the control group mice of B for not injecting the TC-1 cell.The mouse that becomes knurl is divided into PBS group, rAd-LacZ group and adenovirus treatment group at random, every group of 10 mouse, every injected in mice pH of PBS group is 7.4 PBS 0.2ml, rAd-LacZ organizes every injected in mice 1*10 ⁶The adenovirus rAd-LacZ 0.2ml of pfu, every injected in mice 1*10 of adenovirus treatment group ⁶The recombinant adenovirus rAd-HPV16 of pfu embodiment 5 preparations, 18,58mE670.2ml.

Immunely weekly above-mentionedly respectively organize mouse once, continuous two weeks, per two days sizes with the vernier caliper measurement tumour, by formula: the high * 0.52 of the size of tumour=wide * of long * calculates the size of tumour.The measuring result of gross tumor volume as shown in figure 10 shown in.Three repetitions are established in experiment, and the numerical value among Figure 10 is three multiple mean values.The result shows that the mouse tumor growth of adenovirus treatment group obviously is suppressed.

Two, survival rate experiment

To be divided into PBS group, rAd-LacZ group and adenovirus treatment group according to the one-tenth knurl mouse of above-mentioned steps one preparation at random, every group of 10 mouse, immunity once is total to immunity 2 times, the immunogen and all same step 1 of immunizing dose of each group weekly.Observe the survival condition of respectively organizing mouse.Each measuring result of organizing the mouse survival rate as shown in figure 11.Three repetitions are established in experiment, and the survival rate among Figure 11 is three multiple mean values.The result shows, the survival rate in the adenovirus treatment group mouse 42 days is 100%, and the fate that group of PBS in contrast and rAd-LacZ group mouse survive is no more than 32 days.

Three, the study on mechanism of vaccine

The one-tenth knurl mouse of above-mentioned steps one is divided into PBS group, rAd-LacZ group and adenovirus treatment group at random, every group of 10 mouse, every two all immunity once, immunity is 3 times altogether, the immunogen and all same step 1 of immunizing dose of each group.After six weeks, put to death the mouse extracting spleen cell and make ctl response.Concrete experimentation is as follows:

The single-cell suspension of each group mice spleen is cultivated in RPMI1640 (GIBCO/BRL), replenished the gentamicin sulphate (GIBCO/BRL) of 10%FCS, 2mM L-glutaminate, 1mM Sodium.alpha.-ketopropionate (GIBCO/BRL), 50 μ M2-mercaptoethanols and 50 μ g/ml.1 μ M HPV16E7 with the foregoing description 1 preparation stimulates splenocyte again.One week post analysis respectively organize the lytic activity of cell.With the splenocyte action effect cell that stimulates, with the TC-1 cell as target cell, in varing proportions with above-mentioned cell: mixed in 50: 1,25: 1 and 12.5: 1, and cell mixing cultivated in 96 orifice plates 5%CO under 37 ℃ of conditions ₂Cultivated 4 hours in the incubator, (Beckman Coulter Canada, Mississauga is ON) with the centrifugal 5min of 200xg to use BeckmanG5-6R then.Collect the 100ul culture supernatant from each hole, detect the activity of serum lactic dehydrogenase (LDH) with test kit.The calculation formula of lactate dehydrogenase activity is: (LDHtest-LDHspont)/(LDH total-LDHspont)] x 100.LDHtest, LDHspont and LDH total represent test holes, spontaneous release and maximum release respectively in the formula.Three repetitions are established in experiment, and concrete outcome as shown in figure 12.The result shows that adenovirus treatment group can produce the intensive ctl response, and the ctl response of adenovirus treatment group is all higher than PBS group and rAd-LacZ group.

Sequence table

<160>8

<210>1

<211>777

<212>DNA

＜213〉artificial sequence

<400>1

atgcaccaaa agagaactgc aatgtttcag gacccacagg agcgacccag aaagttacca 60

catttatgca cagagctgca aacaactata catgatataa tattagaatg tgtgtactgc 120

aagcaacagt tactgcgacg tgaggtatat gactttgctt ttcgggatgg ttgcatagta 180

tatagagatg ggaatccata tgcagtgtgt gataaatgtt taaagtttta ttctaaaatt 240

agtgagtata gatattattg ttatagtgtg tatggaacaa cattagaaca gcaatacaac 300

aaaccgttgt gtgatttgtt aattaggggt attaactgtc aaaagccact gtgtcctgaa 360

gaaaagcaaa gacatctgga caaaaagcaa agattccata atataagggg tcggtggacc 420

ggtcgatgta tgtcttgttg cagatcatca agaacccgta gagaaaccca gctgaagctt 480

atgcatggag atacacctac attgcatgaa tatatgttag atttgcaacc agagacaact 540

gatggctact gttatgagca attaaatgac agctcagagg aggaagatga aatagatggt 600

ccagctggac aagcagaacc ggacagagcc cattacaata ttgtaacctt ttgttgcaag 660

tgtgactcta cgcttcggtt gtgcgtacaa agcacacacg tagacattcg tactttggaa 720

gacctgttaa tgggcacact aggaattgtg gggcccatct gttctcagaa accataa 777

<210>2

<211>798

<212>DNA

＜213〉artificial sequence

<400>2

atggcgcgct ttgaggatcc aacacggcga ccctacaagc tacctgatct gtgcacggaa 60

ctgaacactt cactgcaaga catagaaata acctgtgtat attgcaagac agtattggaa 120

cttacagagg tatttgaatt tgcatttaaa gatggatttg tagtgtatag agacagtata 180

ccgcatgctg catgccataa aggtatagat ttctattcta gaattagaga attaagatat 240

tattcagact ctgtgtatgg agacacatta gaaaaactaa ctaacactgg gttatacaat 300

ttattaataa ggtgcctgcg gtgccagaaa ccgttgaatc cagcagaaaa acttagacac 360

cttaatgaaa aacgacgatt ccacaaaata gctgggcact atagaggcca gtgccattcg 420

tgctgcaacc gagcacgaca ggagagactc caacgacgca gagaaacaca agtagtcgac 480

atgtatggac ctaaggcaac attgcaagac attgtattgc atttagagcc tcaaaatgaa 540

attccggttg accttctagg tcacgagcaa ttaagcgact cagaggaaga aaacgatgaa 600

atagatggag ttaatcatca acatttacca gcccgacgag ccgaaccaca acgtcacaca 660

atgttgtgta tgtgttgtaa gtgtgaagcc agaattgagc tagtagtaga aagctcagca 720

gacgaccttc gagcattcca gcagctgttt ctgagcaccc tgtcctttgt gggtccgtgg 780

tgtgcatccc agcagtaa 798

<210>3

<211>750

<212>DNA

＜213〉artificial sequence

<400>3

atgttccagg acgcagagga gaaaccacgg acattgcatg atttgtgtca ggcgttggag 60

acatctgtgc atgaaatcga attgaaatgc gttgaatgca aaaagacttt gcagcgatct 120

gaggtatatg actttgtatt tgcagatggc agaatagtgt atagagatgg aaatccattt 180

gcagtatgta aagtgggctt acgattgcta tctaaaataa gtgagtatag acattataat 240

tattcgctat atggagacac attagaacaa acactaaaaa agtgtttaaa tgaaatatta 300

attagatgta ttatttgtca aagaccattg tgtccacaag aaaaaaaaag gcatgtggat 360

ttaaacaaaa ggtttcataa tatttcgggt cgttggacag ggcgctgtgc agtgtgttgg 420

agaccccgac gtagacaaac acaagtggaa ttcatgagag gaaacaaccc aacgctaaga 480

gaatatattt tagatttaca tcctgaacca actgacctat tctgctacgg ccaattatgt 540

gacagctcag acgaggatga aataggcttg gacgggccag atggacaagc acaaccggcc 600

acagctaatt actacattgt aacttgttgt tacacttgtg gcaccacggt tcgtttgtgt 660

atcaacagta caacaaccga cgtacgaacc ctacagcagc tgcttatggg cacatgtacc 720

attgtgggcc ctagctgtgc acagcaataa 750

<210>4

<211>663

<212>DNA

＜213〉artificial sequence

<400>4

gatccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg 60

ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag 120

ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc 180

caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg 240

aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag 300

gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca 360

gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt 420

caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc 480

tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag atgggatctg 540

atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag 600

gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca 660

acc 663

<210>5

<211>3676

<212>DNA

＜213〉artificial sequence

<400>5

tctagaatgc accaaaagag aactgcaatg tttcaggacc cacaggagcg acccagaaag 60

ttaccacatt tatgcacaga gctgcaaaca actatacatg atataatatt agaatgtgtg 120

tactgcaagc aacagttact gcgacgtgag gtatatgact ttgcttttcg ggatggttgc 180

atagtatata gagatgggaa tccatatgca gtgtgtgata aatgtttaaa gttttattct 240

aaaattagtg agtatagata ttattgttat agtgtgtatg gaacaacatt agaacagcaa 300

tacaacaaac cgttgtgtga tttgttaatt aggggtatta actgtcaaaa gccactgtgt 360

cctgaagaaa agcaaagaca tctggacaaa aagcaaagat tccataatat aaggggtcgg 420

tggaccggtc gatgtatgtc ttgttgcaga tcatcaagaa cccgtagaga aacccagctg 480

aagcttatgc atggagatac acctacattg catgaatata tgttagattt gcaaccagag 540

acaactgatg gctactgtta tgagcaatta aatgacagct cagaggagga agatgaaata 600

gatggtccag ctggacaagc agaaccggac agagcccatt acaatattgt aaccttttgt 660

tgcaagtgtg actctacgct tcggttgtgc gtacaaagca cacacgtaga cattcgtact 720

ttggaagacc tgttaatggg cacactagga attgtggggc ccatctgttc tcagaaacca 780

taaggatcct tacctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat 840

aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg 900

tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc 960

tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt 1020

cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg 1080

acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac 1140

cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg 1200

tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg 1260

ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctagatggga 1320

tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt 1380

ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc 1440

caccgaattc atggcgcgct ttgaggatcc aacacggcga ccctacaagc tacctgatct 1500

gtgcacggaa ctgaacactt cactgcaaga catagaaata acctgtgtat attgcaagac 1560

agtattggaa cttacagagg tatttgaatt tgcatttaaa gatggatttg tagtgtatag 1620

agacagtata ccgcatgctg catgccataa aggtatagat ttctattcta gaattagaga 1680

attaagatat tattcagact ctgtgtatgg agacacatta gaaaaactaa ctaacactgg 1740

gttatacaat ttattaataa ggtgcctgcg gtgccagaaa ccgttgaatc cagcagaaaa 1800

acttagacac cttaatgaaa aacgacgatt ccacaaaata gctgggcact atagaggcca 1860

gtgccattcg tgctgcaacc gagcacgaca ggagagactc caacgacgca gagaaacaca 1920

agtagtcgac atgtatggac ctaaggcaac attgcaagac attgtattgc atttagagcc 1980

tcaaaatgaa attccggttg accttctagg tcacgagcaa ttaagcgact cagaggaaga 2040

aaacgatgaa atagatggag ttaatcatca acatttacca gcccgacgag ccgaaccaca 2100

acgtcacaca atgttgtgta tgtgttgtaa gtgtgaagcc agaattgagc tagtagtaga 2160

aagctcagca gacgaccttc gagcattcca gcagctgttt ctgagcaccc tgtcctttgt 2220

gggtccgtgg tgtgcatccc agcagtaagg atccttacct ctccctcccc cccccctaac 2280

gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc 2340

accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg 2400

agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg 2460

aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc 2520

aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa 2580

gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa 2640

agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 2700

ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg 2760

aggttaaaaa aacgtctaga tgggatctga tctggggcct cggtgcacat gctttacatg 2820

tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct 2880

ttgaaaaaca cgatgataat atggccacac tcgagatgtt ccaggacgca gaggagaaac 2940

cacggacatt gcatgatttg tgtcaggcgt tggagacatc tgtgcatgaa atcgaattga 3000

aatgcgttga atgcaaaaag actttgcagc gatctgaggt atatgacttt gtatttgcag 3060

atggcagaat agtgtataga gatggaaatc catttgcagt atgtaaagtg ggcttacgat 3120

tgctatctaa aataagtgag tatagacatt ataattattc gctatatgga gacacattag 3180

aacaaacact aaaaaagtgt ttaaatgaaa tattaattag atgtattatt tgtcaaagac 3240

cattgtgtcc acaagaaaaa aaaaggcatg tggatttaaa caaaaggttt cataatattt 3300

cgggtcgttg gacagggcgc tgtgcagtgt gttggagacc ccgacgtaga caaacacaag 3360

tggaattcat gagaggaaac aacccaacgc taagagaata tattttagat ttacatcctg 3420

aaccaactga cctattctgc tacggccaat tatgtgacag ctcagacgag gatgaaatag 3480

gcttggacgg gccagatgga caagcacaac cggccacagc taattactac attgtaactt 3540

gttgttacac ttgtggcacc acggttcgtt tgtgtatcaa cagtacaaca accgacgtac 3600

gaaccctaca gcagctgctt atgggcacat gtaccattgt gggccctagc tgtgcacagc 3660

aataaacagc ggccgc 3676

<210>6

<211>258

<212>PRT

＜213〉artificial sequence

<400>6

Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro

1 5 10 15

Arg Lys Leu Pro His Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp

20 25 30

Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu

35 40 45

Val Tyr Asp Phe Ala Phe Arg Asp Gly Cys Ile Val Tyr Arg Asp Gly

50 55 60

Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile

65 70 75 80

Ser Glu Tyr Arg Tyr Tyr Cys Tyr Ser Val Tyr Gly Thr Thr Leu Glu

85 90 95

Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Gly Ile Asn

100 105 110

Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys

115 120 125

Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met

130 135 140

Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu Lys Leu

145 150 155 160

Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln

165 170 175

Pro Glu Thr Thr Asp Gly Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser

180 185 190

Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp

195 200 205

Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr

210 215 220

Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu

225 230 235 240

Asp Leu Leu Met Gly Thr Leu Gly Ile Val Gly Pro Ile Cys Ser Gln

245 250 255

Lys Pro

<210>7

<211>265

<212>PRT

＜213〉artificial sequence

<400>7

Met Ala Arg Phe Glu Asp Pro Thr Arg Arg Pro Tyr Lys Leu Pro Asp

1 5 10 15

Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys

20 25 30

Val Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu Phe Ala

35 40 45

Phe Lys Asp Gly Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala Ala

50 55 60

Cys His Lys Gly Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg Tyr

65 70 75 80

Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr

85 90 95

Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro Leu

100 105 110

Asn Pro Ala Glu Lys Leu Arg His Leu Asn Glu Lys Arg Arg Phe His

115 120 125

Lys Ile Ala Gly His Tyr Arg Gly Gln Cys His Ser Cys Cys Asn Arg

130 135 140

Ala Arg Gln Glu Arg Leu Gln Arg Arg Arg Glu Thr Gln Val Val Asp

145 150 155 160

Met Tyr Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu

165 170 175

Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Gly His Glu Gln Leu Ser

180 185 190

Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His

195 200 205

Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met

210 215 220

Cys Cys Lys Cys Glu Ala Arg Ile Glu Leu Val Val Glu Ser Ser Ala

225 230 235 240

Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Ser Thr Leu Ser Phe

245 250 255

Val Gly Pro Trp Cys Ala Ser Gln Gln

260 265

<210>8

<211>249

<212>PRT

＜213〉artificial sequence

<400>8

Met Phe Gln Asp Ala Glu Glu Lys Pro Arg Thr Leu His Asp Leu Cys

1 5 10 15

Gln Ala Leu Glu Thr Ser Val His Glu Ile Glu Leu Lys Cys Val Glu

20 25 30

Cys Lys Lys Thr Leu Gln Arg Ser Glu Val Tyr Asp Phe Val Phe Ala

35 40 45

Asp Gly Arg Ile Val Tyr Arg Asp Gly Asn Pro Phe Ala Val Cys Lys

50 55 60

Val Gly Leu Arg Leu Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn

65 70 75 80

Tyr Ser Leu Tyr Gly Asp Thr Leu Glu Gln Thr Leu Lys Lys Cys Leu

85 90 95

Asn Glu Ile Leu Ile Arg Cys Ile Ile Cys Gln Arg Pro Leu Cys Pro

100 105 110

Gln Glu Lys Lys Arg His Val Asp Leu Asn Lys Arg Phe His Asn Ile

115 120 125

Ser Gly Arg Trp Thr Gly Arg Cys Ala Val Cys Trp Arg Pro Arg Arg

130 135 140

Arg Gln Thr Gln Val Glu Phe Met Arg Gly Asn Asn Pro Thr Leu Arg

145 150 155 160

Glu Tyr Ile Leu Asp Leu His Pro Glu Pro Thr Asp Leu Phe Cys Tyr

165 170 175

Gly Gln Leu Cys Asp Ser Ser Asp Glu Asp Glu Ile Gly Leu Asp Gly

180 185 190

Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asn Tyr Tyr Ile Val Thr

195 200 205

Cys Cys Tyr Thr Cys Gly Thr Thr Val Arg Leu Cys Ile Asn Ser Thr

210 215 220

Thr Thr Asp Val Arg Thr Leu Gln Gln Leu Leu Met Gly Thr Cys Thr

225 230 235 240

Ile Val Gly Pro Ser Cys Ala Gln Gln

245

Claims

1. transcribe and merge three gene fragments for one kind, comprise HPV16mE67, HPV18mE67 and HPV58mE67 and the short nucleotide fragments that is connected the three;

Described HPV16mE67 amino acid sequence coded is a sequence 6 in the sequence table;

Described HPV18mE67 amino acid sequence coded is a sequence 7 in the sequence table;

Described HPV58mE67 amino acid sequence coded is a sequence 8 in the sequence table;

The described short nucleotide fragments that connects HPV16mE67, HPV18mE67 and HPV58mE67 is the ribosome internal entry site.

2. according to claim 1 transcribing merged three gene fragments, and it is characterized in that: the deoxyribonucleotide sequence of described HPV16mE67 is a sequence 1 in the sequence table; The deoxyribonucleotide sequence of described HPV18mE67 is a sequence 2 in the sequence table; The deoxyribonucleotide sequence of described HPV58mE67 is a sequence 3 in the sequence table; The deoxyribonucleotide sequence of described ribosome internal entry site is a sequence 4 in the sequence table.

3. according to claim 1 and 2 transcribing merged three gene fragments, it is characterized in that: described deoxyribonucleotide sequence of transcribing fusion three gene fragments is a sequence 5 in the sequence table.

4. according to claim 3 transcribing merged three gene fragments, it is characterized in that: described HPV16mE67, the HPV18mE67 and the HPV58mE67 that merge in three gene fragments of transcribing all has initiator codon and terminator codon.

5. contain arbitrary described recombinant expression vector that merges three gene fragments of transcribing among the claim 1-4.

6. contain arbitrary described transgenic cell line that merges three gene fragments of transcribing among the claim 1-4.

7. contain arbitrary described reorganization bacterium of merging three gene fragments of transcribing among the claim 1-4.

8. recombinant expression vector according to claim 5 is characterized in that: described recombinant expression vector is a replication defective recombinant adenoviral vector.

9. the described recombinant expression vector of claim 8 prevents and/or treats application in human cervical carcinoma's vaccine in preparation.

10. contain arbitrary described duplicate deficit type recombinant adenovirus that merges three gene fragments of transcribing among the claim 1-4.

11. according to right 10 described duplicate deficit type recombinant adenovirus, it is characterized in that: described duplicate deficit type recombinant adenovirus prepares with the Adeno-X expression system.

12. a kind of therapeutic vaccine of human cervical carcinoma, its activeconstituents are right 10 or 11 described duplicate deficit type recombinant adenovirus.