CN102002105B - Gene, expression vector, expression method, expression cell and application of human papilloma virus (HPV) 16 E7E6 fusion protein - Google Patents

Abstract

The invention provides a gene, an expression vector, an expression method, an expression cell and an application of a human papilloma virus (HPV) 16 E7E6 fusion protein. In the invention, recombinant adenovirus is adopted to fuse E6 and E7 protein genes which have no converting activity after modification, and the mammalian cell codon optimization and transformation is performed based on the amino acid sequences of the fusion protein to lead the codon to be expressed in the mammalian cells in a high level. In the invention, an E7E6 fusion protein mammalian cell codon optimization gene is designed successfully, the recombinant adenovirus capable of expressing the HPV16 E7E6 fusion proteins in a high level is successfully constructed, has strong immunogenic performance, can induce immunoreaction of specific T cells, and completely protects immune mice from attacking of anti-tumor cells. The invention can be used for searching and developing therapeutic vaccines of cervical cancer.

Description

HPV 16 type E7E6 antigen-4 fusion protein genes, expression vector, method, cell and purposes

Technical field

The invention belongs to medical biotechnology field.Particularly, the present invention relates to gene, expression vector, method and the immunotherapy purposes of a kind of expression human papillomavirus (HPV) 16 type E7E6 fusion roteins.

Background technology

A large amount of research datas prove that the Combining high-risk human papillomavirus (humanpapillomavirus, HPV) of reproductive tract infects with cervical cancer very close relationship.90% cervical cancer patient can be found the existence of HPV DNA, and wherein the HPV16 type accounts for 50%, is the main virulence factor that causes cervical cancer.Although early cervical carcinoma corrective surgery and radiotherapy in the treatment effect are better at present, the middle and advanced stage patient must give on the basis of operative treatment radiotherapy and certain chemotherapy.Because the huge side effect of radiotherapy and cervical cancer to the insensitivity of chemotherapy, still have 35% patient to occur there is no at present the serious complication that method is treated, expense is large and recurrence rate is high.Everything shows and is necessary to develop infection and cervical cancer and the related neoplasms that more effective prevention and therapeutic vaccine are prevented and treated HPV.

Because HPV oncogene E6, the E7 early protein is continuous expression in epithelium of cervix uteri, do not exist in healthy tissues, so these two kinds of albumen can be used as the desirable target antigen of the relevant cervical cancer of development HPV and precancerous lesion therapeutic vaccine.Most of tumour specific antigens are cell proteins normal or sudden change, and E6 and E7 are exogenous virus albumen fully, therefore may have better immunogenicity than the cell protein of sudden change.And because the sustainable existence of E6 and E7 is tumour cell transformation and to keep malignant characteristics necessary, so cervical cancer cell can not lose the escape from immune supervision by antigen.Experimentation on animals shows, with E6 and E7 albumen as target antigen, the Immunotherapy that can produce.

The duplicate deficit type recombinant adenovirus preferendum is extensive, is foreign gene is imported one of carrier commonly used in the zooblast.After the inoculation, recombinant adenovirus carries out nucleic acid replication and protein expression in vivo, produce a large amount of antigens, stimulate body to produce specific immune response, and its infection is similar to natural process, the antigen of expressing is processed and present in host cell, can produce strong humoral immunization and cellular immunization, and virus vector itself also can be brought into play adjuvant effect and strengthen immune response; Simultaneously because the recombinant adenovirus unconformability in host genome, has good biological peaceful property; Physico-chemical property is more stable, is easy to separation and purification; The suitable carrier that these characteristics all make adenovirus become tumor vaccine is widely used in the experiments such as gene therapy, outer-gene transfection and gene vaccine preparation and the clinical study.

Adenovirus carrier vaccine has many advantages, but also exist deficiency, its original high background infects the neutralizing antibody that produces and affects its being widely used in new generation vaccine, the antiviral antibody that produces after the immunization in addition may suppress again immunity, thereby affect the immunotherapy effect, but the methods for the treatment of of people by heterology vaccine combined immunization overcome this shortcoming.He Z. in 2000 etc. have delivered at Virology and expressed respectively E6, E7 albumen in adenovirus, and have carried out vaccine.

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

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 (Phosphatebuffered saline);

The present invention utilizes adenovirus carrier, modified E6, the E7 protein gene that loses activity of conversion is merged, and carry out the codon optimized transformation of mammalian cell according to the aminoacid sequence of fusion rotein, and making it can high level expression at mammalian cell.The present invention successfully designs the codon optimized gene of E7E6 fusion rotein mammalian cell; and successfully made up the recombinant adenovirus rAd5HPV16SmE7E6 of high expression level HPV16 type E7E6 fusion rotein; this recombinant adenovirus vaccine has strong immunogenicity; can bring out the Specific T cell immunity reaction, protect the attack of immune mouse opposing tumour cell fully.The present invention can be used for researching and developing treatment and the therapeutic vaccine for cervical cancer of cervical cancer.

Particularly, one object of the present invention is, provides a kind of human papillomavirus (HPV) 16 type E7E6 fusion roteins.Another object of the present invention is, a kind of optimization dna sequence dna be used to aforesaid human papillomavirus (HPV) the 16 type E7E6 fusion roteins of encoding is provided.Another purpose of the present invention is, a kind of expression vector is provided and comprises the recombinant adenovirus of human papillomavirus (HPV) 16 type E7E6 fusion genes.A further object of the present invention is, a kind of cell for the production of aforesaid human papillomavirus (HPV) 16 type E7E6 fusion rotein recombinant adenovirus is provided.

Also purpose of the present invention is, a kind of method of production aforesaid expression human papillomavirus (HPV) 16 type E7E6 fusion roteins is provided.In addition, the present invention also provides the purposes of dna sequence dna, expression vector, adenovirus and the cell of above-mentioned HPV 16 type E7E6 fusion roteins.

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

On the one hand, the invention provides a kind of human papillomavirus (HPV) 16 type E7E6 fusion roteins, the aminoacid sequence of described fusion rotein is the sequence shown in the SQE.ID.NO.1.

On the other hand, the invention provides a kind of optimization dna sequence dna be used to above-mentioned human papillomavirus (HPV) the 16 type E7E6 fusion roteins of encoding, described dna sequence dna is the sequence that is optimized according to the mammalian cell codon, and its nucleotides sequence is classified the sequence shown in the SQE.ID.NO.2 as.

Another aspect the invention provides a kind of expression vector, and described expression vector contains the optimization dna sequence dna of above-mentioned expression human papillomavirus (HPV) 16 type E7E6 fusion roteins; Preferably, described expression vector comprises recombinant adenovirus.Because it is synthetic that above-mentioned optimization dna sequence dna provided by the present invention is optimized according to the mammalian cell codon, in theory, since it is adapted at expressing on the adenovirus carrier, just should efficiently express in other mammalian cell expression system.

Another aspect the invention provides a kind of recombinant adenovirus plasmid, and described plasmid contains optimization dna sequence dna and cloned plasmids or the adenovirus shuttle plasmid of above-mentioned encoding human papilloma virus (HPV) 16 type E7E6 fusion roteins; Preferably, described cloned plasmids is selected from pUC18, pUC19 and pUC57 etc.; Described adenovirus shuttle plasmid is selected from pDC316, pDC311, pDC312 and pDC315 etc.; More preferably, described recombinant adenovirus plasmid is that dna sequence dna with coding above-mentioned human papillomavirus (HPV) 16 type E7E6 fusion roteins is inserted between the Bgl II site of cloned plasmids pUC 18 or adenovirus shuttle plasmid pDC316 and makes up.

In addition, the invention provides a kind of recombinant adenovirus be used to expressing above-mentioned human papillomavirus (HPV) 16 type E7E6 fusion roteins, described recombinant adenovirus is replication-defective adenoviral, this virus carry above-mentioned expression human papillomavirus (HPV) 16 type E7E6 fusion roteins the optimization dna sequence dna, and can express human papillomavirus (HPV) 16 type E7E6 fusion roteins; Preferably, described recombinant adenovirus is occured to form in the human embryo kidney 293 cells internal packing after the homologous recombination in human embryo kidney 293 cells by recombinant adenovirus shuttle plasmid and adenovirus skeleton plasmid, wherein said adenovirus shuttle plasmid is selected from pDC316, pDC311, pDC312 and pDC315 etc., described adenovirus skeleton plasmid is replication-defective vector, be preferably selected from pBHGlox Δ E1,3Cre.

On the one hand, the invention provides a kind of cell for the production of above-mentioned recombinant adenovirus again, described cell is the packing cell of above-mentioned recombinant adenovirus; Preferably, described cell is selected from human embryo kidney 293 cells, human embryo kidney (HEK) 293T cell, human embryo kidney (HEK) 293FT cell and human embryo kidney (HEK) 293F cell etc.

The present invention also provides a kind of mammalian cell be used to expressing above-mentioned human papillomavirus (HPV) 16 type E7E6 fusion roteins, and described cell is infected by above-mentioned expression vector or transfection obtains; Preferably, described expression vector is recombinant adenovirus; More preferably, described mammalian cell is human embryo kidney 293 cells, human embryo kidney (HEK) 293T cell, human embryo kidney (HEK) 293FT cell and human embryo kidney (HEK) 293F cell.

Also on the one hand, the invention provides a kind of method of expression above-mentioned human papillomavirus (HPV) 16 type E7E6 fusion roteins, described method comprises the expression vector that makes up the optimization dna sequence dna that contains above-mentioned encoding human papilloma virus (HPV) 16 type E7E6 fusion roteins, and makes this expression vector obtain expressing in mammalian cell; Preferably, described expression vector is recombinant adenovirus; More preferably, described cell is selected from human embryo kidney 293 cells, human embryo kidney (HEK) 293T cell, human embryo kidney (HEK) 293FT cell and human embryo kidney (HEK) 293F cell etc.

The present invention also provides the method for the recombinant adenovirus of a kind of expression of preparation above-mentioned human papillomavirus (HPV) 16 type E7E6 fusion roteins, said method comprising the steps of: 1) the optimization dna sequence dna with above-mentioned human papillomavirus (HPV) 16 type E7E6 fusion roteins is inserted into adenovirus shuttle plasmid, constructs the recombinant adenovirus shuttle vector; 2) with step 1) constructed recombinant adenovirus shuttle plasmid and adenovirus skeleton plasmid cotransfection human embryo kidney 293 cells; 3) with step 2) resulting cell carry out centrifugal after, with aseptic PBS re-suspended cell precipitation, behind the multigelation, recentrifuge, supernatant liquor are the former generation virus liquid that contains recombinant adenovirus; 4) with step 3) in former generation virus liquid carry out single spot purifying, and be tested and appraised and filter out correct recombinant adenovirus; 5) with step 4) in the recombinant adenovirus amplification of in HEKC, going down to posterity.

In addition, the present invention also provides a kind of and has infected and the method for relative disease (such as cervical cancer) medicine for the preparation for the treatment of human papillomavirus (HPV) 16 types, and described method comprises the optimization dna sequence dna of encoding human papilloma virus (HPV) 16 type E7E6 fusion roteins, the expression vector (comprising recombinant adenovirus) of optimization dna sequence dna that comprises encoding human papilloma virus (HPV) 16 type E7E6 fusion roteins or the packing cell that comprises this recombinant adenovirus are used for expressing human papillomavirus (HPV) 16 type E7E6 fusion roteins.

The present invention also provides above-mentioned human papillomavirus (HPV) 16 type E7E6 fusion roteins, the optimization dna sequence dna of encoding human papilloma virus (HPV) 16 type E7E6 fusion roteins, the expression vector (comprising recombinant adenovirus) that comprises the optimization dna sequence dna of encoding human papilloma virus (HPV) 16 type E7E6 fusion roteins, the cell that perhaps comprises this recombinant adenovirus infect for the preparation for the treatment of human papillomavirus (HPV) 16 types and relative disease (such as cervical cancer) medicine in application; Preferably, described medicine is vaccine.

The present invention also provides a kind of vaccine that is used for the treatment of cervical cancer, and described vaccine comprises above-mentioned fusion rotein, dna sequence dna, expression vector, recombinant adenovirus or cell.

This shows, the present invention adopts the genetic engineering technique method, at first, HPV16 type early protein E7, E6 coding region gene are merged, and E7, E6 coding region gene are transformed modification remove its activity of conversion, keep antigenicity, design its corresponding optimal codon nucleotide sequence to give company synthetic according to the access to your password inclined to one side preferendum of son of mammalian cell again.As everyone knows, 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.Then, with synthetic being inserted among the adenovirus recombination vector plasmid pCD316 through the E7E6 of codon optimized mistake fusion gene, with Ad5 virus vector skeleton plasmid cotransfection 293 cells, purified and carry out the evaluation of E7E6 fusion gene and albumen, filter out the recombinant adenovirus rAd5HPV16SmE7E6 that expresses human papillomavirus HPV16 type E7E6 fusion rotein, the amplification recombinant virus, through the animal immune experimental evaluation, show that prepared recombinant adenovirus rAd5HPV16SmE7E6 has immunocompetence, it can bring out immune animal and produce cellullar immunologic response for the specific T-cells mediation of HPV16E7, and the growth of tumour is had obvious restraining effect.

Description of drawings

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

Fig. 1 shows the structure collection of illustrative plates of the cloned plasmids pUC18-HPV16smE7E6 that has inserted codon optimized HPV16 type E7E6 fusion gene.

Fig. 2 shows the structure collection of illustrative plates of adenovirus shuttle plasmid pDC316.

Fig. 3 shows the structure collection of illustrative plates of the recombinant adenovirus shuttle plasmid pDC-HPV16smE7E6 that has inserted codon optimized HPV16 type E7E6 fusion gene.

Fig. 4 shows that the enzyme of the recombinant adenovirus shuttle plasmid pDC-HPV16SmE7E6 that has inserted codon optimized HPV16 type E7E6 fusion gene is cut and identifies the gel electrophoresis result, wherein M1 is λ-EcoT14I DNA Marker, M2 is DNA Marer III, 1 is the pDC-HPV16SmE7E6 plasmid of cutting with Ava I enzyme, the 2 pDC-HPV16SmE7E6 plasmids of cutting for Bgl II enzyme, the 3 pDC-HPV16SmE7E6 plasmids of cutting for Nsi I enzyme, 4 for not carrying out the pDC-HPV16SmE7E6 plasmid that enzyme is cut.

Fig. 5 shows the structure collection of illustrative plates of the recombinant adenovirus rAd5HPV16SmE7E6 of the recombinant adenovirus shuttle plasmid pDC-HPV16SmE7E6 that has inserted codon optimized HPV16 type E7E6 fusion gene.

Fig. 6 shows the PCR qualification result of recombinant adenovirus rAd5HPV16SmE7E6, and wherein M is λ-EcoT14I DNA Marker, and 1 is blank, and 2 is Ad5 adenovirus negative control, and 3 is recombinant adenovirus rAd5HPV16SmE7E6.

Fig. 7 shows the Western-blot detected result of the E7E6 fusion gene expression product among the recombinant adenovirus rAd5HPV16SmE7E6, wherein M is the pre-dsred protein Marker of wide molecular weight, 1 is Ad5 adenovirus negative control, 2 is recombinant adenovirus rAd5HPV16SmE7E6, A figure is the reaction signal with anti-HPV16E6 antibody test, and B figure is the reaction signal with anti-HPV16E7 antibody test.

Fig. 8 shows behind the recombinant adenovirus rAd5HPV16SmE7E6 immune mouse as a result figure of ELISPOT enzyme linked immunological spot detection.

After Fig. 9 shows recombinant adenovirus rAd5HPV16SmE7E6 immune mouse, the as a result figure of antitumor transplantation experiments.

Figure 10 shows that recombinant adenovirus rAd5HPV16SmE7E6 is to the inhibiting as a result figure of tumor growth.

Embodiment

Referring to specific embodiment the present invention is described.It will be appreciated by those skilled in the art that these embodiment only are used for explanation the present invention, the scope that it does not limit the present invention in any way.

Except as otherwise noted, various experiment materials used in the present invention are originated as follows: restriction enzyme commonly used, T4 ligase enzyme, Taq enzyme, alkaline phosphatase 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; Lipofectamine Lipofectamine ^TM2000 is Invitrogen company product, available from the luxuriant strong couple stars in Beijing Science and Technology Ltd.; Mouse ELISPOT test kit is Dutch U-Cytech company, is Bioisystech Co., Ltd available from reaching section; Mouse-anti HPV16E7 monoclonal antibody is Britain SantaCruz company product; The horseradish peroxidase-labeled sheep anti-mouse igg antibody is available from biotech company of China fir Golden Bridge in Beijing; Nitrocellulose filter is available from Britain Whatman company; The lower molecular weight standard protein is available from the magnificent transduction in Beijing Science and Technology Ltd.; Recombinant adenovirus shuttle plasmid pDC316, recombinant adenovirus skeleton plasmid pBHGlox Δ E1,293 cells are all available from Canadian Microbix Bixosystems company on 3Cre and the adenovirus packaging cell people kidney.

Except as otherwise noted, primer used in the present invention source is as follows: it is synthetic by Shanghai living worker's biotechnology gene engineering company limited to detect primer, upstream primer pDC316F:5 ' acg tgg gta taagag gcg 3 ' wherein, downstream primer pDC316R:5 ' cga tgc tag acg atc cag 3 '; For the specific epitope peptide of HPV16E7CD8+T cell (amino acid 49-57 position: RAHYNIVTF) synthetic by the BeiJing ZhongKe Yaguang Biology Science Co., Ltd.

Except as otherwise noted, laboratory animal used in the present invention source is as follows: the C57BL/6 mouse is available from animal institute of the Chinese Academy of Medical Sciences.

Except as otherwise noted, biological gene engineering operative technique involved in the present invention (cutting digestion, fragment recovery, nucleic acid fragment and plasmid vector ligation and clone and screening etc. such as plasmid extraction, enzyme), see also " molecular biosciences clone guide ", Pehanorm Brooker J etc., second edition (Beijing): Science Press, 1992.

Embodiment 1: design and synthesize human papilloma virus 16 type E 7 E6 fusion rotein and be suitable for the codon optimized fusion gene that mammalian cell is expressed

Present embodiment is for designing and synthesizing human papilloma virus 16 type E 7 E6 fusion rotein, and design is suitable for the codon optimized fusion gene that mammalian cell is expressed, and detailed process is as follows.

Obtain the aminoacid sequence of HPV16 type E7, E6 genes encoding according to the coding sequence (Genebank NC 001526) of HPV16 type E7, E6, the 24th halfcystine of two key amino acids and the 26th L-glutamic acid of E7 albumen and pRB binding site are become glycine, the leucine that the key amino acid in the P53 degrading activity site of E6 albumen is the 57th becomes glycine, to eliminate its tumour activity of conversion.The E7 Argine Monohydrochloride sequence C end of HPV16 is linked to each other with the N end of HPV16E6 protein sequence, unitized design is following fusion rotein again, aminoacid sequence shown in SQE.ID.NO.1, totally 255 amino acid (not containing terminator codon).

Utilize the gene degeneracy, under the prerequisite that the product albumen aminoacid sequence of its coding remains unchanged, design and be suitable for the optimal codon that mammalian cell expresses and optimize nucleotide sequence, the contriver is with this sequence called after SmE7E6, detect proof through experiment, this sequence is suitable for expressing the HPV16SmE7E6 fusion rotein in mammalian cell, its sequence is total to 771bp (containing two termination codon subsequences) shown in SQE.ID.NO.2.

Embodiment 2: the structure of cloned plasmids pUC18-HPV16smE7E6

Present embodiment is for making up cloned plasmids pUC18-HPV16smE7E6.

Sending Beijing to hold up Bioisystech Co., Ltd of section embodiment 1 designed HPV16SmE7E6 fusion gene nucleotide sequence synthesizes, and by company it is cloned among the pUC18, obtain the cloned plasmids pUC18-HPV16smE7E6 of synthetic gene, show that through sequencing composition sequence is consistent with design.Inserted the cloned plasmids pUC18-HPV16smE7E6 structure collection of illustrative plates of codon optimized HPV16 type E7E6 fusion gene as shown in Figure 1, its sequence is total to 3475bp shown in SQE.ID.NO.3.

Employed pUC18 is the plasmid vector of commercially available acquisition in the present embodiment, and available from Beijing six directions trade company limited of stimulating the menstrual flow, its concrete structure is seen document " molecular biosciences clone guide ", Pehanorm Brooker J etc., second edition (Beijing): Science Press, the 9th page, 1992.

Embodiment 3: the structure of recombinant adenovirus 5 type shuttle plasmid pDC-HPV16smE7E6

Present embodiment is for making up recombinant adenovirus 5 type shuttle plasmid pDC-HPV16smE7E6, and detailed process is as follows.

At first, SmE7E6 optimized gene synthetic among the embodiment 2 and that be cloned on the pUC18 was digested 2 hours with 37 ℃ of water-baths of BglII enzyme, then reclaim test kit with sepharose and reclaim SmE7E6 fusion gene fragment.

Secondly, this SmE7E6 fusion gene fragment is inserted with identical restriction endonuclease (Bgl II) digestion process, and carry out among the adenovirus shuttle plasmid pDC316 (its concrete structure collection of illustrative plates is seen Fig. 2) that dephosphorylation processed with alkaline phosphatase, cut evaluation (electrophoresis result as shown in Figure 4) and order-checking screening through Ava I, BglII and NsiI enzyme, acquisition has the recombinant adenovirus shuttle plasmid pDC-HPV16SmE7E6 of correct insertion, its structure collection of illustrative plates as shown in Figure 3, its sequence is total to 4690bp shown in SQE.ID.NO.4.

Embodiment 4: the structure of recombinant adenovirus rAd5HPV16SmE

Present embodiment is the recombinant adenovirus rAd5HPV16SmE7E of construction expression HPV16 type E7E6 fusion rotein, and its structure collection of illustrative plates is seen Fig. 5, and its sequence is total to 31762bp shown in SQE.ID.NO.5.Concrete building process comprises:

(1) adenovirus skeleton plasmid pBHGlox Δ E1, the preparation of 3Cre and recombinant adenovirus shuttle plasmid pDC-HPV16SmE7E6:

Use the large extraction reagent kit of qiagen plasmid (available from Beijing North instrument great waves commerce and trade company limited) to extract respectively pBHGlox Δ E1, the pDC-HPV16SmE7E6 that 3Cre and embodiment 3 are constructed, operation steps is seen QIAGEN Plasmid Midi Kits service manual.

(2) packing of recombinant adenovirus:

Transfection passes 293 cells in 25cm the day before yesterday ²Tissue Culture Flask, the substratum DMEM that does not contain antibiotic 5%FCS, second day selects 60%～80% cell that is paved with for subsequent use.Preparation transfection liquid is as follows:

A liquid: recombinant adenovirus shuttle plasmid pDC-HPV16SmE7E6 and skeleton plasmid pBHGlox Δ E1, each 5 μ l of 3Cre (each 4 μ g) add mixing in the 245 μ l Opti-MEM substratum;

B liquid: 5 μ l LipofectamineTM2000 add mixing in the 245 μ l Opti-MEM substratum.

A liquid and B liquid chamber temperature were placed after 5 minutes, and A is added in the B liquid gently mixing, and room temperature was placed 20 minutes.Old cell culture fluid is abandoned in suction, changes fresh not the containing microbiotic, contain the DMEM of 5%FCS of 2ml, then the A+B mixed solution is added on the culturing cell, in 37 ℃, 5%CO ₂Hatch 8h, replenish fresh substratum 2.5ml.Changing the DMEM that contains 2%FCS behind the 24h keeps.After the transfection 7～10 days, scrape with cell cell is swiped, centrifugal 5 minutes of 800rpm abandons supernatant, with the aseptic PBS re-suspended cell precipitation of 2ml;-70 ℃ and 37 ℃ of multigelations 4 times; Centrifugal 10 minutes of 2000rpm draws supernatant and is former generation virus liquid, gained recombinant adenovirus called after rAd5HPV 16SmE7E6, and-70 ℃ save backup.

Embodiment 5: the purifying of recombinant adenovirus rAd5HPV16SmE7E and PCR identify

Present embodiment is the purification of Recombinant adenovirus and identifies this recombinant adenovirus by PCR that concrete experimental implementation is as follows:

With among the embodiment 4 former generation virus liquid with 10 times of serial dilutions, infect 293 cells, cultivate and spread spot after 2-4 hour, cultivate after 4-6 days, behind single spot purifying, identify for 37 ℃.

After single pinta poison freeze thawing three times, infect and in 24 orifice plates, grow to 90% 293 cells.Virus is put into 37 ℃ and is cultivated after 24-48 hour 37 ℃ of absorption fluid infusion after 1-2 hour, draws the supernatant liquor 50 μ l of cells infected, add 1 μ l Proteinase K (10mg/ml), 37 ℃ of digestion were boiled 10 minutes after 1.5 hours, were placed on rapidly 5 minutes on ice, as the template of PCR detection.When contamination do simultaneously a hole of infecting the Ad5 adenovirus carry out same treatment after as the negative control that detects usefulness.

Get the above-mentioned sample of 0.01 μ l-1 μ l and carry out the PCR reaction.Sample comprises the contrast of recombinant adenovirus poison strain to be measured and Ad5 adenovirus, does simultaneously not add dna profiling and contrast as reagent blank.

The PCR reaction system

Sample 0.2 μ l

10×PCR Buffer 2μl

Primer pDC316F 1 μ l

Primer pDC316R 1 μ l

dNTP 1.6μl

Taq enzyme 0.2 μ l

H ₂O 14μl

Amount to 20 μ l

Loop parameter: 94 ℃ of denaturations 10 minutes; 94 ℃ of sex change 30 seconds, 53 ℃ of annealing 30 seconds, 72 ℃ were extended 30 circulations 1 minute and 30 seconds; 72 ℃ were extended 10 minutes.Get the size of 1.0% agarose gel electrophoresis evaluation amplified production on the 5 μ l PCR reaction solns, the results are shown in Figure 6.Testing sample amplifies the band about 1039bp, conform to expected results, and adenovirus contrast and blank have no amplified band in the corresponding position, send Beijing to hold up the order-checking of Bioisystech Co., Ltd of section the PCR product that amplifies, sequence correctly conforms to design, shows that goal gene correctly inserts in the adenovirus.

Embodiment 6: the amplification of going down to posterity of recombinant adenovirus rAd5HPV16SmE7E6

Present embodiment is the amplification of going down to posterity of recombinant adenovirus rAd5HPV16SmE7E6, and concrete operations are as follows:

Get among the embodiment 5 and identify that through PCR correct recombinant virus and the about 0.2ml of single spot frozen storing liquid that purifying is good grow to 60%～70% 293 cells with the DMEM mixing postoperative infection that 1ml contains 2% foetal calf serum, hatched 2 hours for 37 ℃, add the DMEM maintenance medium 5ml of 2% foetal calf serum, the typical pathologies such as cellular swelling, circle contracting occur to most 293 cells after, collect sick cell, cell precipitation is resuspended in PBS, after-70 ℃ of freezing three times, increases by 1: 10 virus inoculation.In 293 cells, go down to posterity amplification 5-6 generation, until obtain the recombinant virus (＞10 of required titre ⁸Pfu/ml).

Embodiment 7: the detection of expression of E7E6 fusion rotein in 293 cells that recombinant adenovirus rAd5HPV16SmE7E6 infects

Present embodiment is the detection of expression of E7E6 fusion rotein in 293 cells that infect of recombinant adenovirus rAd5HPV16SmE7E6, and concrete operations are as follows:

At 25cm ²293 cells grow up to about 90% in the Tissue Culture Flask, and with the contamination of 1pfu/cel titre, virus is put into 37 ℃ and cultivated 24-48 hour to the full pathology of cell 37 ℃ of absorption fluid infusion after 1-2 hour.Outwell virus liquid, wash gently twice with PBS, with the cell sleaker cell is scraped, 1000-2000 rev/min, centrifugal 5 minutes, collecting cell.In cell, add 50 μ l, 1 * SDS-PAGE sample loading buffer, mixing, 100 ℃ were heated 3 minutes.Get the SDS-PAGE glue of 5-10 μ l loading (sample comprise recombinant adenovirus poison strain to be measured, Ad5 adenovirus do negative control) in 10% and carry out electrophoresis, then electricity turns sample in nitrocellulose filter.Use respectively anti-HPV16E7 mouse monoclonal antibody and anti-HPV16E6 goat polyclonal antibody (to be Santa Cruz company product, available from Beijing North instrument great waves commerce and trade company limited as first antibody, then use the sheep anti-mouse igg of corresponding horseradish peroxidase-labeled and the anti-sheep IgG of rabbit as second antibody, carry out the exposure tests signal with chemical luminescence reagent kit (being the Pierce product, available from the together positive biotech development company limited of Beijing North).The result shows, recombinant adenovirus dyes Marker 40KD place in advance about protein, be equivalent to the non-Marker about 35KD position and HPV16E7 mouse monoclonal antibody and anti-HPV16E6 goat polyclonal antibody of dying in advance of protein one reaction band is all arranged, and adenovirus is to impinging upon the reactionless band in corresponding position, conform to E7E6 fusion rotein estimated molecular weight, the result is shown in Fig. 7 A and B.

Embodiment 8: the animal immune experiment of recombinant adenovirus rAd5HPV16SmE7E6

Present embodiment detects serum specific antibody and cell immune response for after using recombinant adenovirus rAd5HPV16SmE7E6 of the present invention that mouse is carried out immunity, and has carried out antitumor transplanting and suppressed the tumor growth experiment, and is specific as follows.

(1) serum specific antibody and cell immune response detect behind the recombinant adenovirus rAd5HPV16SmE7E6 immune mouse

The recombinant adenovirus rAd5HPV16SmE7E61 that use is prepared by previous embodiment 6 * 10 ⁷The immune C57BL/6 mouse of pfu intramuscular injection, same way as booster immunization one pins after two weeks, the serum specific antibody detection was carried out in the immunity end in rear 14 days and cellular immunization detects.

A. detection of specific antibody: get serum, (ELISA) carries out the detection of E7 antibody and E6 antibody with euzymelinked immunosorbent assay (ELISA), with the negative contrast of reagent set PBS.

The E7 antibody horizontal that brings out of recombinant adenovirus is lower as a result, and titre is 1: 20, and E6 antibody does not detect.

B. cell immune response detects: extracting spleen cell, carrying out E7 albumen 49-57 position peptide with enzyme linked immunological spot (ELISPOT) method stimulates the number of the effector T cell of the specific secretion IFN-γ that produces to detect.

The result as shown in Figure 8, it is that 329, PBS and control group then do not detect corresponding spot number that the effector T cell that produces secretion of gamma-IFN behind the mouse of HPV16E749-57 peptide immune stimulatory recombinant adenovirus is counted the spot number.Both have obvious difference, illustrate that recombinant adenovirus rAd5HPV16SmE7E6 can bring out the mouse generation for the cellullar immunologic response of the specific T-cells mediation of HPV16E7.

The ELISPOT detection method sees also Miyahira Y, et al.Quantification of antigenspecific CD8+T cell using an ELISPOT assay.J Immunol Methods, 1995,181 (1): 45-54. and Murali-Krishna K, et al.Counting antigen-speific CD8 Tcells:a reevaluation of bystander activation during viral infection.Immunity, 1998,8 (2): 1771-87).Concrete operation step sees also ELISPOT test kit specification sheets (the ELISPOT test kit is U-CyTech company product, available from Shenzhen Da Kewei Bioisystech Co., Ltd).

(2) the antitumor transplantation experiments of recombinant adenovirus rAd5HPV16SmE7E6

The recombinant adenovirus rAd5HPV16SmE7E6 1 * 10 for preparing with previous embodiment 6 ⁷Pfu intramuscular injection immunity C57BL/6 mouse, same way as booster immunization after two weeks, two weeks after immunity finishes are with 5 * 10 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 E6, E7 albumen, presented by John Hopkins professor T.C.Wu of university, document sees reference: Lin K Y, Guarnieri FG, Stavelev-o ' Canrroll KF, et al.Treatment of established tumor with a novelvaccine that enhance major histocompatibility class II presentation of tumorantigen.Cancer Res, 1996,56 (1): 21～26) subcutaneous vaccination mouse inguinal region place, observe the tumor growth situation twice weekly.

The result shows that the mouse after recombinant adenovirus rAd5HPV16SmE7E6 immunity has obvious restraining effect to the growth of tumour as shown in Figure 9, can protect mouse to avoid the TC-1 growth of tumour cell fully and attack.

(3) recombinant adenovirus rAd5HPV16SmE7E6 is to the restraining effect of tumor growth

Use first 5 * 10 ⁴TC-1 tumor cell injection C57BL/6 mouse inguinal region place prepares tumor-bearing mice.The recombinant adenovirus rAd5HPV16SmE7E61 that second day prepares with embodiment 6 * 10 ⁷The above-mentioned tumor-bearing mice of pfu intramuscular injection immunity, same dose booster immunization after 10 days is observed the tumor growth situation, 2 times weekly.

The result shows the growth of the obvious inhibition tumor cell of recombinant adenovirus rAd5HPV16SmE7E6 as shown in figure 10, and mouse TC-1 growth of tumour cell suppresses fully, and protection ratio is 100%.

Sequence table

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

＜120〉HPV 16 type E7E6 antigen-4 fusion protein genes, expression vector, method, cell and purposes

<130>DIC09110130

<160>5

<170>PatentIn version 3.3

<210>1

<211>255

<212>PRT

＜213〉HPV16SmE7E6 fusion rotein

<400>1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu

245 250 255

<210>2

<211>771

<212>DNA

＜213〉artificial sequence

<400>2

atgcacggcg acacccccac cctgcacgag tacatgctgg acctgcagcc cgagaccacc 60

gacctgtacg gctacggcca gctgaacgac agcagcgagg aggaggacga gatcgacggc 120

cccgccggcc aggccgagcc cgacagagcc cactacaaca tcgtgacctt ctgctgcaag 180

tgcgacagca ccctgagact gtgcgtgcag agcacccacg tggacatcag aaccctggag 240

gacctgctga tgggcaccct gggcatcgtg tgccccatct gcagccagaa gccccaccag 300

aagagaaccg ccatgttcca ggacccccag gagagaccca gaaagctgcc ccagctgtgc 360

accgagctgc agaccaccat ccacgacatc atcctggagt gcgtgtactg caagcagcag 420

ctgctgagaa gagaggtgta cgacttcgcc ttcagagacg gctgcatcgt gtacagagac 480

ggcaacccct acgccgtgtg cgacaagtgc ctgaagttct acagcaagat cagcgagtac 540

agacactact gctacagcct gtacggcacc accctggagc agcagtacaa caagcccctg 600

tgcgacctgc tgatcagatg catcaactgc cagaagcccc tgtgccccga ggagaagcag 660

agacacctgg acaagaagca gagattccac aacatcagag gcagatggac cggcagatgc 720

atgagctgct gcagaagcag cagaaccaga agagagaccc agctgtgata a 771

<210>3

<211>3475

<212>DNA

＜213〉artificial sequence

<400>3

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg 420

actctagagg atccccggaa gatctatgca cggcgacacc cccaccctgc acgagtacat 480

gctggacctg cagcccgaga ccaccgacct gtacggctac ggccagctga acgacagcag 540

cgaggaggag gacgagatcg acggccccgc cggccaggcc gagcccgaca gagcccacta 600

caacatcgtg accttctgct gcaagtgcga cagcaccctg agactgtgcg tgcagagcac 660

ccacgtggac atcagaaccc tggaggacct gctgatgggc accctgggca tcgtgtgccc 720

catctgcagc cagaagcccc accagaagag aaccgccatg ttccaggacc cccaggagag 780

acccagaaag ctgccccagc tgtgcaccga gctgcagacc accatccacg acatcatcct 840

ggagtgcgtg tactgcaagc agcagctgct gagaagagag gtgtacgact tcgccttcag 900

agacggctgc atcgtgtaca gagacggcaa cccctacgcc gtgtgcgaca agtgcctgaa 960

gttctacagc aagatcagcg agtacagaca ctactgctac agcctgtacg gcaccaccct 1020

ggagcagcag tacaacaagc ccctgtgcga cctgctgatc agatgcatca actgccagaa 1080

gcccctgtgc cccgaggaga agcagagaca cctggacaag aagcagagat tccacaacat 1140

cagaggcaga tggaccggca gatgcatgag ctgctgcaga agcagcagaa ccagaagaga 1200

gacccagctg tgataaagat cttccgggta ccgagctcga attcgtaatc atggtcatag 1260

ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 1320

ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 1380

tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 1440

cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 1500

ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 1560

ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 1620

gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 1680

gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 1740

taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 1800

accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 1860

tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 1920

cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 1980

agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 2040

gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 2100

gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 2160

tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 2220

acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2280

cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 2340

acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 2400

acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 2460

tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 2520

ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 2580

ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 2640

tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 2700

aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 2760

ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 2820

ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 2880

gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 2940

gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 3000

cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 3060

actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 3120

ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 3180

tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 3240

ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 3300

agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 3360

aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 3420

attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc 3475

<210>4

<211>4690

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_ftature

<222>(454)..(454)

<223>nis a，c，g，or t

<400>4

ttcatcaata atatacctta ttttggattg aagccaatat gataatgagg gggtggagtt 60

tgtgacgtgg cgcggggcgt gggaacgggg cgggtgacgt agtagtgtgg cggaagtgtg 120

atgttgcaag tgtggcggaa cacatgtaag cgacggatgt ggcaaaagtg acgtttttgg 180

tgtgcgccgg tgtacacagg aagtgacaat tttcgcgcgg ttttaggcgg atgttgtagt 240

aaatttgggc gtaaccgagt aagatttggc cattttcgcg ggaaaactga ataagaggaa 300

gtgaaatctg aataattttg tgttactcat agcgcgtaat atttgtctag ggccgcgggg 360

actttgaccg tttacgtgga gactcgccca ggtgtttttc tcaggtgttt tccgcgttcc 420

gggtcaaagt tggcgtttta ttattatagt cagntctaga gatatactga gtcattaggg 480

actttccaat gggttttgcc cagtacataa ggtcaatagg ggtgaatcaa caggaaagtc 540

ccattggagc caagtacact gagtcaatag ggactttcca ttgggttttg cccagtacaa 600

aaggtcaata gggggtgagt caatgggttt ttcccattat tggcacgtac ataaggtcaa 660

taggggtgag tcattgggtt tttccagcca tttaattaaa acgccatgta ctttcccacc 720

attgacgtca atgggctatt gaaactaatg caacgtgacc tttaaacggt actttcccat 780

agctgattaa tgggaaagta ccgttctcga gccaatacac gtcaatggga agtgaaaggg 840

cagccaaaac gtaacaccgc cccggttttc ccctggaaat tccatattgg cactcattct 900

attggctgag ctgcgttcta cgtgggtata agaggcgcga ccagcgtcgg taccgtcgca 960

gtcttcggtc tgaccaccgt agaacgcaga tcgaattccc cgggagatct atgcacggcg 1020

acacccccac cctgcacgag tacatgctgg acctgcagcc cgagaccacc gacctgtacg 1080

gctacggcca gctgaacgac agcagcgagg aggaggacga gatcgacggc cccgccggcc 1140

aggccgagcc cgacagagcc cactacaaca tcgtgacctt ctgctgcaag tgcgacagca 1200

ccctgagact gtgcgtgcag agcacccacg tggacatcag aaccctggag gacctgctga 1260

tgggcaccct gggcatcgtg tgccccatct gcagccagaa gccccaccag aagagaaccg 1320

ccatgttcca ggacccccag gagagaccca gaaagctgcc ccagctgtgc accgagctgc 1380

agaccaccat ccacgacatc atcctggagt gcgtgtactg caagcagcag ctgctgagaa 1440

gagaggtgta cgacttcgcc ttcagagacg gctgcatcgt gtacagagac ggcaacccct 1500

acgccgtgtg cgacaagtgc ctgaagttct acagcaagat cagcgagtac agacactact 1560

gctacagcct gtacggcacc accctggagc agcagtacaa caagcccctg tgcgacctgc 1620

tgatcagatg catcaactgc cagaagcccc tgtgccccga ggagaagcag agacacctgg 1680

acaagaagca gagattccac aacatcagag gcagatggac cggcagatgc atgagctgct 1740

gcagaagcag cagaaccaga agagagaccc agctgtgata aagatctaag cttgagctcg 1800

tcgacttcga gcaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 1860

acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 1920

atcaatgtat cttatcatgt ctggatcgtc tagcatcgaa gatccaataa cttcgtatag 1980

catacattat acgaagttat aagtagcttg gcgtaatcat ggtcatagct gtttcctgtg 2040

tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 2100

gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 2160

ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 2220

ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 2280

gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 2340

tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 2400

aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 2460

aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 2520

ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 2580

tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc 2640

agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 2700

gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 2760

tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 2820

acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 2880

tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 2940

caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 3000

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 3060

aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 3120

ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 3180

agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 3240

atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 3300

cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 3360

aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 3420

cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 3480

aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 3540

ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 3600

gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 3660

ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 3720

tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 3780

tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg 3840

ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 3900

tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 3960

agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 4020

acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 4080

ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 4140

gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg 4200

acattaacct ataaaaatag gcgtatcact ctaggcaaaa tagcaccctc ccgctccaga 4260

acaacataca gcgcttcaca gcggcagcct aacagtcagc cttaccagta aaaaagaaaa 4320

cctattaaaa aaacaccact cgacacggca ccagctcaat cagtcacagt gtaaaaaagg 4380

gccaagtgca gagcgagtat atataggact aaaaaatgac gtaacggtta aagtccacaa 4440

aaaacaccca gaaaaccgca cgcgaaccta cgcccagaaa cgaaagccaa aaaacccaca 4500

acttcctcaa atcgtcactt ccgttttccc acgttacgta acttcccatt ttaagaaaac 4560

tacaattccc aacacataca agttactccg ccctaaaacc tacgtcaccc gccccgttcc 4620

cacgccccgc gccacgtcac aaactccacc ccctcattat catattggct tcaatccaaa 4680

ataaggtata 4690

<210>5

<211>31762

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(454)..(454)

<223>nis a，c，g，ort

<400>5

ttcatcaata atatacctta ttttggattg aagccaatat gataatgagg gggtggagtt 60

tgtgacgtgg cgcggggcgt gggaacgggg cgggtgacgt agtagtgtgg cggaagtgtg 120

atgttgcaag tgtggcggaa cacatgtaag cgacggatgt ggcaaaagtg acgtttttgg 180

tgtgcgccgg tgtacacagg aagtgacaat tttcgcgcgg ttttaggcgg atgttgtagt 240

aaatttgggc gtaaccgagt aagatttggc cattttcgcg ggaaaactga ataagaggaa 300

gtgaaatctg aataattttg tgttactcat agcgcgtaat atttgtctag ggccgcgggg 360

actttgaccg tttacgtgga gactcgccca ggtgtttttc tcaggtgttt tccgcgttcc 420

gggtcaaagt tggcgtttta ttattatagt cagntctaga gatatactga gtcattaggg 480

actttccaat gggttttgcc cagtacataa ggtcaatagg ggtgaatcaa caggaaagtc 540

ccattggagc caagtacact gagtcaatag ggactttcca ttgggttttg cccagtacaa 600

aaggtcaata gggggtgagt caatgggttt ttcccattat tggcacgtac ataaggtcaa 660

taggggtgag tcattgggtt tttccagcca tttaattaaa acgccatgta ctttcccacc 720

attgacgtca atgggctatt gaaactaatg caacgtgacc tttaaacggt actttcccat 780

agctgattaa tgggaaagta ccgttctcga gccaatacac gtcaatggga agtgaaaggg 840

cagccaaaac gtaacaccgc cccggttttc ccctggaaat tccatattgg cactcattct 900

attggctgag ctgcgttcta cgtgggtata agaggcgcga ccagcgtcgg taccgtcgca 960

gtcttcggtc tgaccaccgt agaacgcaga tcgaattccc cgggagatct atgcacggcg 1020

acacccccac cctgcacgag tacatgctgg acctgcagcc cgagaccacc gacctgtacg 1080

gctacggcca gctgaacgac agcagcgagg aggaggacga gatcgacggc cccgccggcc 1140

aggccgagcc cgacagagcc cactacaaca tcgtgacctt ctgctgcaag tgcgacagca 1200

ccctgagact gtgcgtgcag agcacccacg tggacatcag aaccctggag gacctgctga 1260

tgggcaccct gggcatcgtg tgccccatct gcagccagaa gccccaccag aagagaaccg 1320

ccatgttcca ggacccccag gagagaccca gaaagctgcc ccagctgtgc accgagctgc 1380

agaccaccat ccacgacatc atcctggagt gcgtgtactg caagcagcag ctgctgagaa 1440

gagaggtgta cgacttcgcc ttcagagacg gctgcatcgt gtacagagac ggcaacccct 1500

acgccgtgtg cgacaagtgc ctgaagttct acagcaagat cagcgagtac agacactact 1560

gctacagcct gtacggcacc accctggagc agcagtacaa caagcccctg tgcgacctgc 1620

tgatcagatg catcaactgc cagaagcccc tgtgccccga ggagaagcag agacacctgg 1680

acaagaagca gagattccac aacatcagag gcagatggac cggcagatgc atgagctgct 1740

gcagaagcag cagaaccaga agagagaccc agctgtgata aagatctaag cttgagctcg 1800

tcgacttcga gcaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 1860

acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 1920

atcaatgtat cttatcatgt ctggatcgtc tagcatcgaa gatccaataa cttcgtatag 1980

catacattat acgaagttat aagtactgaa ttcggatctg ggcgtggtta agggtgggaa 2040

agaatatata aggtgggggt cttatgtagt tttgtatctg ttttgcagca gccgccgccg 2100

ccatgagcac caactcgttt gatggaagca ttgtgagctc atatttgaca acgcgcatgc 2160

ccccatgggc cggggtgcgt cagaatgtga tgggctccag cattgatggt cgccccgtcc 2220

tgcccgcaaa ctctactacc ttgacctacg agaccgtgtc tggaacgccg ttggagactg 2280

cagcctccgc cgccgcttca gccgctgcag ccaccgcccg cgggattgtg actgactttg 2340

ctttcctgag cccgcttgca agcagtgcag cttcccgttc atccgcccgc gatgacaagt 2400

tgacggctct tttggcacaa ttggattctt tgacccggga acttaatgtc gtttctcagc 2460

agctgttgga tctgcgccag caggtttctg ccctgaaggc ttcctcccct cccaatgcgg 2520

tttaaaacat aaataaaaaa ccagactctg tttggatttg gatcaagcaa gtgtcttgct 2580

gtctttattt aggggttttg cgcgcgcggt aggcccggga ccagcggtct cggtcgttga 2640

gggtcctgtg tattttttcc aggacgtggt aaaggtgact ctggatgttc agatacatgg 2700

gcataagccc gtctctgggg tggaggtagc accactgcag agcttcatgc tgcggggtgg 2760

tgttgtagat gatccagtcg tagcaggagc gctgggcgtg gtgcctaaaa atgtctttca 2820

gtagcaagct gattgccagg ggcaggccct tggtgtaagt gtttacaaag cggttaagct 2880

gggatgggtg catacgtggg gatatgagat gcatcttgga ctgtattttt aggttggcta 2940

tgttcccagc catatccctc cggggattca tgttgtgcag aaccaccagc acagtgtatc 3000

cggtgcactt gggaaatttg tcatgtagct tagaaggaaa tgcgtggaag aacttggaga 3060

cgcccttgtg acctccaaga ttttccatgc attcgtccat aatgatggca atgggcccac 3120

gggcggcggc ctgggcgaag atatttctgg gatcactaac gtcatagttg tgttccagga 3180

tgagatcgtc ataggccatt tttacaaagc gcgggcggag ggtgccagac tgcggtataa 3240

tggttccatc cggcccaggg gcgtagttac cctcacagat ttgcatttcc cacgctttga 3300

gttcagatgg ggggatcatg tctacctgcg gggcgatgaa gaaaacggtt tccggggtag 3360

gggagatcag ctgggaagaa agcaggttcc tgagcagctg cgacttaccg cagccggtgg 3420

gcccgtaaat cacacctatt accgggtgca actggtagtt aagagagctg cagctgccgt 3480

catccctgag caggggggcc acttcgttaa gcatgtccct gactcgcatg ttttccctga 3540

ccaaatccgc cagaaggcgc tcgccgccca gcgatagcag ttcttgcaag gaagcaaagt 3600

ttttcaacgg tttgagaccg tccgccgtag gcatgctttt gagcgtttga ccaagcagtt 3660

ccaggcggtc ccacagctcg gtcacctgct ctacggcatc tcgatccagc atatctcctc 3720

gtttcgcggg ttggggcggc tttcgctgta cggcagtagt cggtgctcgt ccagacgggc 3780

cagggtcatg tctttccacg ggcgcagggt cctcgtcagc gtagtctggg tcacggtgaa 3840

ggggtgcgct ccgggctgcg cgctggccag ggtgcgcttg aggctggtcc tgctggtgct 3900

gaagcgctgc cggtcttcgc cctgcgcgtc ggccaggtag catttgacca tggtgtcata 3960

gtccagcccc tccgcggcgt ggcccttggc gcgcagcttg cccttggagg aggcgccgca 4020

cgaggggcag tgcagacttt tgagggcgta gagcttgggc gcgagaaata ccgattccgg 4080

ggagtaggca tccgcgccgc aggccccgca gacggtctcg cattccacga gccaggtgag 4140

ctctggccgt tcggggtcaa aaaccaggtt tcccccatgc tttttgatgc gtttcttacc 4200

tctggtttcc atgagccggt gtccacgctc ggtgacgaaa aggctgtccg tgtccccgta 4260

tacagacttg agaggcctgt cctcgagcgg tgttccgcgg tcctcctcgt atagaaactc 4320

ggaccactct gagacaaagg ctcgcgtcca ggccagcacg aaggaggcta agtgggaggg 4380

gtagcggtcg ttgtccacta gggggtccac tcgctccagg gtgtgaagac acatgtcgcc 4440

ctcttcggca tcaaggaagg tgattggttt gtaggtgtag gccacgtgac cgggtgttcc 4500

tgaagggggg ctataaaagg gggtgggggc gcgttcgtcc tcactctctt ccgcatcgct 4560

gtctgcgagg gccagctgtt ggggtgagta ctccctctga aaagcgggca tgacttctgc 4620

gctaagattg tcagtttcca aaaacgagga ggatttgata ttcacctggc ccgcggtgat 4680

gcctttgagg gtggccgcat ccatctggtc agaaaagaca atctttttgt tgtcaagctt 4740

ggtggcaaac gacccgtaga gggcgttgga cagcaacttg gcgatggagc gcagggtttg 4800

gtttttgtcg cgatcggcgc gctccttggc cgcgatgttt agctgcacgt attcgcgcgc 4860

aacgcaccgc cattcgggaa agacggtggt gcgctcgtcg ggcaccaggt gcacgcgcca 4920

accgcggttg tgcagggtga caaggtcaac gctggtggct acctctccgc gtaggcgctc 4980

gttggtccag cagaggcggc cgcccttgcg cgagcagaat ggcggtaggg ggtctagctg 5040

cgtctcgtcc ggggggtctg cgtccacggt aaagaccccg ggcagcaggc gcgcgtcgaa 5100

gtagtctatc ttgcatcctt gcaagtctag cgcctgctgc catgcgcggg cggcaagcgc 5160

gcgctcgtat gggttgagtg ggggacccca tggcatgggg tgggtgagcg cggaggcgta 5220

catgccgcaa atgtcgtaaa cgtagagggg ctctctgagt attccaagat atgtagggta 5280

gcatcttcca ccgcggatgc tggcgcgcac gtaatcgtat agttcgtgcg agggagcgag 5340

gaggtcggga ccgaggttgc tacgggcggg ctgctctgct cggaagacta tctgcctgaa 5400

gatggcatgt gagttggatg atatggttgg acgctggaag acgttgaagc tggcgtctgt 5460

gagacctacc gcgtcacgca cgaaggaggc gtaggagtcg cgcagcttgt tgaccagctc 5520

ggcggtgacc tgcacgtcta gggcgcagta gtccagggtt tccttgatga tgtcatactt 5580

atcctgtccc ttttttttcc acagctcgcg gttgaggaca aactcttcgc ggtctttcca 5640

gtactcttgg atcggaaacc cgtcggcctc cgaacggtaa gagcctagca tgtagaactg 5700

gttgacggcc tggtaggcgc agcatccctt ttctacgggt agcgcgtatg cctgcgcggc 5760

cttccggagc gaggtgtggg tgagcgcaaa ggtgtccctg accatgactt tgaggtactg 5820

gtatttgaag tcagtgtcgt cgcatccgcc ctgctcccag agcaaaaagt ccgtgcgctt 5880

tttggaacgc ggatttggca gggcgaaggt gacatcgttg aagagtatct ttcccgcgcg 5940

aggcataaag ttgcgtgtga tgcggaaggg tcccggcacc tcggaacggt tgttaattac 6000

ctgggcggcg agcacgatct cgtcaaagcc gttgatgttg tggcccacaa tgtaaagttc 6060

caagaagcgc gggatgccct tgatggaagg caatttttta agttcctcgt aggtgagctc 6120

ttcaggggag ctgagcccgt gctctgaaag ggcccagtct gcaagatgag ggttggaagc 6180

gacgaatgag ctccacaggt cacgggccat tagcatttgc aggtggtcgc gaaaggtcct 6240

aaactggcga cctatggcca ttttttctgg ggtgatgcag tagaaggtaa gcgggtcttg 6300

ttcccagcgg tcccatccaa ggttcgcggc taggtctcgc gcggcagtca ctagaggctc 6360

atctccgccg aacttcatga ccagcatgaa gggcacgagc tgcttcccaa aggcccccat 6420

ccaagtatag gtctctacat cgtaggtgac aaagagacgc tcggtgcgag gatgcgagcc 6480

gatcgggaag aactggatct cccgccacca attggaggag tggctattga tgtggtgaaa 6540

gtagaagtcc ctgcgacggg ccgaacactc gtgctggctt ttgtaaaaac gtgcgcagta 6600

ctggcagcgg tgcacgggct gtacatcctg cacgaggttg acctgacgac cgcgcacaag 6660

gaagcagagt gggaatttga gcccctcgcc tggcgggttt ggctggtggt cttctacttc 6720

ggctgcttgt ccttgaccgt ctggctgctc gaggggagtt acggtggatc ggaccaccac 6780

gccgcgcgag cccaaagtcc agatgtccgc gcgcggcggt cggagcttga tgacaacatc 6840

gcgcagatgg gagctgtcca tggtctggag ctcccgcggc gtcaggtcag gcgggagctc 6900

ctgcaggttt acctcgcata gacgggtcag ggcgcgggct agatccaggt gatacctaat 6960

ttccaggggc tggttggtgg cggcgtcgat ggcttgcaag aggccgcatc cccgcggcgc 7020

gactacggta ccgcgcggcg ggcggtgggc cgcgggggtg tccttggatg atgcatctaa 7080

aagcggtgac gcgggcgagc ccccggaggt agggggggct ccggacccgc cgggagaggg 7140

ggcaggggca cgtcggcgcc gcgcgcgggc aggagctggt gctgcgcgcg taggttgctg 7200

gcgaacgcga cgacgcggcg gttgatctcc tgaatctggc gcctctgcgt gaagacgacg 7260

ggcccggtga gcttgagcct gaaagagagt tcgacagaat caatttcggt gtcgttgacg 7320

gcggcctggc gcaaaatctc ctgcacgtct cctgagttgt cttgataggc gatctcggcc 7380

atgaactgct cgatctcttc ctcctggaga tctccgcgtc cggctcgctc cacggtggcg 7440

gcgaggtcgt tggaaatgcg ggccatgagc tgcgagaagg cgttgaggcc tccctcgttc 7500

cagacgcggc tgtagaccac gcccccttcg gcatcgcggg cgcgcatgac cacctgcgcg 7560

agattgagct ccacgtgccg ggcgaagacg gcgtagtttc gcaggcgctg aaagaggtag 7620

ttgagggtgg tggcggtgtg ttctgccacg aagaagtaca taacccagcg tcgcaacgtg 7680

gattcgttga tatcccccaa ggcctcaagg cgctccatgg cctcgtagaa gtccacggcg 7740

aagttgaaaa actgggagtt gcgcgccgac acggttaact cctcctccag aagacggatg 7800

agctcggcga cagtgtcgcg cacctcgcgc tcaaaggcta caggggcctc ttcttcttct 7860

tcaatctcct cttccataag ggcctcccct tcttcttctt ctggcggcgg tgggggaggg 7920

gggacacggc ggcgacgacg gcgcaccggg aggcggtcga caaagcgctc gatcatctcc 7980

ccgcggcgac ggcgcatggt ctcggtgacg gcgcggccgt tctcgcgggg gcgcagttgg 8040

aagacgccgc ccgtcatgtc ccggttatgg gttggcgggg ggctgccatg cggcagggat 8100

acggcgctaa cgatgcatct caacaattgt tgtgtaggta ctccgccgcc gagggacctg 8160

agcgagtccg catcgaccgg atcggaaaac ctctcgagaa aggcgtctaa ccagtcacag 8220

tcgcaaggta ggctgagcac cgtggcgggc ggcagcgggc ggcggtcggg gttgtttctg 8280

gcggaggtgc tgctgatgat gtaattaaag taggcggtct tgagacggcg gatggtcgac 8340

agaagcacca tgtccttggg tccggcctgc tgaatgcgca ggcggtcggc catgccccag 8400

gcttcgtttt gacatcggcg caggtctttg tagtagtctt gcatgagcct ttctaccggc 8460

acttcttctt ctccttcctc ttgtcctgca tctcttgcat ctatcgctgc ggcggcggcg 8520

gagtttggcc gtaggtggcg ccctcttcct cccatgcgtg tgaccccgaa gcccctcatc 8580

ggctgaagca gggctaggtc ggcgacaacg cgctcggcta atatggcctg ctgcacctgc 8640

gtgagggtag actggaagtc atccatgtcc acaaagcggt ggtatgcgcc cgtgttgatg 8700

gtgtaagtgc agttggccat aacggaccag ttaacggtct ggtgacccgg ctgcgagagc 8760

tcggtgtacc tgagacgcga gtaagccctc gagtcaaata cgtagtcgtt gcaagtccgc 8820

accaggtact ggtatcccac caaaaagtgc ggcggcggct ggcggtagag gggccagcgt 8880

agggtggccg gggctccggg ggcgagatct tccaacataa ggcgatgata tccgtagatg 8940

tacctggaca tccaggtgat gccggcggcg gtggtggagg cgcgcggaaa gtcgcggacg 9000

cggttccaga tgttgcgcag cggcaaaaag tgctccatgg tcgggacgct ctggccggtc 9060

aggcgcgcgc aatcgttgac gctctaccgt gcaaaaggag agcctgtaag cgggcactct 9120

tccgtggtct ggtggataaa ttcgcaaggg tatcatggcg gacgaccggg gttcgagccc 9180

cgtatccggc cgtccgccgt gatccatgcg gttaccgccc gcgtgtcgaa cccaggtgtg 9240

cgacgtcaga caacggggga gtgctccttt tggcttcctt ccaggcgcgg cggctgctgc 9300

gctagctttt ttggccactg gccgcgcgca gcgtaagcgg ttaggctgga aagcgaaagc 9360

attaagtggc tcgctccctg tagccggagg gttattttcc aagggttgag tcgcgggacc 9420

cccggttcga gtctcggacc ggccggactg cggcgaacgg gggtttgcct ccccgtcatg 9480

caagaccccg cttgcaaatt cctccggaaa cagggacgag cccctttttt gcttttccca 9540

gatgcatccg gtgctgcggc agatgcgccc ccctcctcag cagcggcaag agcaagagca 9600

gcggcagaca tgcagggcac cctcccctcc tcctaccgcg tcaggagggg cgacatccgc 9660

ggttgacgcg gcagcagatg gtgattacga acccccgcgg cgccgggccc ggcactacct 9720

ggacttggag gagggcgagg gcctggcgcg gctaggagcg ccctctcctg agcggtaccc 9780

aagggtgcag ctgaagcgtg atacgcgtga ggcgtacgtg ccgcggcaga acctgtttcg 9840

cgaccgcgag ggagaggagc ccgaggagat gcgggatcga aagttccacg cagggcgcga 9900

gctgcggcat ggcctgaatc gcgagcggtt gctgcgcgag gaggactttg agcccgacgc 9960

gcgaaccggg attagtcccg cgcgcgcaca cgtggcggcc gccgacctgg taaccgcata 10020

cgagcagacg gtgaaccagg agattaactt tcaaaaaagc tttaacaacc acgtgcgtac 10080

gcttgtggcg cgcgaggagg tggctatagg actgatgcat ctgtgggact ttgtaagcgc 10140

gctggagcaa aacccaaata gcaagccgct catggcgcag ctgttcctta tagtgcagca 10200

cagcagggac aacgaggcat tcagggatgc gctgctaaac atagtagagc ccgagggccg 10260

ctggctgctc gatttgataa acatcctgca gagcatagtg gtgcaggagc gcagcttgag 10320

cctggctgac aaggtggccg ccatcaacta ttccatgctt agcctgggca agttttacgc 10380

ccgcaagata taccataccc cttacgttcc catagacaag gaggtaaaga tcgaggggtt 10440

ctacatgcgc atggcgctga aggtgcttac cttgagcgac gacctgggcg tttatcgcaa 10500

cgagcgcatc cacaaggccg tgagcgtgag ccggcggcgc gagctcagcg accgcgagct 10560

gatgcacagc ctgcaaaggg ccctggctgg cacgggcagc ggcgatagag aggccgagtc 10620

ctactttgac gcgggcgctg acctgcgctg ggccccaagc cgacgcgccc tggaggcagc 10680

tggggccgga cctgggctgg cggtggcacc cgcgcgcgct ggcaacgtcg gcggcgtgga 10740

ggaatatgac gaggacgatg agtacgagcc agaggacggc gagtactaag cggtgatgtt 10800

tctgatcaga tgatgcaaga cgcaacggac ccggcggtgc gggcggcgct gcagagccag 10860

ccgtccggcc ttaactccac ggacgactgg cgccaggtca tggaccgcat catgtcgctg 10920

actgcgcgca atcctgacgc gttccggcag cagccgcagg ccaaccggct ctccgcaatt 10980

ctggaagcgg tggtcccggc gcgcgcaaac cccacgcacg agaaggtgct ggcgatcgta 11040

aacgcgctgg ccgaaaacag ggccatccgg cccgacgagg ccggcctggt ctacgacgcg 11100

ctgcttcagc gcgtggctcg ttacaacagc ggcaacgtgc agaccaacct ggaccggctg 11160

gtgggggatg tgcgcgaggc cgtggcgcag cgtgagcgcg cgcagcagca gggcaacctg 11220

ggctccatgg ttgcactaaa cgccttcctg agtacacagc ccgccaacgt gccgcgggga 11280

caggaggact acaccaactt tgtgagcgca ctgcggctaa tggtgactga gacaccgcaa 11340

agtgaggtgt accagtctgg gccagactat tttttccaga ccagtagaca aggcctgcag 11400

accgtaaacc tgagccaggc tttcaaaaac ttgcaggggc tgtggggggt gcgggctccc 11460

acaggcgacc gcgcgaccgt gtctagcttg ctgacgccca actcgcgcct gttgctgctg 11520

ctaatagcgc ccttcacgga cagtggcagc gtgtcccggg acacatacct aggtcacttg 11580

ctgacactgt accgcgaggc cataggtcag gcgcatgtgg acgagcatac tttccaggag 11640

attacaagtg tcagccgcgc gctggggcag gaggacacgg gcagcctgga ggcaacccta 11700

aactacctgc tgaccaaccg gcggcagaag atcccctcgt tgcacagttt aaacagcgag 11760

gaggagcgca ttttgcgcta cgtgcagcag agcgtgagcc ttaacctgat gcgcgacggg 11820

gtaacgccca gcgtggcgct ggacatgacc gcgcgcaaca tggaaccggg catgtatgcc 11880

tcaaaccggc cgtttatcaa ccgcctaatg gactacttgc atcgcgcggc cgccgtgaac 11940

cccgagtatt tcaccaatgc catcttgaac ccgcactggc taccgccccc tggtttctac 12000

accgggggat tcgaggtgcc cgagggtaac gatggattcc tctgggacga catagacgac 12060

agcgtgtttt ccccgcaacc gcagaccctg ctagagttgc aacagcgcga gcaggcagag 12120

gcggcgctgc gaaaggaaag cttccgcagg ccaagcagct tgtccgatct aggcgctgcg 12180

gccccgcggt cagatgctag tagcccattt ccaagcttga tagggtctct taccagcact 12240

cgcaccaccc gcccgcgcct gctgggcgag gaggagtacc taaacaactc gctgctgcag 12300

ccgcagcgcg aaaaaaacct gcctccggca tttcccaaca acgggataga gagcctagtg 12360

gacaagatga gtagatggaa gacgtacgcg caggagcaca gggacgtgcc aggcccgcgc 12420

ccgcccaccc gtcgtcaaag gcacgaccgt cagcggggtc tggtgtggga ggacgatgac 12480

tcggcagacg acagcagcgt cctggatttg ggagggagtg gcaacccgtt tgcgcacctt 12540

cgccccaggc tggggagaat gttttaaaaa aaaaaaagca tgatgcaaaa taaaaaactc 12600

accaaggcca tggcaccgag cgttggtttt cttgtattcc ccttagtatg cggcgcgcgg 12660

cgatgtatga ggaaggtcct cctccctcct acgagagtgt ggtgagcgcg gcgccagtgg 12720

cggcggcgct gggttctccc ttcgatgctc ccctggaccc gccgtttgtg cctccgcggt 12780

acctgcggcc taccgggggg agaaacagca tccgttactc tgagttggca cccctattcg 12840

acaccacccg tgtgtacctg gtggacaaca agtcaacgga tgtggcatcc ctgaactacc 12900

agaacgacca cagcaacttt ctgaccacgg tcattcaaaa caatgactac agcccggggg 12960

aggcaagcac acagaccatc aatcttgacg accggtcgca ctggggcggc gacctgaaaa 13020

ccatcctgca taccaacatg ccaaatgtga acgagttcat gtttaccaat aagtttaagg 13080

cgcgggtgat ggtgtcgcgc ttgcctacta aggacaatca ggtggagctg aaatacgagt 13140

gggtggagtt cacgctgccc gagggcaact actccgagac catgaccata gaccttatga 13200

acaacgcgat cgtggagcac tacttgaaag tgggcagaca gaacggggtt ctggaaagcg 13260

acatcggggt aaagtttgac acccgcaact tcagactggg gtttgacccc gtcactggtc 13320

ttgtcatgcc tggggtatat acaaacgaag ccttccatcc agacatcatt ttgctgccag 13380

gatgcggggt ggacttcacc cacagccgcc tgagcaactt gttgggcatc cgcaagcggc 13440

aacccttcca ggagggcttt aggatcacct acgatgatct ggagggtggt aacattcccg 13500

cactgttgga tgtggacgcc taccaggcga gcttgaaaga tgacaccgaa cagggcgggg 13560

gtggcgcagg cggcagcaac agcagtggca gcggcgcgga agagaactcc aacgcggcag 13620

ccgcggcaat gcagccggtg gaggacatga acgatcatgc cattcgcggc gacacctttg 13680

ccacacgggc tgaggagaag cgcgctgagg ccgaagcagc ggccgaagct gccgcccccg 13740

ctgcgcaacc cgaggtcgag aagcctcaga agaaaccggt gatcaaaccc ctgacagagg 13800

acagcaagaa acgcagttac aacctaataa gcaatgacag caccttcacc cagtaccgca 13860

gctggtacct tgcatacaac tacggcgacc ctcagaccgg aatccgctca tggaccctgc 13920

tttgcactcc tgacgtaacc tgcggctcgg agcaggtcta ctggtcgttg ccagacatga 13980

tgcaagaccc cgtgaccttc cgctccacgc gccagatcag caactttccg gtggtgggcg 14040

ccgagctgtt gcccgtgcac tccaagagct tctacaacga ccaggccgtc tactcccaac 14100

tcatccgcca gtttacctct ctgacccacg tgttcaatcg ctttcccgag aaccagattt 14160

tggcgcgccc gccagccccc accatcacca ccgtcagtga aaacgttcct gctctcacag 14220

atcacgggac gctaccgctg cgcaacagca tcggaggagt ccagcgagtg accattactg 14280

acgccagacg ccgcacctgc ccctacgttt acaaggccct gggcatagtc tcgccgcgcg 14340

tcctatcgag ccgcactttt tgagcaagca tgtccatcct tatatcgccc agcaataaca 14400

caggctgggg cctgcgcttc ccaagcaaga tgtttggcgg ggccaagaag cgctccgacc 14460

aacacccagt gcgcgtgcgc gggcactacc gcgcgccctg gggcgcgcac aaacgcggcc 14520

gcactgggcg caccaccgtc gatgacgcca tcgacgcggt ggtggaggag gcgcgcaact 14580

acacgcccac gccgccacca gtgtccacag tggacgcggc cattcagacc gtggtgcgcg 14640

gagcccggcg ctatgctaaa atgaagagac ggcggaggcg cgtagcacgt cgccaccgcc 14700

gccgacccgg cactgccgcc caacgcgcgg cggcggccct gcttaaccgc gcacgtcgca 14760

ccggccgacg ggcggccatg cgggccgctc gaaggctggc cgcgggtatt gtcactgtgc 14820

cccccaggtc caggcgacga gcggccgccg cagcagccgc ggccattagt gctatgactc 14880

agggtcgcag gggcaacgtg tattgggtgc gcgactcggt tagcggcctg cgcgtgcccg 14940

tgcgcacccg ccccccgcgc aactagattg caagaaaaaa ctacttagac tcgtactgtt 15000

gtatgtatcc agcggcggcg gcgcgcaacg aagctatgtc caagcgcaaa atcaaagaag 15060

agatgctcca ggtcatcgcg ccggagatct atggcccccc gaagaaggaa gagcaggatt 15120

acaagccccg aaagctaaag cgggtcaaaa agaaaaagaa agatgatgat gatgaacttg 15180

acgacgaggt ggaactgctg cacgctaccg cgcccaggcg acgggtacag tggaaaggtc 15240

gacgcgtaaa acgtgttttg cgacccggca ccaccgtagt ctttacgccc ggtgagcgct 15300

ccacccgcac ctacaagcgc gtgtatgatg aggtgtacgg cgacgaggac ctgcttgagc 15360

aggccaacga gcgcctcggg gagtttgcct acggaaagcg gcataaggac atgctggcgt 15420

tgccgctgga cgagggcaac ccaacaccta gcctaaagcc cgtaacactg cagcaggtgc 15480

tgcccgcgct tgcaccgtcc gaagaaaagc gcggcctaaa gcgcgagtct ggtgacttgg 15540

cacccaccgt gcagctgatg gtacccaagc gccagcgact ggaagatgtc ttggaaaaaa 15600

tgaccgtgga acctgggctg gagcccgagg tccgcgtgcg gccaatcaag caggtggcgc 15660

cgggactggg cgtgcagacc gtggacgttc agatacccac taccagtagc accagtattg 15720

ccaccgccac agagggcatg gagacacaaa cgtccccggt tgcctcagcg gtggcggatg 15780

ccgcggtgca ggcggtcgct gcggccgcgt ccaagacctc tacggaggtg caaacggacc 15840

cgtggatgtt tcgcgtttca gccccccggc gcccgcgcgg ttcgaggaag tacggcgccg 15900

ccagcgcgct actgcccgaa tatgccctac atccttccat tgcgcctacc cccggctatc 15960

gtggctacac ctaccgcccc agaagacgag caactacccg acgccgaacc accactggaa 16020

cccgccgccg ccgtcgccgt cgccagcccg tgctggcccc gatttccgtg cgcagggtgg 16080

ctcgcgaagg aggcaggacc ctggtgctgc caacagcgcg ctaccacccc agcatcgttt 16140

aaaagccggt ctttgtggtt cttgcagata tggccctcac ctgccgcctc cgtttcccgg 16200

tgccgggatt ccgaggaaga atgcaccgta ggaggggcat ggccggccac ggcctgacgg 16260

gcggcatgcg tcgtgcgcac caccggcggc ggcgcgcgtc gcaccgtcgc atgcgcggcg 16320

gtatcctgcc cctccttatt ccactgatcg ccgcggcgat tggcgccgtg cccggaattg 16380

catccgtggc cttgcaggcg cagagacact gattaaaaac aagttgcatg tggaaaaatc 16440

aaaataaaaa gtctggactc tcacgctcgc ttggtcctgt aactattttg tagaatggaa 16500

gacatcaact ttgcgtctct ggccccgcga cacggctcgc gcccgttcat gggaaactgg 16560

caagatatcg gcaccagcaa tatgagcggt ggcgccttca gctggggctc gctgtggagc 16620

ggcattaaaa atttcggttc caccgttaag aactatggca gcaaggcctg gaacagcagc 16680

acaggccaga tgctgaggga taagttgaaa gagcaaaatt tccaacaaaa ggtggtagat 16740

ggcctggcct ctggcattag cggggtggtg gacctggcca accaggcagt gcaaaataag 16800

attaacagta agcttgatcc ccgccctccc gtagaggagc ctccaccggc cgtggagaca 16860

gtgtctccag aggggcgtgg cgaaaagcgt ccgcgccccg acagggaaga aactctggtg 16920

acgcaaatag acgagcctcc ctcgtacgag gaggcactaa agcaaggcct gcccaccacc 16980

cgtcccatcg cgcccatggc taccggagtg ctgggccagc acacacccgt aacgctggac 17040

ctgcctcccc ccgccgacac ccagcagaaa cctgtgctgc caggcccgac cgccgttgtt 17100

gtaacccgtc ctagccgcgc gtccctgcgc cgcgccgcca gcggtccgcg atcgttgcgg 17160

cccgtagcca gtggcaactg gcaaagcaca ctgaacagca tcgtgggtct gggggtgcaa 17220

tccctgaagc gccgacgatg cttctgaata gctaacgtgt cgtatgtgtg tcatgtatgc 17280

gtccatgtcg ccgccagagg agctgctgag ccgccgcgcg cccgctttcc aagatggcta 17340

ccccttcgat gatgccgcag tggtcttaca tgcacatctc gggccaggac gcctcggagt 17400

acctgagccc cgggctggtg cagtttgccc gcgccaccga gacgtacttc agcctgaata 17460

acaagtttag aaaccccacg gtggcgccta cgcacgacgt gaccacagac cggtcccagc 17520

gtttgacgct gcggttcatc cctgtggacc gtgaggatac tgcgtactcg tacaaggcgc 17580

ggttcaccct agctgtgggt gataaccgtg tgctggacat ggcttccacg tactttgaca 17640

tccgcggcgt gctggacagg ggccctactt ttaagcccta ctctggcact gcctacaacg 17700

ccctggctcc caagggtgcc ccaaatcctt gcgaatggga tgaagctgct actgctcttg 17760

aaataaacct agaagaagag gacgatgaca acgaagacga agtagacgag caagctgagc 17820

agcaaaaaac tcacgtattt gggcaggcgc cttattctgg tataaatatt acaaaggagg 17880

gtattcaaat aggtgtcgaa ggtcaaacac ctaaatatgc cgataaaaca tttcaacctg 17940

aacctcaaat aggagaatct cagtggtacg aaactgaaat taatcatgca gctgggagag 18000

tccttaaaaa gactacccca atgaaaccat gttacggttc atatgcaaaa cccacaaatg 18060

aaaatggagg gcaaggcatt cttgtaaagc aacaaaatgg aaagctagaa agtcaagtgg 18120

aaatgcaatt tttctcaact actgaggcga ccgcaggcaa tggtgataac ttgactccta 18180

aagtggtatt gtacagtgaa gatgtagata tagaaacccc agacactcat atttcttaca 18240

tgcccactat taaggaaggt aactcacgag aactaatggg ccaacaatct atgcccaaca 18300

ggcctaatta cattgctttt agggacaatt ttattggtct aatgtattac aacagcacgg 18360

gtaatatggg tgttctggcg ggccaagcat cgcagttgaa tgctgttgta gatttgcaag 18420

acagaaacac agagctttca taccagcttt tgcttgattc cattggtgat agaaccaggt 18480

acttttctat gtggaatcag gctgttgaca gctatgatcc agatgttaga attattgaaa 18540

atcatggaac tgaagatgaa cttccaaatt actgctttcc actgggaggt gtgattaata 18600

cagagactct taccaaggta aaacctaaaa caggtcagga aaatggatgg gaaaaagatg 18660

ctacagaatt ttcagataaa aatgaaataa gagttggaaa taattttgcc atggaaatca 18720

atctaaatgc caacctgtgg agaaatttcc tgtactccaa catagcgctg tatttgcccg 18780

acaagctaaa gtacagtcct tccaacgtaa aaatttctga taacccaaac acctacgact 18840

acatgaacaa gcgagtggtg gctcccgggt tagtggactg ctacattaac cttggagcac 18900

gctggtccct tgactatatg gacaacgtca acccatttaa ccaccaccgc aatgctggcc 18960

tgcgctaccg ctcaatgttg ctgggcaatg gtcgctatgt gcccttccac atccaggtgc 19020

ctcagaagtt ctttgccatt aaaaacctcc ttctcctgcc gggctcatac acctacgagt 19080

ggaacttcag gaaggatgtt aacatggttc tgcagagctc cctaggaaat gacctaaggg 19140

ttgacggagc cagcattaag tttgatagca tttgccttta cgccaccttc ttccccatgg 19200

cccacaacac cgcctccacg cttgaggcca tgcttagaaa cgacaccaac gaccagtcct 19260

ttaacgacta tctctccgcc gccaacatgc tctaccctat acccgccaac gctaccaacg 19320

tgcccatatc catcccctcc cgcaactggg cggctttccg cggctgggcc ttcacgcgcc 19380

ttaagactaa ggaaacccca tcactgggct cgggctacga cccttattac acctactctg 19440

gctctatacc ctacctagat ggaacctttt acctcaacca cacctttaag aaggtggcca 19500

ttacctttga ctcttctgtc agctggcctg gcaatgaccg cctgcttacc cccaacgagt 19560

ttgaaattaa gcgctcagtt gacggggagg gttacaacgt tgcccagtgt aacatgacca 19620

aagactggtt cctggtacaa atgctagcta actacaacat tggctaccag ggcttctata 19680

tcccagagag ctacaaggac cgcatgtact ccttctttag aaacttccag cccatgagcc 19740

gtcaggtggt ggatgatact aaatacaagg actaccaaca ggtgggcatc ctacaccaac 19800

acaacaactc tggatttgtt ggctaccttg cccccaccat gcgcgaagga caggcctacc 19860

ctgctaactt cccctatccg cttataggca agaccgcagt tgacagcatt acccagaaaa 19920

agtttctttg cgatcgcacc ctttggcgca tcccattctc cagtaacttt atgtccatgg 19980

gcgcactcac agacctgggc caaaaccttc tctacgccaa ctccgcccac gcgctagaca 20040

tgacttttga ggtggatccc atggacgagc ccacccttct ttatgttttg tttgaagtct 20100

ttgacgtggt ccgtgtgcac cggccgcacc gcggcgtcat cgaaaccgtg tacctgcgca 20160

cgcccttctc ggccggcaac gccacaacat aaagaagcaa gcaacatcaa caacagctgc 20220

cgccatgggc tccagtgagc aggaactgaa agccattgtc aaagatcttg gttgtgggcc 20280

atattttttg ggcacctatg acaagcgctt tccaggcttt gtttctccac acaagctcgc 20340

ctgcgccata gtcaatacgg ccggtcgcga gactgggggc gtacactgga tggcctttgc 20400

ctggaacccg cactcaaaaa catgctacct ctttgagccc tttggctttt ctgaccagcg 20460

actcaagcag gtttaccagt ttgagtacga gtcactcctg cgccgtagcg ccattgcttc 20520

ttcccccgac cgctgtataa cgctggaaaa gtccacccaa agcgtacagg ggcccaactc 20580

ggccgcctgt ggactattct gctgcatgtt tctccacgcc tttgccaact ggccccaaac 20640

tcccatggat cacaacccca ccatgaacct tattaccggg gtacccaact ccatgctcaa 20700

cagtccccag gtacagccca ccctgcgtcg caaccaggaa cagctctaca gcttcctgga 20760

gcgccactcg ccctacttcc gcagccacag tgcgcagatt aggagcgcca cttctttttg 20820

tcacttgaaa aacatgtaaa aataatgtac tagagacact ttcaataaag gcaaatgctt 20880

ttatttgtac actctcgggt gattatttac ccccaccctt gccgtctgcg ccgtttaaaa 20940

atcaaagggg ttctgccgcg catcgctatg cgccactggc agggacacgt tgcgatactg 21000

gtgtttagtg ctccacttaa actcaggcac aaccatccgc ggcagctcgg tgaagttttc 21060

actccacagg ctgcgcacca tcaccaacgc gtttagcagg tcgggcgccg atatcttgaa 21120

gtcgcagttg gggcctccgc cctgcgcgcg cgagttgcga tacacagggt tgcagcactg 21180

gaacactatc agcgccgggt ggtgcacgct ggccagcacg ctcttgtcgg agatcagatc 21240

cgcgtccagg tcctccgcgt tgctcagggc gaacggagtc aactttggta gctgccttcc 21300

caaaaagggc gcgtgcccag gctttgagtt gcactcgcac cgtagtggca tcaaaaggtg 21360

accgtgcccg gtctgggcgt taggatacag cgcctgcata aaagccttga tctgcttaaa 21420

agccacctga gcctttgcgc cttcagagaa gaacatgccg caagacttgc cggaaaactg 21480

attggccgga caggccgcgt cgtgcacgca gcaccttgcg tcggtgttgg agatctgcac 21540

cacatttcgg ccccaccggt tcttcacgat cttggccttg ctagactgct ccttcagcgc 21600

gcgctgcccg ttttcgctcg tcacatccat ttcaatcacg tgctccttat ttatcataat 21660

gcttccgtgt agacacttaa gctcgccttc gatctcagcg cagcggtgca gccacaacgc 21720

gcagcccgtg ggctcgtgat gcttgtaggt cacctctgca aacgactgca ggtacgcctg 21780

caggaatcgc cccatcatcg tcacaaaggt cttgttgctg gtgaaggtca gctgcaaccc 21840

gcggtgctcc tcgttcagcc aggtcttgca tacggccgcc agagcttcca cttggtcagg 21900

cagtagtttg aagttcgcct ttagatcgtt atccacgtgg tacttgtcca tcagcgcgcg 21960

cgcagcctcc atgcccttct cccacgcaga cacgatcggc acactcagcg ggttcatcac 22020

cgtaatttca ctttccgctt cgctgggctc ttcctcttcc tcttgcgtcc gcataccacg 22080

cgccactggg tcgtcttcat tcagccgccg cactgtgcgc ttacctcctt tgccatgctt 22140

gattagcacc ggtgggttgc tgaaacccac catttgtagc gccacatctt ctctttcttc 22200

ctcgctgtcc acgattacct ctggtgatgg cgggcgctcg ggcttgggag aagggcgctt 22260

ctttttcttc ttgggcgcaa tggccaaatc cgccgccgag gtcgatggcc gcgggctggg 22320

tgtgcgcggc accagcgcgt cttgtgatga gtcttcctcg tcctcggact cgatacgccg 22380

cctcatccgc ttttttgggg gcgcccgggg aggcggcggc gacggggacg gggacgacac 22440

gtcctccatg gttgggggac gtcgcgccgc accgcgtccg cgctcggggg tggtttcgcg 22500

ctgctcctct tcccgactgg ccatttcctt ctcctatagg cagaaaaaga tcatggagtc 22560

agtcgagaag aaggacagcc taaccgcccc ctctgagttc gccaccaccg cctccaccga 22620

tgccgccaac gcgcctacca ccttccccgt cgaggcaccc ccgcttgagg aggaggaagt 22680

gattatcgag caggacccag gttttgtaag cgaagacgac gaggaccgct cagtaccaac 22740

agaggataaa aagcaagacc aggacaacgc agaggcaaac gaggaacaag tcgggcgggg 22800

ggacgaaagg catggcgact acctagatgt gggagacgac gtgctgttga agcatctgca 22860

gcgccagtgc gccattatct gcgacgcgtt gcaagagcgc agcgatgtgc ccctcgccat 22920

agcggatgtc agccttgcct acgaacgcca cctattctca ccgcgcgtac cccccaaacg 22980

ccaagaaaac ggcacatgcg agcccaaccc gcgcctcaac ttctaccccg tatttgccgt 23040

gccagaggtg cttgccacct atcacatctt tttccaaaac tgcaagatac ccctatcctg 23100

ccgtgccaac cgcagccgag cggacaagca gctggccttg cggcagggcg ctgtcatacc 23160

tgatatcgcc tcgctcaacg aagtgccaaa aatctttgag ggtcttggac gcgacgagaa 23220

gcgcgcggca aacgctctgc aacaggaaaa cagcgaaaat gaaagtcact ctggagtgtt 23280

ggtggaactc gagggtgaca acgcgcgcct agccgtacta aaacgcagca tcgaggtcac 23340

ccactttgcc tacccggcac ttaacctacc ccccaaggtc atgagcacag tcatgagtga 23400

gctgatcgtg cgccgtgcgc agcccctgga gagggatgca aatttgcaag aacaaacaga 23460

ggagggccta cccgcagttg gcgacgagca gctagcgcgc tggcttcaaa cgcgcgagcc 23520

tgccgacttg gaggagcgac gcaaactaat gatggccgca gtgctcgtta ccgtggagct 23580

tgagtgcatg cagcggttct ttgctgaccc ggagatgcag cgcaagctag aggaaacatt 23640

gcactacacc tttcgacagg gctacgtacg ccaggcctgc aagatctcca acgtggagct 23700

ctgcaacctg gtctcctacc ttggaatttt gcacgaaaac cgccttgggc aaaacgtgct 23760

tcattccacg ctcaagggcg aggcgcgccg cgactacgtc cgcgactgcg tttacttatt 23820

tctatgctac acctggcaga cggccatggg cgtttggcag cagtgcttgg aggagtgcaa 23880

cctcaaggag ctgcagaaac tgctaaagca aaacttgaag gacctatgga cggccttcaa 23940

cgagcgctcc gtggccgcgc acctggcgga catcattttc cccgaacgcc tgcttaaaac 24000

cctgcaacag ggtctgccag acttcaccag tcaaagcatg ttgcagaact ttaggaactt 24060

tatcctagag cgctcaggaa tcttgcccgc cacctgctgt gcacttccta gcgactttgt 24120

gcccattaag taccgcgaat gccctccgcc gctttggggc cactgctacc ttctgcagct 24180

agccaactac cttgcctacc actctgacat aatggaagac gtgagcggtg acggtctact 24240

ggagtgtcac tgtcgctgca acctatgcac cccgcaccgc tccctggttt gcaattcgca 24300

gctgcttaac gaaagtcaaa ttatcggtac ctttgagctg cagggtccct cgcctgacga 24360

aaagtccgcg gctccggggt tgaaactcac tccggggctg tggacgtcgg cttaccttcg 24420

caaatttgta cctgaggact accacgccca cgagattagg ttctacgaag accaatcccg 24480

cccgccaaat gcggagctta ccgcctgcgt cattacccag ggccacattc ttggccaatt 24540

gcaagccatc aacaaagccc gccaagagtt tctgctacga aagggacggg gggtttactt 24600

ggacccccag tccggcgagg agctcaaccc aatccccccg ccgccgcagc cctatcagca 24660

gcagccgcgg gcccttgctt cccaggatgg cacccaaaaa gaagctgcag ctgccgccgc 24720

cacccacgga cgaggaggaa tactgggaca gtcaggcaga ggaggttttg gacgaggagg 24780

aggaggacat gatggaagac tgggagagcc tagacgagga agcttccgag gtcgaagagg 24840

tgtcagacga aacaccgtca ccctcggtcg cattcccctc gccggcgccc cagaaatcgg 24900

caaccggttc cagcatggct acaacctccg ctcctcaggc gccgccggca ctgcccgttc 24960

gccgacccaa ccgtagatgg gacaccactg gaaccagggc cggtaagtcc aagcagccgc 25020

cgccgttagc ccaagagcaa caacagcgcc aaggctaccg ctcatggcgc gggcacaaga 25080

acgccatagt tgcttgcttg caagactgtg ggggcaacat ctccttcgcc cgccgctttc 25140

ttctctacca tcacggcgtg gccttccccc gtaacatcct gcattactac cgtcatctct 25200

acagcccata ctgcaccggc ggcagcggca gcggcagcaa cagcagcggc cacacagaag 25260

caaaggcgac cggatagcaa gactctgaca aagcccaaga aatccacagc ggcggcagca 25320

gcaggaggag gagcgctgcg tctggcgccc aacgaacccg tatcgacccg cgagcttaga 25380

aacaggattt ttcccactct gtatgctata tttcaacaga gcaggggcca agaacaagag 25440

ctgaaaataa aaaacaggtc tctgcgatcc ctcacccgca gctgcctgta tcacaaaagc 25500

gaagatcagc ttcggcgcac gctggaagac gcggaggctc tcttcagtaa atactgcgcg 25560

ctgactctta aggactagtt tcgcgccctt tctcaaattt aagcgcgaaa actacgtcat 25620

ctccagcggc cacacccggc gccagcacct gtcgtcagcg ccattatgag caaggaaatt 25680

cccacgccct acatgtggag ttaccagcca caaatgggac ttgcggctgg agctgcccaa 25740

gactactcaa cccgaataaa ctacatgagc gcgggacccc acatgatatc ccgggtcaac 25800

ggaatccgcg cccaccgaaa ccgaattctc ttggaacagg cggctattac caccacacct 25860

cgtaataacc ttaatccccg tagttggccc gctgccctgg tgtaccagga aagtcccgct 25920

cccaccactg tggtacttcc cagagacgcc caggccgaag ttcagatgac taactcaggg 25980

gcgcagcttg cgggcggctt tcgtcacagg gtgcggtcgc ccgggcaggg tataactcac 26040

ctgacaatca gagggcgagg tattcagctc aacgacgagt cggtgagctc ctcgcttggt 26100

ctccgtccgg acgggacatt tcagatcggc ggcgccggcc gtccttcatt cacgcctcgt 26160

caggcaatcc taactctgca gacctcgtcc tctgagccgc gctctggagg cattggaact 26220

ctgcaattta ttgaggagtt tgtgccatcg gtctacttta accccttctc gggacctccc 26280

ggccactatc cggatcaatt tattcctaac tttgacgcgg taaaggactc ggcggacggc 26340

tacgactgaa tgttaagtgg agaggcagag caactgcgcc tgaaacacct ggtccactgt 26400

cgccgccaca agtgctttgc ccgcgactcc ggtgagtttt gctactttga attgcccgag 26460

gatcatatcg agggcccggc gcacggcgtc cggcttaccg cccagggaga gcttgcccgt 26520

agcctgattc gggagtttac ccagcgcccc ctgctagttg agcgggacag gggaccctgt 26580

gttctcactg tgatttgcaa ctgtcctaac cttggattac atcaagatcc tctagttaat 26640

taactagagt acccggggat cttattccct ttaactaata aaaaaaaata ataaagcatc 26700

acttacttaa aatcagttag caaatttctg tccagtttat tcagcagcac ctccttgccc 26760

tcctcccagc tctggtattg cagcttcctc ctggctgcaa actttctcca caatctaaat 26820

ggaatgtcag tttcctcctg ttcctgtcca tccgcaccca ctatcttcat gttgttgcag 26880

atgaagcgcg caagaccgtc tgaagatacc ttcaaccccg tgtatccata tgacacggaa 26940

accggtcctc caactgtgcc ttttcttact cctccctttg tatcccccaa tgggtttcaa 27000

gagagtcccc ctggggtact ctctttgcgc ctatccgaac ctctagttac ctccaatggc 27060

atgcttgcgc tcaaaatggg caacggcctc tctctggacg aggccggcaa ccttacctcc 27120

caaaatgtaa ccactgtgag cccacctctc aaaaaaacca agtcaaacat aaacctggaa 27180

atatctgcac ccctcacagt tacctcagaa gccctaactg tggctgccgc cgcacctcta 27240

atggtcgcgg gcaacacact caccatgcaa tcacaggccc cgctaaccgt gcacgactcc 27300

aaacttagca ttgccaccca aggacccctc acagtgtcag aaggaaagct agccctgcaa 27360

acatcaggcc ccctcaccac caccgatagc agtaccctta ctatcactgc ctcaccccct 27420

ctaactactg ccactggtag cttgggcatt gacttgaaag agcccattta tacacaaaat 27480

ggaaaactag gactaaagta cggggctcct ttgcatgtaa cagacgacct aaacactttg 27540

accgtagcaa ctggtccagg tgtgactatt aataatactt ccttgcaaac taaagttact 27600

ggagccttgg gttttgattc acaaggcaat atgcaactta atgtagcagg aggactaagg 27660

attgattctc aaaacagacg ccttatactt gatgttagtt atccgtttga tgctcaaaac 27720

caactaaatc taagactagg acagggccct ctttttataa actcagccca caacttggat 27780

attaactaca acaaaggcct ttacttgttt acagcttcaa acaattccaa aaagcttgag 27840

gttaacctaa gcactgccaa ggggttgatg tttgacgcta cagccatagc cattaatgca 27900

ggagatgggc ttgaatttgg ttcacctaat gcaccaaaca caaatcccct caaaacaaaa 27960

attggccatg gcctagaatt tgattcaaac aaggctatgg ttcctaaact aggaactggc 28020

cttagttttg acagcacagg tgccattaca gtaggaaaca aaaataatga taagctaact 28080

ttgtggacca caccagctcc atctcctaac tgtagactaa atgcagagaa agatgctaaa 28140

ctcactttgg tcttaacaaa atgtggcagt caaatacttg ctacagtttc agttttggct 28200

gttaaaggca gtttggctcc aatatctgga acagttcaaa gtgctcatct tattataaga 28260

tttgacgaaa atggagtgct actaaacaat tccttcctgg acccagaata ttggaacttt 28320

agaaatggag atcttactga aggcacagcc tatacaaacg ctgttggatt tatgcctaac 28380

ctatcagctt atccaaaatc tcacggtaaa actgccaaaa gtaacattgt cagtcaagtt 28440

tacttaaacg gagacaaaac taaacctgta acactaacca ttacactaaa cggtacacag 28500

gaaacaggag acacaactcc aagtgcatac tctatgtcat tttcatggga ctggtctggc 28560

cacaactaca ttaatgaaat atttgccaca tcctcttaca ctttttcata cattgcccaa 28620

gaataaagaa tcgtttgtgt tatgtttcaa cgtgtttatt tttcaattgc agaaaatttc 28680

aagtcatttt tcattcagta gtatagcccc accaccacat agcttataca gatcaccgta 28740

ccttaatcaa actcacagaa ccctagtatt caacctgcca cctccctccc aacacacaga 28800

gtacacagtc ctttctcccc ggctggcctt aaaaagcatc atatcatggg taacagacat 28860

attcttaggt gttatattcc acacggtttc ctgtcgagcc aaacgctcat cagtgatatt 28920

aataaactcc ccgggcagct cacttaagtt catgtcgctg tccagctgct gagccacagg 28980

ctgctgtcca acttgcggtt gcttaacggg cggcgaagga gaagtccacg cctacatggg 29040

ggtagagtca taatcgtgca tcaggatagg gcggtggtgc tgcagcagcg cgcgaataaa 29100

ctgctgccgc cgccgctccg tcctgcagga atacaacatg gcagtggtct cctcagcgat 29160

gattcgcacc gcccgcagca taaggcgcct tgtcctccgg gcacagcagc gcaccctgat 29220

ctcacttaaa tcagcacagt aactgcagca cagcaccaca atattgttca aaatcccaca 29280

gtgcaaggcg ctgtatccaa agctcatggc ggggaccaca gaacccacgt ggccatcata 29340

ccacaagcgc aggtagatta agtggcgacc cctcataaac acgctggaca taaacattac 29400

ctcttttggc atgttgtaat tcaccacctc ccggtaccat ataaacctct gattaaacat 29460

ggcgccatcc accaccatcc taaaccagct ggccaaaacc tgcccgccgg ctatacactg 29520

cagggaaccg ggactggaac aatgacagtg gagagcccag gactcgtaac catggatcat 29580

catgctcgtc atgatatcaa tgttggcaca acacaggcac acgtgcatac acttcctcag 29640

gattacaagc tcctcccgcg ttagaaccat atcccaggga acaacccatt cctgaatcag 29700

cgtaaatccc acactgcagg gaagacctcg cacgtaactc acgttgtgca ttgtcaaagt 29760

gttacattcg ggcagcagcg gatgatcctc cagtatggta gcgcgggttt ctgtctcaaa 29820

aggaggtaga cgatccctac tgtacggagt gcgccgagac aaccgagatc gtgttggtcg 29880

tagtgtcatg ccaaatggaa cgccggacgt agtcatattt cctgaagcaa aaccaggtgc 29940

gggcgtgaca aacagatctg cgtctccggt ctcgccgctt agatcgctct gtgtagtagt 30000

tgtagtatat ccactctctc aaagcatcca ggcgccccct ggcttcgggt tctatgtaaa 30060

ctccttcatg cgccgctgcc ctgataacat ccaccaccgc agaataagcc acacccagcc 30120

aacctacaca ttcgttctgc gagtcacaca cgggaggagc gggaagagct ggaagaacca 30180

tgtttttttt tttattccaa aagattatcc aaaacctcaa aatgaagatc tattaagtga 30240

acgcgctccc ctccggtggc gtggtcaaac tctacagcca aagaacagat aatggcattt 30300

gtaagatgtt gcacaatggc ttccaaaagg caaacggccc tcacgtccaa gtggacgtaa 30360

aggctaaacc cttcagggtg aatctcctct ataaacattc cagcaccttc aaccatgccc 30420

aaataattct catctcgcca ccttctcaat atatctctaa gcaaatcccg aatattaagt 30480

ccggccattg taaaaatctg ctccagagcg ccctccacct tcagcctcaa gcagcgaatc 30540

atgattgcaa aaattcaggt tcctcacaga cctgtataag attcaaaagc ggaacattaa 30600

caaaaatacc gcgatcccgt aggtcccttc gcagggccag ctgaacataa tcgtgcaggt 30660

ctgcacggac cagcgcggcc acttccccgc caggaacctt gacaaaagaa cccacactga 30720

ttatgacacg catactcgga gctatgctaa ccagcgtagc cccgatgtaa gctttgttgc 30780

atgggcggcg atataaaatg caaggtgctg ctcaaaaaat caggcaaagc ctcgcgcaaa 30840

aaagaaagca catcgtagtc atgctcatgc agataaaggc aggtaagctc cggaaccacc 30900

acagaaaaag acaccatttt tctctcaaac atgtctgcgg gtttctgcat aaacacaaaa 30960

taaaataaca aaaaaacatt taaacattag aagcctgtct tacaacagga aaaacaaccc 31020

ttataagcat aagacggact acggccatgc cggcgtgacc gtaaaaaaac tggtcaccgt 31080

gattaaaaag caccaccgac agctcctcgg tcatgtccgg agtcataatg taagactcgg 31140

taaacacatc aggttgattc atcggtcagt gctaaaaagc gaccgaaata gcccggggga 31200

atacataccc gcaggcgtag agacaacatt acagccccca taggaggtat aacaaaatta 31260

ataggagaga aaaacacata aacacctgaa aaaccctcct gcctaggcaa aatagcaccc 31320

tcccgctcca gaacaacata cagcgcttca cagcggcagc ctaacagtca gccttaccag 31380

taaaaaagaa aacctattaa aaaaacacca ctcgacacgg caccagctca atcagtcaca 31440

gtgtaaaaaa gggccaagtg cagagcgagt atatatagga ctaaaaaatg acgtaacggt 31500

taaagtccac aaaaaacacc cagaaaaccg cacgcgaacc tacgcccaga aacgaaagcc 31560

aaaaaaccca caacttcctc aaatcgtcac ttccgttttc ccacgttacg taacttccca 31620

ttttaagaaa actacaattc ccaacacata caagttactc cgccctaaaa cctacgtcac 31680

ccgccccgtt cccacgcccc gcgccacgtc acaaactcca ccccctcatt atcatattgg 31740

cttcaatcca aaataaggta ta 31762

Claims (11)

1. a dna sequence dna that is used for encoding human papilloma virus (HPV) 16 type E7E6 fusion roteins is characterized in that the nucleotides sequence of described dna sequence dna is classified the sequence shown in the SEQ.ID.NO.2 as.

2. the expression vector for human papillomavirus (HPV) the 16 type E7E6 fusion roteins of expressing dna sequence encoding claimed in claim 1 is characterized in that described expression vector contains dna sequence dna claimed in claim 1.

3. expression vector according to claim 2 is characterized in that, described expression vector is recombinant adenovirus.

4. expression vector according to claim 3 is characterized in that, described recombinant adenovirus is replication-defective adenoviral, and it is obtained by adenovirus shuttle plasmid and adenovirus skeleton plasmid cotransfection adenovirus packaging cell.

5. expression vector according to claim 4, it is characterized in that, described adenovirus shuttle plasmid is selected from pDC316, pDC311, pDC312 and pDC315, and it is by making up between the Bgl II site that dna sequence dna claimed in claim 1 is inserted into described adenovirus shuttle plasmid; And/or described adenovirus skeleton plasmid is pBHGlox Δ E1,3Cre.

6. each described expression vector in 5 according to claim 3 is characterized in that described recombinant adenovirus packing cell is selected from human embryo kidney 293 cells, human embryo kidney (HEK) 293T cell, human embryo kidney (HEK) 293FT cell and human embryo kidney (HEK) 293F cell.

Dna sequence dna claimed in claim 1 infect for the preparation for the treatment of human papillomavirus (HPV) 16 types and the relative disease medicine in application.

In the claim 2 to 6 each described expression vector infect for the preparation for the treatment of human papillomavirus (HPV) 16 types and the relative disease medicine in application.

9. according to claim 7 or 8 described application, it is characterized in that described relative disease is cervical cancer.

10. according to claim 7 or 8 described application, it is characterized in that described medicine is vaccine.

11. a vaccine that is used for the treatment of cervical cancer is characterized in that, described vaccine comprises each described expression vector in dna sequence dna claimed in claim 1 or the claim 2 to 6.

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