CN101885778B - HPV (Human Papilloma Virus) 16 type L2N120E7E6 fusion protein, gene, preparation method and application - Google Patents

Abstract

The invention relates to a codon optimization gene of an HPV (Human Papilloma Virus) 16 type L2N120E7E6 recombinant protein efficiently expressed in escherichia coli. The optimization gene is inserted into a prokaryotic expression vector pET9a and can obtain efficient expression, and the expression level occupies 50 percent of the whole escherichia coli; and after the expressed protein is passivated, the expressed protein can immunize a mouse. Proved by an experimental result, the protein has the same immunogenicity as the protein not optimized through the optimization gene and can be used for inducing specific IFN (Interferon)-gamma to release. Proved by a tumor growth inhibition experiment, protein immunization has obvious inhibition effect on the growth of tumors, wherein 90 percent ofthe growth of mouse tumor cells is completely inhibited. An L2N120E7E6 recombinant protein prokaryotic efficient expression system established by the optimization gene can be used for the prevention and the treatment of HPV 16 infections and relevant cervical cancer, the pilot scale production of vaccines and the research and the development of new medicines.

Description

HPV16 type L2N120E7E6 fusion rotein, gene, Preparation method and use

Technical field

The invention belongs to technical field of bioengineering.Particularly, the present invention relates to a kind of human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins, its encoding gene, plasmid vector, expression strain, preparation method and immunoprophylaxis and therepic use.

Background technology

Cervical cancer is one of principal element that causes in the our times scope woman cancer death.In developing country, cervical cancer then is modal type in women's malignant tumour, and its examination and treatment have brought huge economical load to people.The means of seeking effectively prevention and treatment cervical cancer and precancerous lesion thereof become the task of top priority.A large amount of research confirms that fully the generation of cervical cancer and precancerous lesion thereof and human papillomavirus (Human Papillomavirus, HPV) infect very close relationship.Surpass at present more than 100 kind of HPV and be identified, wherein 50 kinds relevant with genital tract infection, passability is handed over propagation.15 kinds are considered to high-risk HPV, and HPV16 is exactly modal type wherein.Chronic, the persistent infection of high-risk HPV is confirmed to be the major cause of cervical cancer and precancerous lesion thereof.Evidence suggests that host's immunne response, especially cellular immune level are to affect the principal element that the HPV relative disease forms.Just because of HPV relative disease and the body immune response substantial connection to HPV, so that people might prevent and treat with the strategy of vaccine immunity the infection of HPV, thereby further reach the purpose of prevention and treatment cervical cancer and precancerous lesions of uterine cervix.HPV is preventative just to begin development with therapeutic vaccine as far back as the early 1990s.Preventative vaccine has been obtained impressive progress at present, and the HPV preventative vaccine Gardix of 06 year Merk company comes into the market by drugs approved by FDA.The immunization strategy of preventative vaccine mainly concentrates on high titre neutralizing antibody and the preventing infection that how to induce for the HPV associated epitope.In this respect, with the HPV16 capsid protein L 1, L2 is the virus-like particle (virus-like particles, VLPs) of target antigen, all shows the effect that good prevention HPV infects in experimentation on animals and clinical trial.Therapeutic vaccine mainly produces cellular immunization for virus antigen by bringing out body, thereby removes focus and by the cell of virus infection.Because HPV16 early protein E6, E7 induces and to keep the cancer cells malignant phenotype necessary, and they just become the desirable target antigen of therapeutic vaccine.Various therapeutic vaccines take them as target antigen all show good cellular immunization in experimentation on animals.The T cell immunogenicity that shows E6 albumen in the human body according to the clinical research data of delivering in the recent period is better than E7 albumen.The target antigen that designs in our early stage research does not contain E6.Therefore in this research, we are on the basis of original research (referring to the pending application of same Applicant: Chinese patent application number 200810227047.6), E6 antigen will be connected behind the L2E7 fusion rotein again, the molecule of considering formation is too large, can image expression level, we will have the L2 PROTEIN C end disappearance of carrier proteins effect and only stay 120 amino acid of N end, in the hope of the high expression level of the new fusion rotein that obtains, in the human clinical trial in future, show strong immunogenicity.And improve that it is water-soluble.Experimental results show that by this design and obtained expected result, and the antineoplastic immune effect of having observed it in animal experiment is suitable with the L2E7 fusion rotein, the Specific T cell immunity reaction level that E6 antigen brings out the C57 mouse is lower, and this is consistent with other bibliographical informations of delivering.

Summary of the invention

The below discusses preparation and the use of the various technical schemes of the present invention in detail, but should be appreciated that, the invention provides many applicable inventive concepts, and it can be embodied on the various concrete aspects.

For helping to understand the present invention, the below has defined some terms.The term of this paper definition has the implication that those of ordinary skill in the related art of the present invention understand usually.The general category of the specific examples that term can be used for illustrating, but their use does not limit the present invention, except summarizing in the claim.

Unless otherwise noted, " HPV " as herein described refers to human papillomavirus (Humanpapillomavirus);

Unless otherwise noted, " bp " as herein described refers to base pair (base pair);

Unless otherwise noted, " ELISPOT " as herein described refers to enzyme linked immunological spot (Enzyme-linked immunospot);

Unless otherwise noted, " IPTG " as herein described refers to isopropylthio-β-D galactoside (Isopropylthio-β-D-galactoside);

Unless otherwise noted, " SDS-PAGE " as herein described refers to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sodium dodecyl sulfate polyacrylamide gel electrophoresis);

Unless otherwise noted, " HRP " as herein described refers to horseradish peroxidase (Horseradishperoxidase);

Unless otherwise noted, " IFN-γ " as herein described refers to gamma-interferon (Interfern gamma);

Unless otherwise noted, " PBS " as herein described refers to phosphate buffered saline buffer (Phosphate bufferedsaline);

The present invention aim to provide a kind of energy simple, economical, effectively prepare the optimized gene nucleotide sequence of human papillomavirus HPV16L2N120E7E6 fusion rotein, this optimized gene can obtain great expression in intestinal bacteria, expressing protein can be used for prevention and the treatment of HPV16 infection and relevant cervical cancer.

Particularly, one object of the present invention is, provides a kind of human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins.Another object of the present invention is, a kind of dna sequence dna for aforesaid human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins is provided.Another object of the present invention is, a kind of colibacillus expression plasmid carrier is provided.Another object of the present invention is, a kind of coli strain for the production of aforesaid human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins is provided.Another object of the present invention is, a kind of method for preparing aforesaid human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins is provided.

For the foregoing invention purpose, the invention provides following technical scheme:

On the one hand, the invention provides the fusion rotein of a kind of human papillomavirus (HPV) 16 type L2N120, E7 and E6, the aminoacid sequence of described fusion rotein is the sequence shown in the SQE ID NO.2.

On the other hand, the invention provides a kind of dna sequence dna for aforesaid human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins, the nucleotides sequence of described dna sequence dna is classified the sequence shown in the SQE IDNO.1 as.

Again on the one hand, the invention provides a kind of colibacillus expression plasmid carrier, described plasmid vector is that the L2N120E7E6 gene fragment by aforesaid HPV16 is inserted between the Nde I of plasmid pET9a and the BamH I site and makes up.

Preferably, in the aforementioned colibacillus expression plasmid carrier, described intestinal bacteria are BL21 (DE3).

Another aspect the invention provides a kind of coli strain of the L2N120E7E6 fusion rotein for the production of aforesaid human papillomavirus (HPV) 16 types, and described coli strain contains aforesaid plasmid vector.

Preferably, aforesaid coli strain is BL21 (DE3).

Also on the one hand, the invention provides a kind of method for preparing aforesaid human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins, said method comprising the steps of:

1) aforesaid HPV16L2N120E7E6 gene fragment is inserted Nde I and the BamH I site of coli expression carrier pET9a, this plasmid transformation escherichia coli is obtained to contain the bacterial strain of pET9a16L2N120E7E6, inoculation culture, IPTG abduction delivering;

2) express thalline after centrifugal cracking, the results inclusion body hangs with 8M urea, and is centrifugal, gets supernatant CM column purification expressing protein.

Preferably, prepare in the method for aforesaid human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins in the present invention, described intestinal bacteria are BL21 (DE3).

On the other hand, the invention provides a kind of method for the preparation for the treatment of HPV 16 infection and relative disease (such as cervical cancer) medicine thereof, described method comprises that the coli strain that will contain pET9a16L2N120E7E6 is used for pilot scale fermentation and produces the L2N120E7E6 fusion rotein.

On the other hand, the invention provides aforesaid human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins, the dna sequence dna of aforesaid human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins, aforesaid colibacillus expression plasmid carrier or aforesaid coli strain for the preparation of the application in the medicine for the treatment of HPV 16 infection and relative disease (such as cervical cancer) thereof.Preferably, described medicine is vaccine; Most preferably, described vaccine possesses prevention and treatment dual function.

The below according to of the present invention one preferred embodiment, in conjunction with Figure of description technical scheme of the present invention is further described in detail:

Genetic engineering technique is adopted in this experiment, at first will separate the HPV16 early protein E7 of order-checking acquisition and the peptide sequence of late protein L2 from the Patients with Cervical Cancer sample merges, secondary structure analysis-by-synthesis according to intestinal bacteria optimal codon and mRNA, simultaneously the 24th halfcystine of two key amino acids of E7 albumen and pRB binding site and the codon of the 26th L-glutamic acid are sported respectively codon glycine, to eliminate its tumour activity of conversion, design the nucleotide sequence of coding HPV16L2E7 fusion rotein polypeptide, synthetic (this part is the content of the applicant's Chinese patent application co-pending, and denomination of invention and application number are respectively: the HPV fusion rotein by company of sincere section, gene, carrier, bacterial strain, Preparation method and use application number: 200810227047.6).

To from the Patients with Cervical Cancer sample, separate the HPV16 early protein E6 albumen that order-checking obtains among the present invention, secondary structure analysis-by-synthesis according to intestinal bacteria optimal codon and mRNA, leucic codon mutation with the 57th of the key amino acid in the p53 degrading activity site of E6 albumen is codon glycine simultaneously, to eliminate its tumour activity of conversion.Design the nucleotide sequence of coding HPV16E6 protein polypeptide, the while is at the 5 ' nucleotide sequence of having optimized of holding with upper E7 240-294 position of E6 gene, and the terminator codon of removal E7 gene:

AGATCTGCTGATGGGCACCCTGGGCATTGTGTGCCCGATTTGCAGCCAGAAACCG (nucleotide sequence of E7 240-294 position, underscore are Bgl II restriction enzyme site), synthetic through company of sincere section.

Utilize the Bgl II restriction enzyme site on the E7 in the HPV16L2E7 fusion gene, synthetic HPV16L2E7 fusion gene is connected by Bgl II restriction enzyme site with the HPV16E6 gene that synthesizes constructs the plasmid that contains the HPV16L2E7E6 fusion gene.

Then take the plasmid that contains the HPV16L2E7E6 fragment as template, amplify respectively two goal gene of HPV16sL2N120 and HPV16sE7E6, amplify the L2N120E7E6 fusion gene that contains 1131bp with overlapping PCR method again.The purpose fragment is cloned into the pMD18-T carrier, after digestion with restriction enzyme evaluation and PCR evaluation correctly, send and hold up the order-checking of company of section.Obtained the pMD18T-HPV16sL2N120E7E6 cloned plasmids that conforms to implementation sequence.

The HPV16sL2N120E7E6 fusion gene is inserted prokaryotic expression carrier pET9a, modifying gene obtains to efficiently express, expression level accounts for 50% of full bacterium, and the purified rear immune mouse of expressing protein is with the immunogenicity of animal immune experimental evaluation synthetic gene expression albumen.

The present invention comes the method for production HPV 16 L2N120E7E6 fusion rotein may further comprise the steps according to the gene order of optimizing codon:

The codon optimized HPV16sL2N120E7E6 gene fragment of design is inserted Nde I and the BamH I site of coli expression carrier pET9a, this plasmid transformation escherichia coli is obtained to contain the bacterial strain of pET9a16sL2N120E7E6, inoculation culture, the IPTG abduction delivering;

Express thalline after centrifugal cracking, the results inclusion body hangs with 8M urea, and is centrifugal, gets supernatant CM column purification expressing protein.

Preferably, produce in the method for HPV16sL2N120E7E6 fusion rotein in the present invention, described intestinal bacteria are BL21 (DE3) (BL21 (DE3) are the escherichia coli expression bacterial strains of commonly using, the general strongly expressed that carries out goal gene with the strongly expressed carrier that cooperates).

The coli strain that contains recombinant plasmid pET9a16sL2N120E7E6 of the present invention can be used for pilot scale fermentation and produce the L2N120E7E6 fusion rotein, and development is treated HPV 16 infection and infected therewith relevant disease (such as cervical cancer) medicine.

As everyone knows, same amino acid has several groups of codons, biologically several groups of codons are represented the degeneracy that a kind of amino acid whose phenomenon is called codon, and degeneracy mainly is because the 3rd base generation dancing of codon forms, the specificity that is to say codon is mainly determined by the first two base, even the 3rd base undergone mutation and also can be translated correct amino acid, this is for guaranteeing that fixity of species has the certain significance; For example: GCU, GCC, GCA, GCG all represent L-Ala.Except tryptophane and methionine(Met), other amino acid whose codons are all more than 1 (2～6).Degeneracy does not also mean that the password imperfection, and each codon is corresponding 1 seed amino acid only.Degeneracy can make the harmful effect of sudden change reduce to minimum.Most of codon has degeneracy, i.e. two or more codons same monoamino-acid of encoding.The codon of degeneracy only has the 3rd bit base different usually, for example, and GAA and the GAG glutamine of all encoding.If no matter which kind of Nucleotide the 3rd of codon is, the same amino acid of all encoding then is referred to as the quadruple degeneracy; If the 3rd has two kinds among four kinds of possible Nucleotide, and coding same amino acid, then being referred to as double degenerate, the Nucleotide of general the 3rd upper two kinds of equivalences is all purine (A/G) or pyrimidine (C/T).Only have two seed amino acids only by a codon coding, one is methionine(Met), by the AUG coding, also is initiator codon simultaneously; Another is tryptophane, is encoded by UGG.These character of genetic code can make gene more tolerate point mutation.For example, quadruple degenerate codon can be tolerated the tertiary any variation of codon; The double degenerate codon makes 1/3rd possible tertiary variations not affect protein sequence.Because conversion variation (purine becomes purine or pyrimidine becomes pyrimidine) is larger than the possibility of transversion variation (purine becomes pyrimidine or pyrimidine becomes purine), so the double degenerate codon also has the very strong ability to anti-mutation.This sudden change that does not affect aminoacid sequence with gene on the genetics is called silent mutation.Yet, in specific expression strain, under the prerequisite of the aminoacid sequence that does not change albumen, use different codons the output of proteins encoded to be had the impact of highly significant.Therefore, from multiple codon, select specific codon to obtain being called " codon optimized " than high protein output.Larger for molecular weight, as namely the to form more albumen of amino acid number is sought and definite codon optimized nucleotide sequence has suitable difficulty.

120 amino acid polypeptide sequences of the HPV16 early protein E7 albumen that inventor's selection will separate and E6 albumen and late protein L2N end are fused into the nucleotide sequence of HPV16sL2N120E7E6 fusion rotein, according to the intestinal bacteria optimal codon, Bgl II restriction enzyme site in the nucleotide sequence of the coding HPV16L2E7 fusion rotein that utilization designed and synthesized originally, the HPV16E6 sequence that contains E7 240-294 position nucleotide sequence with the synthetic 5 ' end of design in this research merges, construct the HPV16sL2N120E7E6 fusion gene, be inserted into prokaryotic expression carrier pET9a, modifying gene obtains to efficiently express, expression level accounts for about 50% of full bacterium, and carried out the purifying of this albumen, behind the immune animal, can be observed the antineoplastic immune effect suitable with the L2E7 fusion rotein, the mouse after ELISPOT detects immunity can produce obviously higher for HPV16E7 than L2E7 fusion rotein _49-57The specific t cell immune response of CTL epitope peptide can detect the Specific T cell immunity reaction for HPV16E6 peptide storehouse of lower level simultaneously.The present invention can be used for research and development prevention and therapeutic vaccine for cervical cancer.

Compared with prior art, the present invention has following obvious advantage:

Contain 120 amino acid of HPV16 L2N end that can produce neutralizing antibody among the present invention, and contain simultaneously E7 and the E6 albumen that can cause cell immune response, use optimized gene of the present invention can obtain to efficiently express, expression level accounts for 50% of full bacterium, the purified immune mouse of expressing protein, experimental result shows that this albumen has good immunogenicity, can produce for HPV16E7 _49-57The specific t cell immune response of CTL epitope peptide and HPV16E6 peptide storehouse, tumor growth suppresses experimentation on animals and shows that protein immunization has obvious restraining effect to the growth of tumour, and wherein the growth of 90% mouse tumor cell suppresses fully.The L2N120E7E6 fusion rotein High level prokaryotic expression system that this optimized gene is set up can be used for HPV16 and infect and the prevention of relevant cervical cancer and trial production and the new drug development for the treatment of vaccine.

Description of drawings

Below, describe by reference to the accompanying drawings embodiments of the invention in detail, wherein:

Fig. 1 is the structure diagram according to the cloned plasmids pMD18sL2N120E7E6 of the method structure of the embodiment of the invention 2;

Fig. 2 is the structure diagram according to the recombined pronucleus expression plasmid pET9asL2N120E7E6 of the method structure of the embodiment of the invention 3;

Fig. 3 is the prokaryotic expression SDS-PAGE of HPV16L2N120E7E6 fusion rotein, is specially the result at the SDS-PAGE of expression in escherichia coli gel analysis according to the codon optimized gene sL2N120E7E6 of the carrying out of the embodiment of the invention 4; Wherein, M is low molecular weight protein Marker, and 1 is the empty carrier contrast, and 2 for inducing front optimized gene expression product, and 3 for inducing rear optimized gene expression product;

Fig. 4 is the Western blot qualification result of HPV16L2N120E7E6 fusion rotein, is specially the codon optimized gene sL2N120E7E6 that carries out according to the embodiment of the invention 5 at the Western-blot of expression in escherichia coli albumen qualification result; Wherein, M is low molecular weight protein Marker, and 1 is the empty carrier contrast, 2 lysates for the optimized gene expression;

Fig. 5 is the HPV16L2N120E7E6 fusion protein S DS-PAGE purity check behind the purifying, be specially the codon optimized gene sL2N120E7E6 that carries out according to the embodiment of the invention 6 and express the result of the SDS-PAGE gel analysis behind the ion exchange chromatography purifying at prokaryotic system, wherein, M is low molecular weight protein Marker; 1 is purified L2N120E7E6 albumen;

Fig. 6 is the humoral immune reaction that the HPV16L2N120E7E6 fusion rotein brings out in Mice Body, and it is purified to be specially the optimized gene expressing protein that carries out according to the embodiment of the invention 7, the result that the antibody titers behind the immune mouse detects;

Fig. 7 for the codon optimized gene sL2N120E7E6 expressing protein immune mouse that carries out according to the embodiment of the invention 7 after enzyme linked immunological spot detection result;

Fig. 8 is the codon optimized gene sL2N120E7E6 expressing protein that carries out according to the embodiment of the invention 7 restraining effect to tumor growth.

Fig. 9 is employed pMD18-T carrier spectrogram in the embodiment of the invention 2.

Figure 10 is employed pET9a carrier spectrogram in the embodiment of the invention 3.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Be not used in but these embodiment only limit to the present invention is described and limit the scope of the invention.The experimental technique of unreceipted concrete experiment condition in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

Embodiment 1: design and synthesize HPV 16 L2N120E7E6 fusion rotein intestinal bacteria table The optimizing codon gene that reaches

Will be from the Patients with Cervical Cancer sample HPV16 late gene L2 and early gene E7 and the E6 order-checking of separating clone, polypeptid acid sequence according to the coding of the HPV16 late gene L2 that obtains and early gene E7, and the leucic codon of the 57th of the key amino acid in the p53 degrading activity site of the codon of the 24th halfcystine of two key amino acids of E7 albumen and pRB binding site and the 26th L-glutamic acid and E6 albumen sported respectively codon glycine, to eliminate its tumour activity of conversion.E7 and the fusion of E6 protein sequence of 120 amino acid of N end of the less important capsid protein sequence L2 of HPV16 virus and HPV16 virus are designed to following fusion rotein:

MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGKTI

ADQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGTRPPTATDT

LAPVRPPLTVDPVGPSDPSIVSLVEETSFIDAGAPMHGDTPTLHEY

MLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIV

TFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPM

HQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLL

RREVYDFAFRDGCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSL

YGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFH

NIRGRWTGRCMSCCRSSRTRRETQL. (376 amino acid)

Utilize the gene degeneracy, under the prerequisite that the product albumen aminoacid sequence of its coding remains unchanged, design the optimizing codon nucleotide sequence, the contriver is with this sequence called after sL2N120E7E6, detect proof through experiment, this is suitable for the fusion rotein at the HPV16 of expression in escherichia coli type L2N120E7E6, and its sequence is specific as follows:

ATGCGTCATAAACGTTCTGCGAAACGTACCAAACGTGCGAGC

GCGACCCAGTTATATAAAACGTGTAAACAGGCCGGTACCTGCCCG

CCGGATATTATTCCGAAAGTGGAAGGCAAAACCATTGCGGATCAGA

TTCTGCAGTATGGCAGCATGGGCGTGTTCTTTGGCGGCCTGGGCA

TTGGCACCGGCAGCGGCACCGGCGGCCGTACCGGCTATATTCCGC

TGGGCACCCGTCCGCCGACCGCGACCGATACCCTGGCGCCGGTG

CGTCCGCCGCTGACCGTTGATCCGGTGGGCCCGAGCGATCCGAG

CATTGTGAGCCTGGTGGAAGAAACCAGCTTTATTGATGCGGGCGC

GCCGATGCATGGCGATACCCCGACCCTGCATGAATATATGCTGGAT

CTGCAGCCGGAAACCACCGATCTGTATGGCTATGGCCAGCTGAAT

GATAGCAGCGAAGAAGAAGATGAAATTGATGGCCCGGCGGGCCAG

GCGGAACCGGATCGTGCGCATTATAATATTGTGACCTTTTGCTGCA

AATGCGATAGCACCCTGCGTCTGTGCGTGCAGAGCACCCATGTGG

ATATTCGTACCCTGGA AGATCTGCTGATGGGCACCCTGGGCATTGT

GTGCCCGATTTGCAGCCAGAAACCGATGCATCAGAAGCGTACCGC

GATGTTTCAAGATCCGCAGGAACGTCCGCGTAAACTGCCGCAGTT

ATGCACCGAACTGCAGACCACGATCCATGATATTATTTTAGAATGC

GTGTATTGCAAACAGCAGCTGCTGCGTCGTGAAGTGTATGATTTT

GCGTTTCGTGATGGCTGCATTGTGTATCGTGATGGCAATCCGTATG

CGGTGTGCGATAAATGCCTGAAATTTTATTCTAAAATTAGCGAATAT

CGTCATTATTGCTATAGCCTGTATGGCACCACCCTGGAACAGCAGT

ATAATAAACCGCTGTGCGATCTGCTGATTCGTTGCATTAATTGCCA

GAAACCGCTGTGCCCGGAAGAAAAACAGCGTCATCTGGATAAAAA

ACAGCGTTTTCATAATATTCGTGGCCGTTGGACCGGCCGTTGCATG

AGCTGCTGCCGTAGCAGCCGTACCCGTCGTGAAACCCAGCTGTAA

(1131bp)

Embodiment 2:

Utilize the applicant's unsettled Chinese patent application number: the Bgl II restriction enzyme site on the E7 in the designed synthetic HPV16 type sL2E7 fusion gene in 200810227047.6, design the nucleotide sequence of codon optimized coding HPV16E6 protein polypeptide, and the E6 gene 5 ' end add up except the E7 240-294 position nucleotide sequence of terminator codon

Concrete sequence is as follows:

AGATCTGCTGATGGGCACCCTGGGCATTGTGTGCCCGATTTG

CAGCCAGAAACCGATGCATCAGAAGCGTACCGCGATGTTTCAAGA

TCCGCAGGAACGTCCGCGTAAACTGCCGCAGTTATGCACCGAACT

GCAGACCACGATCCATGATATTATTTTAGAATGCGTGTATTGCAAA

CAGCAGCTGCTGCGTCGTGAAGTGTATGATTTTGCGTTTCGTGAT

GGCTGCATTGTGTATCGTGATGGCAATCCGTATGCGGTGTGCGATA

AATGCCTGAAATTTTATTCTAAAATTAGCGAATATCGTCATTATTGC

TATAGCCTGTATGGCACCACCCTGGAACAGCAGTATAATAAACCGC

TGTGCGATCTGCTGATTCGTTGCATTAATTGCCAGAAACCGCTGTG

CCCGGAAGAAAAACAGCGTCATCTGGATAAAAAACAGCGTTTTCA

TAATATTCGTGGCCGTTGGACCGGCCGTTGCATGAGCTGCTGCCG

TAGCAGCCGTACCCGTCGTGAAACCCAGCTGTAA。

Wherein underscore is that E7 240-294 position nucleotide sequence contains Bgl II restriction enzyme site, and is synthetic through company of sincere section.

Synthetic HPV16L2E7 fusion gene is connected with BamH I restriction enzyme site by Bgl II with the HPV16E6 gene that synthesizes constructs the plasmid that contains the HPV16L2E7E6 fusion gene.

Then take the plasmid that contains the HPV16L2E7E6 fragment as template, the design primer is as shown in the table, utilize clone primer P1/P2 and P3/P4, amplify respectively two goal gene fragments that contain HPV16sL2N120 (360bp) and HPV16sE7E6 (771bp).

The concrete sequence of HPV16sL2N120 (360bp) is as follows:

ATGCGTCATAAACGTTCTGCGAAACGTACCAAACGTGCGAGCGCG

ACCCAGTTATATAAAACGTGTAAACAGGCCGGTACCTGCCCGCCG

GATATTATTCCGAAAGTGGAAGGCAAAACCATTGCGGATCAGATTC

TGCAGTATGGCAGCATGGGCGTGTTCTTTGGCGGCCTGGGCATTG

GCACCGGCAGCGGCACCGGCGGCCGTACCGGCTATATTCCGCTGG

GCACCCGTCCGCCGACCGCGACCGATACCCTGGCGCCGGTGCGT

CCGCCGCTGACCGTTGATCCGGTGGGCCCGAGCGATCCGAGCATT

GTGAGCCTGGTGGAAGAAACCAGCTTTATTGATGCGGGCGCGCCG

The concrete sequence of HPV16sE7E6 (771bp) is as follows:

ATGCATGGCGATACCCCGACCCTGCATGAATATATGCTGGATC

TGCAGCCGGAAACCACCGATCTGTATGGCTATGGCCAGCTGAATG

ATAGCAGCGAAGAAGAAGATGAAATTGATGGCCCGGCGGGCCAGG

CGGAACCGGATCGTGCGCATTATAATATTGTGACCTTTTGCTGCAA

ATGCGATAGCACCCTGCGTCTGTGCGTGCAGAGCACCCATGTGGA

TATTCGTACCCTGGAAGATCTGCTGATGGGCACCCTGGGCATTGT

GTGCCCGATTTGCAGCCAGAAACCGATGCATCAGAAGCGTACCGC

GATGTTTCAAGATCCGCAGGAACGTCCGCGTAAACTGCCGCAGTT

ATGCACCGAACTGCAGACCACGATCCATGATATTATTTTAGAATGC

GTGTATTGCAAACAGCAGCTGCTGCGTCGTGAAGTGTATGATTTT

GCGTTTCGTGATGGCTGCATTGTGTATCGTGATGGCAATCCGTATG

CGGTGTGCGATAAATGCCTGAAATTTTATTCTAAAATTAGCGAATAT

CGTCATTATTGCTATAGCCTGTATGGCACCACCCTGGAACAGCAGT

ATAATAAACCGCTGTGCGATCTGCTGATTCGTTGCATTAATTGCCA

GAAACCGCTGTGCCCGGAAGAAAAACAGCGTCATCTGGATAAAAA

ACAGCGTTTTCATAATATTCGTGGCCGTTGGACCGGCCGTTGCATG

AGCTGCTGCCGTAGCAGCCGTACCCGTCGTGAAACCCAGCTGTAA

Amplification condition is: 94 ℃ of 3 minutes denaturations, loop body be 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 50 seconds, 30 circulations, then 72 ℃ 10 minutes.SL2N120 (360bp) and sE7E6 (771bp) gene PCR amplified fragments are carried out after gel reclaims, take mixed these two sections PCR products as template, forward clone's primer (P1) and reverse cloning primer (P4) carry out overlapping pcr amplification.Amplification condition is 94 ℃ of 4 minutes denaturations, loop body be 94 ℃ 50 seconds, 65 ℃ 40 seconds, 72 ℃ 1 minute, 30 circulations, then 72 ℃ 10 minutes.Amplify the fragment that contains goal gene sL2N120E7E6 (1131bp).

The purpose fragment is cloned on the pMD18-T carrier, after digestion with restriction enzyme evaluation and PCR evaluation correctly, send and hold up the order-checking of company of section.Obtained the pMD18T-HPV16sL2N120E7E6 cloned plasmids that conforms to implementation sequence, the structure of this cloned plasmids is seen Fig. 1.

Numbering	Title	Sequence
			P1	16sL2F2	5’＞GGGAATTC CATATGCGTCATAAACGTTCT＜3 ' (underscore Nde I restriction enzyme site)
P2	16sL2(360) E7E6R	5’＞ GGTATCGCCATGCATCGGCGCGCCCGCATC ＜3’
			P3	16sL2(360) E7E6F	5’＞GATGCGGGCGCGCCGATGCATGGCGAT ACC＜3’
P4	16sE6R	5’＞GC GGATCCTTACAGCTGGGTTTCACGACG＜3 ' (underscore BamH I restriction enzyme site)

Employed pMD18-T carrier is available from Beijing six directions trade company limited of stimulating the menstrual flow in the present embodiment, article No. D103A, and its concrete structure is as shown in Figure 9.

Embodiment 3:

SL2N120E7E6 optimized gene synthetic among the embodiment 2 and that be cloned on the pMD18-T carrier is cut digestion with enzyme, it is specific as follows that described enzyme is cut the operation of digestion: first with 37 ℃ of water-baths digestion of Nde I enzyme 2 hours, then reclaiming test kit with sepharose reclaims, 37 ℃ of water-bath digestion of BamH I enzyme are 2 hours again, reclaim test kit with sepharose at last and reclaim fragment, wherein:

Employed Nde I and BamH I enzyme are Biolabs company product, available from Beijing North instrument great waves commerce and trade company limited;

It is sky, Beijing root biochemical technology company limited product that sepharose reclaims test kit.

Then be inserted into Nde I and the BamH I site of colibacillus expression plasmid carrier pET9a (its concrete structure collection of illustrative plates is seen Figure 10), cut through Nde I and BamH I enzyme and to identify and the order-checking screening obtains to have the prokaryotic expression recombinant plasmid pET9asL2N120E7E6 of correct insertion that its structure diagram as shown in Figure 2.

Employed pET9a is the expression plasmid carrier of commercially available acquisition, for Novagen company product, available from Huamei Bio-Engrg Co.,'s Beijing Company.

Embodiment 4:

The recombinant plasmid pET9asL2N120E7E6 that uses embodiment 3 to obtain transforms BL21 (DE3) intestinal bacteria, be coated with plate after, 37 ℃ of incubated overnight.Choose single spot at LB substratum (known substratum, this culture medium prescription document that sees reference: Pehanorm Brooker J, Ritchie EF not, with Manny A Disi T, molecular cloning experiment guide .1992. second edition (Beijing): Science Press: the 908th page) cultivates in, work as OD=0.8, the adding final concentration is that the IPTG of 0.8mM (is Promega company product, available from vast Tyke, Beijing biological gene technology limited liability company), 25 ℃ of abduction deliverings 8 hours, get an amount of thalline and carry out the SDS-PAGE gel electrophoresis analysis, the result shows, at the about 45KD of molecular weight place one newly-increased protein band is arranged, this protein band accounts for 50% of whole bacterial protein ultimate production, and electrophoresis result as shown in Figure 3.

The Western-blot of embodiment 5:HPV16sL2N120E7E6 optimized gene prokaryotic expression detects

Get the abduction delivering bacterium liquid 200 μ l that embodiment 4 obtains, centrifugal results thalline, be resuspended in SDS-PAGE sample loading buffer (the known damping fluid of 100 μ l, the concrete prescription of this damping fluid, compound method are seen Pehanorm Brooker J, Ritchie EF not, with Manny A Disi T, molecular cloning experiment guide .1992. second edition (Beijing): Science Press: the 935th page) mixing, 100 ℃ were heated 3 minutes.Get 5-10 μ l after centrifugal and be splined on 10% SDS-PAGE glue, carry out electrophoresis, then electricity turns nitrocellulose filter.Use anti-HPV16L2 self-control cavy to resist (how anti-anti-HPV16L2 is for the self-control cavy more, (the pQE30HPV16L2 prokaryotic expression plasmid is expressed in the JM109 bacterial strain with the HPV16L2 prokaryotic system, the bacterial classification that obtains to be inoculated in 300ml 2 * YT substratum at 1: 1000,37 ℃ of lower shaking culture to OD600=0.8, add IPTG to final concentration be the 0.8mM/ liter, 25 ℃ of lower abduction deliverings 6 hours, collect bacterial sediment; (be Amersham Biosciences (peace agate West Asia) company's product with the StreamlineTMChelating medium, close poly-economy and trade company limited available from Central Plains, Beijing, article No.: 17-1280-01)), the albumen of purifying adds freund's adjuvant (Sigma company product, glad through Bioisystech Co., Ltd of section available from Beijing), subcutaneous multi-point injection immune guinea pig, twice rear heart blood sampling of immunity, centrifuging and taking serum.) as first antibody, the albumin A/G of horseradish peroxidase-labeled (known albumen, for the second antibody of the how anti-reaction of anti-HPV16L2 cavy, available from Beijing North with positive biotech development company), carry out the specificity identification that target protein is expressed.The result shows, the L2N120E7E6 fusion protein molecule amount of being expressed by optimized gene is about the 45KD place, and the result is referring to Fig. 4.

The purifying of embodiment 6:L2N120E7E6 optimized gene Expression in Escherichia coli and albumen

A. the bacterial classification that embodiment 4 is obtained to be inoculated in 300ml2 * YT substratum (known substratum at 1: 1000, document sees reference: Pehanorm Brooker J, Ritchie EF not, and Manny A Disi T, molecular cloning experiment guide .1992. second edition (Beijing): Science Press: the 909th page), 37 ℃ of lower shaking culture to OD ₆₀₀=0.8, add IPTG (be Promega company product, available from vast Tyke, Beijing biological gene technology limited liability company) to final concentration be the 0.8mM/ liter, 25 ℃ of lower abduction deliverings 8 hours, the collection bacterial sediment;

B. the expression thalline that results among the abovementioned steps a is obtained is dissolved in 10mL lysis buffer (50mMTris, 0.5%TritonX-100, PH8.0) add N,O-Diacetylmuramidase in, after placing 30 minutes under the room temperature, supersound process in ice bath (400 watts 25 times) 5 seconds, interval 10 seconds.Centrifugal (12,000rmp, 20 minutes, 4 ℃) collecting precipitation;

C. the precipitation that abovementioned steps b is obtained be suspended in the damping fluid that 20mL contains 2M urea (50mMTris, 2 mol/L urea, PH8.0), supersound process in ice bath (400 watts, 25 times) 5 seconds, every minor tick 10 seconds.Centrifugal (12000rmp, 20 minutes, 4 ℃) collecting precipitation;

D. the precipitation Eddy diffusion that abovementioned steps c is obtained in 10mL contain the 8M urea buffer solution (50mMTris, 8 mol/L urea, PH7.0), supersound process in ice bath (400 watts, 25 times) 5 seconds, every minor tick 10 seconds.Centrifugal (12000rmp, 20 minutes, 4 ℃), it is for subsequent use to collect supernatant liquor.

E. ion exchange chromatography: the supernatant liquor that abovementioned steps d is obtained is through CM ion exchange column (CM:CM SepharoseTM Fast Flow, be Amersham Biosciences (peace agate West Asia) company's product, closing poly-economy and trade company limited available from Central Plains, Beijing) purifying obtains target protein, purity reaches more than 95%, and the SDS-PAGE gel electrophoresis analysis of gained purifying protein the results are shown in Figure 5.

To reclaim albumen through dialysis tubing (available from Huamei Bio-Engrg Co.,'s Beijing Company, specification DM-49, the aperture is 12-14KD) carry out Urea Gradient dialysis, dialyse at last to phosphate buffered saline(PBS) (PBS, its prescription is seen Pehanorm Brooker J, not Ritchie EF, and Manny A Disi T, molecular cloning experiment guide .1992. second edition (Beijing): Science Press: the 927th page) the middle storage for immune animal.

The experiment of embodiment 7:HPV16L2N120E7E6 fusion rotein animal immune

Serum specific antibody and cell immune response detect behind the a.L2N120E7E6 fusion protein immunization mouse:

The L2N120E7E6 fusion rotein 50 μ g that use is prepared by previous embodiment 6, add 10ugCpG (1826) adjuvant, immunity C57BL/6 mouse (C57BL/6 mouse, age in 6-8 week, female, breed the center available from Sinology academy of sciences laboratory animal, raise at SPF2 level Animal House), reinforcement one pin after two weeks (dosage is 50 μ g, adds 10ugCpG (1826) adjuvant) carried out the serum specific antibody detection in 10-14 days after immunity finishes and cellular immunization detects.

(1) detection of specific antibody: get serum, carry out the detection of L2 antibody, E7 antibody and E6 antibody with euzymelinked immunosorbent assay (ELISA) (ELISA), with the negative contrast of reagent set PBS, experimental result as shown in Figure 6.As can be seen from Figure 6, the L2N120E7E6 fusion rotein can be induced the L2 antibody 1: 200000 that produces high titre, E7 antibody 1: 116960 and E6 antibody 1: 20319.

(2) cell immune response detects: extracting spleen cell, carry out the number detection that E7 albumen 49-57 position peptide and E6 protein peptide storehouse stimulate the effector T cell of the specific secretion IFN-γ that produces with enzyme linked immunological spot (ELISPOT) method.

Experimental result as shown in Figure 7, HPV16E7 _49-57The effector T cell that produces secretion of gamma-IFN behind the mouse of peptide and HPV16E6 peptide storehouse immune stimulatory L2N120E7E6 fusion rotein is counted the spot number and is respectively: 464 and 47, PBS control group then do not detect corresponding spot number.Both have obvious difference, illustrate that the L2N120E7E6 fusion rotein can bring out the mouse generation for the cellullar immunologic response of the specific T-cells mediation of HPV16E7 and E6 albumen.

It is that method according to people such as the people such as Miyahira and Murali-Krishna is (referring to Miyahira Y that ELISPOT detects, Murata K, Rodriguez D, Deng people Quantification of antigen specificCD8+T cell using an ELISPOT assay.J Immunol Methods, 1995,181 (1): 45-54.Murali-Krishna K, Altman JD, Suresh M, et al.Counting antigen-speific CD8 Tcells:a reevaluation of bystander activation during viral infection.Immunity, 1998,8 (2): 1771-87.) operation is referring to ELISPOT test kit specification sheets (the ELISPOT test kit is U-CyTech company product, available from Shenzhen Da Kewei Bioisystech Co., Ltd).

B.HPV16L2N120E7E6 is to the restraining effect of tumor growth:

Use first 1x10 ⁴TC-1 tumour cell (TC-1 tumor cell line HPV16-E6, the C57BL/6 mouse lung epithelial cell of E7 and ras gene transformation, but stably express and submission E7 albumen, presented by John Hopkins professor T.C.Wu of university, document sees reference: Lin KY, Guarnieri FG, Stavelev-o ' CanrrollKF, et al.Treatment of established tumor with a novel vaccine that enhance majorhistocompatibility class II presentation of tumor antigen.Cancer Res, 1996,56 (1): 21～26) inguinal region subcutaneous injection (injected dose is 100 μ l) C57BL/6 mouse (C57BL/6 mouse, age in 6-8 week, female, breed the center available from Sinology academy of sciences laboratory animal, raise at SPF2 level Animal House) locate, the L2N120E7E6 fusion rotein of second day intramuscular injection 50 μ g (the L2N120E7E6 fusion rotein that is prepared by previous embodiment 6), booster immunization after 10 days (dosage is the L2N120E7E6 fusion rotein of 50 μ g) is observed tumor growth situation (2 times weekly).The result shows that the growth to tumour has obvious restraining effect behind protein immunization, and wherein 90% mouse TC-1 growth of tumour cell suppresses fully.Experimental result as shown in Figure 8.

Sequence table

＜110〉China Sickness Prevention Control Center Virus Disease Prevention Control Institute

＜120〉HPV16 type L2N120E7E6 fusion rotein, gene, Preparation method and use

<130>DIC09110073

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<170>PatentIn version 3.3

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＜213〉artificial sequence

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ggcaccggca gcggcaccgg cggccgtacc ggctatattc cgctgggcac ccgtccgccg 240

accgcgaccg ataccctggc gccggtgcgt ccgccgctga ccgttgatcc ggtgggcccg 300

agcgatccga gcattgtgag cctggtggaa gaaaccagct ttattgatgc gggcgcgccg 360

atgcatggcg ataccccgac cctgcatgaa tatatgctgg atctgcagcc ggaaaccacc 420

gatctgtatg gctatggcca gctgaatgat agcagcgaag aagaagatga aattgatggc 480

ccggcgggcc aggcggaacc ggatcgtgcg cattataata ttgtgacctt ttgctgcaaa 540

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gatctgctga tgggcaccct gggcattgtg tgcccgattt gcagccagaa accgatgcat 660

cagaagcgta ccgcgatgtt tcaagatccg caggaacgtc cgcgtaaact gccgcagtta 720

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cagctgctgc gtcgtgaagt gtatgatttt gcgtttcgtg atggctgcat tgtgtatcgt 840

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tatcgtcatt attgctatag cctgtatggc accaccctgg aacagcagta taataaaccg 960

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cagcgtcatc tggataaaaa acagcgtttt cataatattc gtggccgttg gaccggccgt 1080

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＜213〉HPV16L2N120E7E6 fusion rotein

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Met Arg His Lys Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr

1 5 10 15

Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile

20 25 30

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

35 40 45

Gly Ser Met Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly Ser

50 55 60

Gly Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu Gly Thr Arg Pro Pro

65 70 75 80

Thr Ala Thr Asp Thr Leu Ala Pro Val Arg Pro Pro Leu Thr Val Asp

85 90 95

Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser Leu Val Glu Glu Thr

100 105 110

Ser Phe Ile Asp Ala Gly Ala Pro Met His Gly Asp Thr Pro Thr Leu

115 120 125

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

130 135 140

Tyr Gly Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly

145 150 155 160

Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr

165 170 175

Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr

180 185 190

His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly

195 200 205

Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Met His Gln Lys Arg Thr

210 215 220

Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn

340 345 350

Ile Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser

355 360 365

Arg Thr Arg Arg Glu Thr Gln Leu

370 375

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atgcaccgaa ctgcagacca cgatccatga tattatttta gaatgcgtgt attgcaaaca 180

gcagctgctg cgtcgtgaag tgtatgattt tgcgtttcgt gatggctgca ttgtgtatcg 240

tgatggcaat ccgtatgcgg tgtgcgataa atgcctgaaa ttttattcta aaattagcga 300

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gctgtgcgat ctgctgattc gttgcattaa ttgccagaaa ccgctgtgcc cggaagaaaa 420

acagcgtcat ctggataaaa aacagcgttt tcataatatt cgtggccgtt ggaccggccg 480

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ggcaccggca gcggcaccgg cggccgtacc ggctatattc cgctgggcac ccgtccgccg 240

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ccggcgggcc aggcggaacc ggatcgtgcg cattataata ttgtgacctt ttgctgcaaa 180

tgcgatagca ccctgcgtct gtgcgtgcag agcacccatg tggatattcg taccctggaa 240

gatctgctga tgggcaccct gggcattgtg tgcccgattt gcagccagaa accgatgcat 300

cagaagcgta ccgcgatgtt tcaagatccg caggaacgtc cgcgtaaact gccgcagtta 360

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cagctgctgc gtcgtgaagt gtatgatttt gcgtttcgtg atggctgcat tgtgtatcgt 480

gatggcaatc cgtatgcggt gtgcgataaa tgcctgaaat tttattctaa aattagcgaa 540

tatcgtcatt attgctatag cctgtatggc accaccctgg aacagcagta taataaaccg 600

ctgtgcgatc tgctgattcg ttgcattaat tgccagaaac cgctgtgccc ggaagaaaaa 660

cagcgtcatc tggataaaaa acagcgtttt cataatattc gtggccgttg gaccggccgt 720

tgcatgagct gctgccgtag cagccgtacc cgtcgtgaaa cccagctgta a 771

Claims (22)

1. a human papillomavirus (HPV) 16 type L2N120E7E6 fusion roteins is characterized in that, the aminoacid sequence of described fusion rotein is the sequence shown in the SQE ID NO.2.

2. the gene for human papillomavirus claimed in claim 1 (HPV) the 16 type L2N120E7E6 fusion roteins of encoding is characterized in that the nucleotides sequence of described gene is classified the sequence shown in the SQE ID NO.1 as.

3. a colibacillus expression plasmid carrier is characterized in that, described plasmid vector is to be inserted between the Nde I of plasmid pET9a and the BamH I site by HPV16 type L2N120E7E6 gene claimed in claim 2 to make up.

4. colibacillus expression plasmid carrier according to claim 3 is characterized in that, described intestinal bacteria are BL21(DE3).

5. the coli strain for the production of human papillomavirus claimed in claim 1 (HPV) 16 type L2N120E7E6 fusion roteins is characterized in that described coli strain contains plasmid vector claimed in claim 3.

6. coli strain according to claim 5 is characterized in that, described intestinal bacteria are BL21(DE3).

7. a method for preparing human papillomavirus claimed in claim 1 (HPV) 16 type L2N120E7E6 fusion roteins is characterized in that, said method comprising the steps of:

1) HPV16L2N120E7E6 gene claimed in claim 2 is inserted NdeI and the BamHI site of coli expression carrier pET9a, this plasmid transformation escherichia coli is obtained to contain the bacterial strain of pET9a16L2N120E7E6, inoculation culture, IPTG abduction delivering;

2) express thalline after centrifugal cracking, the results inclusion body hangs with 8M urea, and is centrifugal, gets supernatant CM column purification expressing protein.

8. method according to claim 7 is characterized in that, described intestinal bacteria are BL21(DE3).

9. the method for the preparation of the medicine for the treatment of HPV 16 infection and cervical cancer is characterized in that, described method comprises, claim 5 or 6 described coli strains are used for fermentative production L2N120E7E6 fusion rotein.

10. human papillomavirus claimed in claim 1 (HPV) 16 type L2N120E7E6 fusion roteins are for the preparation of the application in the medicine for the treatment of HPV 16 infection and cervical cancer.

11. application according to claim 10 is characterized in that described medicine is vaccine.

12. application according to claim 11 is characterized in that described vaccine has therapeutic action.

13. the gene of human papillomavirus claimed in claim 2 (HPV) 16 type L2N120E7E6 fusion roteins is for the preparation of the application in the medicine for the treatment of HPV 16 infection and cervical cancer.

14. application according to claim 13 is characterized in that described medicine is vaccine.

15. application according to claim 14 is characterized in that described vaccine has therapeutic action.

16. each described colibacillus expression plasmid carrier of claim 3-4 is for the preparation of the application in the medicine for the treatment of HPV 16 infection and cervical cancer.

17. application according to claim 16 is characterized in that described medicine is vaccine.

18. application according to claim 17 is characterized in that described vaccine has therapeutic action.

19. each described coli strain of claim 5-6 is for the preparation of the application in the medicine for the treatment of HPV 16 infection and cervical cancer.

20. application according to claim 19 is characterized in that described medicine is vaccine.

21. application according to claim 20 is characterized in that described vaccine has therapeutic action.

22. a vaccine that is used for the treatment of cervical cancer is characterized in that, described vaccine comprises human papillomavirus claimed in claim 1 (HPV) 16 type L2N120E7E6 fusion roteins.

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