CN1171636C - Hepatitis B DNA vaccine - Google Patents

Abstract

The present invention relates to a hepatitis b DNA vaccine having a therapeutic effect, which belongs to the technical field of biomedicine. The vaccine comprises a hepatitis B virus envelope protein gene preS1-preS2-HBsAg and a fusion gene of a core protein gene HBc, wherein the 5' tail end of the fusion gene is led with a section of coding signal peptide and a nucleotide sequence of universal helper T lymphocyte (TH) epitope. The vaccine also comprises a human interleukin-12 gene hIL-12. The vaccine comprises a plurality of elements capable of powerfully stimulating the immune response of human T-cells, the immune response of the T-cells of chronic hepatitis b patients can be activated, and the present invention has the therapeutic effect on chronic hepatitis b.

Description

A kind of hepatitis B DNA vaccine

The present invention relates to the biological medicine technology field, is the hepatitis B DNA vaccine with therapeutical effect.

Hepatitis B virus (HBV) is very harmful to human health, at present the whole world person 3.5 hundred million that has the chronic HBV infection approximately.With of the use of reorganization hbs antigen, can effectively prevent HBV to infect, but, still lack the medicine that definite curative effect is arranged at present for numerous infected chronic patients as vaccine.Dna vaccination is also referred to as gene vaccine or nucleic acid vaccine, it is the eukaryotic expression recombination plasmid of expressing microbial antigen, the experiment of some animal and human's bodies shows that dna vaccination can induce strong cellullar immunologic response, so the development of hepatitis B DNA vaccine has become a research focus of domestic and international microorganism and field of immunology.

The structural protein gene of HBV comprises envelope protein gene preS1, preS2, HBsAg and core protein gene HBc, the corresponding therewith hepatitis B DNA vaccine of report mainly contains four kinds at present, the gene that comprises is HBsAg expression, preS1-preS2-HBsAg, preS2-HBsAg or HBc respectively, all only express a kind of antigen in envelope protein or the core protein, thereby can not induce comprehensive anti-HBV immunne response.The dna vaccination inoculation mice of HBsAg expression, chimpanzee all can induce antibody and cellullar immunologic response, and U.S. PowerJect vaccine company report can induce low-level antibody and T cellullar immunologic response with this dna vaccination inoculation normal adults.It is poorer than the dna vaccination that comprises HBsAg that the dna vaccination that comprises preS1-preS2-HBsAg induces the efficient of immunne response.The dna vaccination that comprises preS2-HBsAg shows that through the toy experiment its immune effect is similar to the dna vaccination that comprises HBsAg.The dna vaccination that comprises HBc also only can just induce immunne response on one's body at toy preferably.

Concerning the chronic HBV infection person, owing to continue to produce HBV antigen in its body, body immune system has formed immune tolerance state for HBV antigen, thereby inoculation the antigenic immunoreactivity of HBV that dna vaccination produced is significantly less than the normal person, so above-mentioned hepatitis B DNA vaccine is for the immune tolerance state that reverses chronic hepatitis B patient with to activate the effect possibility of HBV T cells with antigenic specificity immunne response relatively poor.The research of the chimpanzee that infects HBV being inoculated the dna vaccination of HBsAg expression has also proved this point.

The purpose of this invention is to provide the dna vaccination that a kind of immune efficacy is strong, chronic hepatitis B patient had therapeutical effect.

Above-mentioned hepatitis B DNA vaccine has a common deficiency, promptly only expresses a kind of antigen in envelope protein and the core protein, if its associating just can effectively be strengthened cellullar immunologic response intensity.Cytokine interleukin element-12 (IL-12) plays a crucial role in inducing HBV antigenic specificity cellullar immunologic response, can further promote inducing of cellullar immunologic response so add the IL-12 gene.Some can greatly promote the immunogenicity of vaccine with the bonded helper T cell epi-position of human leucocyte antigen (HLA) HLAII molecule.Given this, we are with the HBV envelope protein gene, the common constructed dna vaccine of core protein gene and people IL-12 gene, and hepatitis B virus peplos-core protein antigen gene modified, promptly introduced the nucleotide sequence ATGGGAATGCAGGTGCAGATCCAGAGCCTGTTTCTGCTCCTCCTGTGGGTGCCCGG GTCCAGAGGAGCCAAGTTCGTGGCTGCCTGGACCCTGAAGGCTGCCGCTGGATCC of one section coding human normal immunoglobulin's IgG kappa chain signal peptide and universal helper T lymphocyte epi-position PADRE gene ST, in the further activation of chronic hepatitis b patient HBV envelope protein and core protein are shown as the T lymphocyte of hypoergia at its 5 ' end.

The hepatitis B DNA vaccine that the present invention makes up contains signal peptide gene and universal helper T lymphocyte epitope gene ST, HBV envelope protein gene preS1-preS2-HBsAg (abbreviating S1S2S as), HBV core protein gene HBc and people IL-12 gene hIL-12.The transcriptional control structure of this dna vaccination comprises the transcription stop signals gene order SV40 polyA of human cytomegalic inclusion disease virus early promoter and enhancer PCMV, one section synthetic intron intron, internal ribosome entry site sequence IVS-IRES and monkey disease poison SV40.

Description of drawings:

Fig. 1 is the structural representation of hepatitis B DNA vaccine of the present invention.

Coding HBV envelope protein and the recombiant plasmid pUC-S1S2S of core protein and the structural representation of pUC-HBc that Fig. 2 makes up for this laboratory.

Fig. 3 is the preparation flow figure of the dna fragmentation ST of coding human normal immunoglobulin's IgG kappa chain signal sequence and universal helper T lymphocyte epi-position.

Fig. 4 is the structure flow chart of recombiant plasmid pcDNA-ST.

Fig. 5 is the structure flow chart of recombiant plasmid pcDNA-ST-HBc.

Fig. 6 is the structure flow chart of recombiant plasmid pcDNA-ST-S1S2S-HBc.

Fig. 7 is the structure flow chart of recombiant plasmid pCI-hIL12.

Fig. 8 is the structure flow chart of recombiant plasmid pCI-IRES-hIL12.

Fig. 9 is the structure flow chart of recombiant plasmid pCI-ST-HBV-hIL12.

The present invention adopts overlapping extension PCR, PCR, enzyme to cut, connect equimolecular biological experimental method constructed dna vaccine, Can be with reference to U.S.'s Pehanorm Brooker work " molecular cloning " second edition.

Now in conjunction with the accompanying drawings and embodiments the present invention is described in detail.

The basic material that makes up this dna vaccination comprises: the recombiant plasmid pUC-S1S2S and the pUC-HBc (this laboratory makes up, and structure is seen accompanying drawing 3) that contain HBV adr hypotype total length envelope glycoprotein gene and core protein antigen gene, the recombiant plasmid pORF-hIL12 (American I nvivogen company product) of coding human interleukin-12p35 and two subunit cDNA of p40, the recombiant plasmid pIRES-neo (U.S. Clontech company product) that contains the internal ribosome entry site IVS-IRES (being called for short IRES) of encephalomyocarditis virus, eukaryon expression plasmid pcDNA3.1 (American I nvitrogen company product), eukaryon expression plasmid pCI-neo (U.S. Promega company product).

One, the preparation of ST gene:

1. synthetic four sections single stranded oligonucleotide fragment P1, P2, P3, P4, sequence is respectively:

P1：AAGCTTGCCGCCACCATGGGAATGCAGGTGCAGATCCAGAGC

P2：GACCCGGGCACCCACAGGAGGAGCAGAAACAGGCTCTGGATCTGCAC

P3：GTGGGTGCCCGGGTCCAGAGGAGCCAAGTTCGTGGCTGCCTGGACC

P4：GGATCCAGCGGCAGCCTTCAGGGTCCAGGCAGCCAC

2. splicing single stranded oligonucleotide fragment is a double chain DNA fragment ST (see figure 4):

(1) each component below a centrifuge tube adds, (used PCR reagent comprises mononucleotide mixture dNTP, 10 * PCR buffer and pfu archaeal dna polymerase with overlap extension PCR method amplifying doulbe-chain dna fragmentation, be Canadian Songon biological engineering company limited product, down together).

P1 (20μM) 2μl

P2 (20μM) 2μl

P3 (20μM) 2μl

P4 (20μM) 2μl

dNTP (10mM) 2μl

10 * PCR buffer, 2.5 μ l

Pfu archaeal dna polymerase (2U/ μ l) 1 μ l

Sterilization distilled water 6.5 μ l

With the said components mixing, on the PCR thermal cycler, carry out following reaction with pipettor: 94 ℃ of degeneration in 2 minutes, again 94 ℃ 50 seconds, 55 ℃ 50 seconds, 70 ℃ were carried out 10 circulations in 40 seconds.

(2) get above-mentioned reactant liquor 2 μ l, add following reacted constituent:

P1 (20μM) 1μl

P4 (20μM) 1μl

dNTP (10mM) 2μl

10 * PCR buffer, 5 μ l

Pfu archaeal dna polymerase (2U/ μ l) 1 μ l

Sterilization distilled water 37 μ l

With the said components mixing, on the PCR thermal cycler, carry out following reaction with pipettor: 94 ℃ of degeneration in 2 minutes, again 94 ℃ 50 seconds, 55 ℃ 50 seconds, 70 ℃ were carried out 35 circulations in 40 seconds, 70 ℃ were finished reaction in 10 minutes.The PCR product is carried out the agarose gel electrophoresis purification, the dna fragmentation ST of obtain encoding human normal immunoglobulin IgG kappa chain signal peptide and universal helper T lymphocyte epi-position PADRE.

Two, the structure (see figure 5) of recombiant plasmid pcDNA-ST:

Plasmid vector pcDNA3.1 restriction endonuclease HindIII, BamHI enzyme action (U.S. Promega company product, down together), product carries out the agarose gel electrophoresis purification.Simultaneously, with PCR product ST restriction endonuclease HindIII, the BamHI enzyme action of above-mentioned purification, product carries out the agarose gel electrophoresis purification and reclaims.Then two kinds of purified products are connected with T4 phage DNA ligase (U.S. Promega company product, down together), obtain recombiant plasmid pcDNA-ST.

The component of DNA coupled reaction comprises:

pcDNA3.1/HindIII+BamHI(50ng/μl) 1μl

PCR product S T/HindIII+BamHI (20ng/ μ l) 5 μ l

5 * dna ligase buffer, 2 μ l

T4 phage DNA ligase (2U/ μ l) 1 μ l

Sterilization distilled water 1 μ l

With pipettor with the said components mixing, 16 ℃ of water-baths 16 hours.The transformed competence colibacillus e. coli tg1 extracts plasmid and carries out the enzyme action evaluation, is defined as pcDNA-ST.

Three, the pcr amplification of HBV total length envelope glycoprotein antigen preS1-preS2-HBsAg (abbreviating S1S2S as) and core protein antigen HBc gene:

Synthetic two S1S2S amplimers are respectively Y1 and Y2:

Y1：GGATCCACCATGGGAGGTTGGTCTTC

Y2：GGATCCAATGTATACCCAAAGAC

Each component below a centrifuge tube adds:

Plasmid pUC-S1S2S (20ng/ μ l) 1 μ l

Y1(10μM) 1μl

Y2(10μM) 1μl

dNTP(10mM) 2μl

10 * PCR buffer, 5 μ l

Pfu archaeal dna polymerase (2U/ μ l) 1 μ l

Sterilization distilled water 39 μ l

With the said components mixing, on the PCR thermal cycler, carry out following reaction with pipettor: 94 ℃ of degeneration in 2 minutes, again 94 ℃ 50 seconds, 58 ℃ 50 seconds, 70 ℃ were carried out 30 circulations in 80 seconds, 70 ℃ were finished reaction after 10 minutes.The S1S2S PCR product of gained is carried out the agarose gel electrophoresis purification and is reclaimed.

Synthetic two HBc amplimers are respectively Y3 and Y4:

Y3：GGATCCATGGACATTGACCCGTA

Y4：GAATTCTAACATTGAGATTCCCGA

Each component below a centrifuge tube adds:

Plasmid pUC-HBc (10ng/ μ l) 1 μ l

Y3(10μM) 1μl

Y4(10μM) 1μl

dNTP(10mM) 2μl

10 * PCR buffer, 5 μ l

Pfu archaeal dna polymerase (2U/ μ l) 1 μ l

Sterilization distilled water 39 μ l

With the said components mixing, on the PCR thermal cycler, carry out following reaction with pipettor: 94 ℃ of degeneration in 2 minutes, again 94 ℃ 50 seconds, 62 ℃ 50 seconds, 70 ℃ were carried out 30 circulations in 50 seconds, 70 ℃ were finished reaction in 10 minutes.The HBc PCR product of gained is carried out the agarose gel electrophoresis purification and is reclaimed.

Four, the structure of HBV fusion antigen gene expression plasmid pcDNA-ST-S1S2S-HBc (seeing Fig. 6, Fig. 7):

1. plasmid pcDNA-ST is with restriction endonuclease BamHI, EcoRI enzyme action, simultaneously with the HBc pcr amplification product with restriction endonuclease BamHI, EcoRI enzyme action, pcDNA-ST with enzyme action is connected with HBc again, recombiant plasmid is identified through restriction endonuclease BamHI, EcoRI enzyme action, is defined as pcDNA-ST-HBc.

2. plasmid pcDNA-ST-HBc removes the phosphoric acid of DNA 5 ' end again with restriction endonuclease BamHI enzyme action with calf intestinal alkaline phosphatase; Simultaneously with the S1S2S pcr amplification product with restriction endonuclease BamHI enzyme action, two kinds of enzyme action products are connected obtain recombiant plasmid again, identify with restriction endonuclease BamHI, XhoI enzyme action, determine that this plasmid is pcDNA-ST-S1S2S-HBc.

Five, the amplification of human interleukin-11 2 (hIL12) gene:

Synthetic two hIL12 amplimers are respectively Y5 and Y6:

Y5：GCGGCCGCCACCATGGGTCACCAGCAG

Y6：GCGGCCGCCTCGAGTTAGGAAGCATTCAG

Each component below a centrifuge tube adds:

Plasmid pORF-hIL12 (25ng/ μ l) 3 μ l

Y5(10μM) 2μl

Y6(10μM) 2μl

dNTP(10mM) 2μl

10 * PCR buffer, 5 μ l

Pfu archaeal dna polymerase (2U/ μ l) 1 μ l

Sterilization distilled water 35 μ l

With the said components mixing, on the PCR thermal cycler, carry out following reaction with pipettor: 94 ℃ of degeneration in 2 minutes, again 94 ℃ 50 seconds, 58 ℃ 50 seconds, 70 ℃ were carried out 30 circulations in 1 minute 40 seconds, 70 ℃ were finished reaction in 10 minutes.The PCR product is carried out the agarose gel electrophoresis purification, obtains the hIL12 gene.

Six, the structure (see figure 8) of recombiant plasmid pCI-hIL12:

Plasmid pCI-neo removes the terminal phosphoric acid of DNA5 ' with calf intestinal alkaline phosphatase (BCIP) again with restriction endonuclease NotI enzyme action; Simultaneously with the hIL12 pcr amplification product with restriction endonuclease NotI enzyme action, two kinds of enzyme action products are connected obtain recombiant plasmid again, identify with restriction endonuclease NotI, XbaI enzyme cutting, be defined as pCI-hIL12.

Seven, the structure of pHBV/hIL12 (seeing Fig. 9, Figure 10):

1. plasmid pCI-hIL12 carries out agarose gel electrophoresis and reclaims dna fragmentation with restriction endonuclease EcoRI, SmaI enzyme action; Simultaneously with plasmid pIRES-neo with restriction endonuclease EcoRI, SmaI enzyme action, carry out agarose gel electrophoresis and reclaim the IRES fragment cut out; Again the IRES fragment is connected with the pCI-hIL12 fragment and obtains recombiant plasmid pCI-IRES-hIL12.

2. recombiant plasmid pCI-IRES-hIL12 carries out agarose gel electrophoresis and reclaims the pCI-IRES-hIL12 fragment with restriction endonuclease NheI, EcoRI enzyme action; Simultaneously with plasmid pcDNA-ST-S1S2S-HBc with restriction endonuclease NheI, EcoRI enzyme action, carry out agarose gel electrophoresis and reclaim the ST-S1S2S-HBc fragment cut out; Again the ST-S1S2S-HBc fragment is connected with dna ligase with the pCI-IRES-hIL12 fragment, obtains recombiant plasmid pCI-ST-S1S2S-HBc-IRES-hIL12, abbreviate pHBV/hIL12 as.

Eight, the ST-S1S2S-HBc-IRES-hIL12 among the recombiant plasmid pHBV/hIL12 is carried out sequencing, result and expectation in full accord.The complete nucleotide sequence of pHBV/hIL12 is seen Fig. 2, and wherein setting-out partly is the nucleotide sequence of ST-S1S2S-HBc-IRES-hIL12.

The structure of pHBV/hIL12 can be brought into play pivotal role for the person's that overcomes the chronic HBV infection immune tolerance state, clinical important potential using value is arranged.

(1) with approach such as intramuscular injection, intradermal injection inoculations pHBV/hIL12, be immunization method easily, can induce many antigenic T cellullar immunologic responses of anti-HBV and neutralizing antibody.On the basis of direct injection, unite use with the immunological adjuvant that approved is used at human body, can further improve the inductive immunne response of pHBV/hIL12.

(2) pHBV/hIL12 is transformed the endotrophic antibacterial of attenuation, as attenuation bacillus bifidus, lactobacillus, attenuation people Salmonella typhi, attenuation Listerella, attenuation enteroinvasive E etc., the inoculation of approach such as available oral, nasal mucosa, because antibacterial can be carried plasmid immunoadjuvant function into human body cell and antibacterial itself, this represents a kind of more effective and immunotherapy method easily.

(3) based on synthetic fusion gene ST-S1S2S-HBc, carry out suitable modification, transformation again, can carry out the specific aim immunization therapy the hepatitis B patient in different course of disease stage.

(4) above immunization method overcomes the reactionless and hypoergia of part crowd counterweight group hepatitis B vaccine may obviously surpassing present recombinant hepatitis B vaccine aspect the prevention HBV infection, these crowds great using value is arranged.

(5) the HBV antigen fusion gene is carried out protokaryon or eukaryotic expression; as recombiant vaccine, its immanoprotection action will and can be induced strong T cellullar immunologic response at chronic hepatitis B patient above present already used recombinant hepatitis B vaccine with the recombinant antigen of purification.

Zoopery PRELIMINARY RESULTS of the present invention:

We have compared pHBV/hIL12, pCI-HBs (expressing HBV surface antigen HBsAg), at the immune effect of mice, the result is as follows for pCI-HBc (expressing HBV core protein HBc):

Laboratory animal is female Balb/C mice, 6 ages in week, 30, be divided into three groups, 10 every group, respectively intramuscular injection plasmid pHBV/hIL12, pCI-HBs,, each 100 μ g, every three weeks once, inject altogether three times.Get mice serum after the per injection 2 weeks and detect antibody, 4 weeks of last injection back are detected mouse boosting cell to the antigenic breeder reaction of reorganization HBV.

After injecting for the first time, injection pHBV/hIL12, pCI-HBs organize mice serum, and anti--HBs antibody male rotary rate is respectively 5/10,3/10, and injection pHBV/hIL12, pCI-HBc organize mice serum, and anti--HBc antibody male rotary rate is respectively 8/10,5/10.

After injecting for the second time, injection pHBV/hIL12, pCI-HBs organize mice serum, and anti--HBs antibody male rotary rate is respectively 9/10,6/10, and injection pHBV/hIL12, pCI-HBc organize mice serum, and anti--HBc antibody male rotary rate is respectively 10/10,7/10.

After injecting for the third time, injection pHBV/hIL12, pCI-HBs organize mice serum, and anti--HBs antibody male rotary rate is respectively 10/10,7/10, and injection pHBV/hIL12, pCI-HBc organize mice serum, and anti--HBc antibody male rotary rate is respectively 10/10,8/10.

Last injection 4 weeks of back stimulate mouse boosting cell with reorganization HBV envelope protein HBsAg, and the stimulation index of injection pHBV/hIL12, pCI-HBs group mice is respectively 5.4,3.1; HBc stimulates mouse boosting cell with reorganization HBV core protein albumen, and the stimulation index of injection pHBV/hIL12, pCI-HBc group mice is respectively 6.3,3.8.

Above result shows that the dna vaccination pHBV/hIL12 that we design, make up induces the efficient of body fluid and cellullar immunologic response all obviously to be better than the dna vaccination of simple expression HBV envelope protein or core protein.

PHBV/hIL12 comprises the element of the strong stimulation of a plurality of energy human T-cell immunne response, and the T cellullar immunologic response of energy activation of chronic hepatitis B patient has therapeutical effect to chronic hepatitis B.

Advantage of the present invention and good effect:

Hepatitis B DNA vaccine of the present invention comprises the element of the strong stimulation of a plurality of energy human T-cell immune response, can activate slow The T cellullar immunologic response of property hepatitis B patient has therapeutic action to chronic hepatitis B.

The nucleotides sequence tabulation

TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATAC

GTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATT

GACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACT

TACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT

AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACA

TCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCA

GTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATACCATGGTGATGCGG

TTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTC

AATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGT

TGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCA

CTAGAAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGA

ACTTAAGCTGCAGTGACTCTCTTAAGGTAGCCTTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAG

GTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTTTGCGTTTCTGATAGG

CACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTT

AAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCCTCGAGAATTCACGCGTGGTACCTCTAGA GTCGACCC

GGGAAGCTTGCCGCCACCATGGGAATGCAGGTGCAGATCCAGAGCCTGTTTCTGCTCCTCCTGTGGGTGCCCGGG

TCCAGAGGAGCCAAGTTCGTGGCTGCCTGGACCCTGAAGGCTGCCGCTGGATCCACCATGGGAGGTTGGTCTTCC

AAACCTCGACAAGGCATGGGGACGAATCTTTCTGTTCCCAATCCTCTGGGATTCTTTCCCGATCACCAGTTGGAC

CCTGCGTTCGGAGCCAACTCAAACAATCCAGATTGGGACTTCAACCCCAACAAGGATCACTGGCCAGAGGCAAAT

CAGGTAGGAGCGGGAGCATTCGGGCCAGGGTTCACCCCACCACACGGCGGTCTTTTGGGGTGGAGCCCTCAGGCT

CAGGGCATATTGACAACAGTGCCAGTAGCACCTCCTCCTGCCTCCACCAATCGGCAGTCAGGAAGACAGCCTACT

CCCATCTCTCCACCTCTAAGAGACAGTCATCCTCAGGCCATGCAGTGGAACTCCACAACATTCCACCAAGCTCTG

CTAGACCCCAGAGTGAGGGGCCTATACTTTCCTGCTGGTGGCTCCAGTTCCGGAACAGTAAACCCTGTTCCGACT

ACTGCCTCACCCATATCGTCAATCTTCTCGAGGACTGGGGACCCTGCACCGAACATGGAGAACACAACATCAGGA

TTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTA

GACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCACCCACGTGTCTTGGCCAAAATTCGCAGTCCCCAACC

TCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGTTATCGTTGGATGTGTCTGCGGCGTTTTATCATA

TTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTTGCCCGTTTGT

CCTCTACTTCCAAGAACATCAACTACCAGCACGGGACCATGCAAGACCTGCACGATTCCTGCTCAAGGAACCTCT

ATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTTGTATTCCCATCCCATCATCTTGGGCT

TTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGG

TTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAAC

ATCTTGAGTCCCTTTTTACCTCTATTACCAATTTTCTTTTGTCTTTGGGTATACATTGGATCCACCATGATGGAC

ATTGACCCGTATAAAGAATTTGGAGCTTCTGTGGAGTTACTCTCTTTTTTGCCTTCTGACTTCTTTCCTTCTATT

CGAGATCTCCTTGACACCGCCTCAGCTCTGTATCGGGAGGCCTTAGAGTCTCCGGAACATTGCTCACCTCACCAT

ACCGCACTCAGGCAAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTGGCCACCTGGGTGGGAAGTAATTTGGAA

GACCCAGCATCCAGGGAATTAGTAGTCAGCTATGTCAATGTTAATATGGGCCTAAAAATCAGACAACTACTGTGG

TTTCACATTTCCTGTCTTACTTTTGGAAGAGAAACTGTTCTTGAGTATTTGGTGTCTTTTGGAGTGTGGATTCGC

ACTCCTCCTGCTTACAGACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTAGACGACGA

GGACGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTCAA

TCTCGGGAATCTCAATGTTAGGAATTCAGTGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTAATTCGCTG

TCTGCGAGGGCCGGCTGTTGGGGTGAGTACTCCCTCTCAAAAGCGGGCATGACTTCTGCGCTAAGATTGTCAGTT

TCCAAAAACGAGGAGGATTTGATATTCACCTGGCCCGCGGTGATGCCTTTGAGGGTGGCCGCGTCCATCTGGTCA

GAAAAGACAATCTTTTTGTTGTCAAGCTTGAGGTGTGGCAGGCTTGAGATCTGGCCATACACTTGAGTGACAATG

ACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTCCAACTGCAGGTCGATCGAGCATGCATCTAGG

GCGGCCAATTCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGT

GTTTGTCTATATGTGATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTT

CTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGC

AGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGG

CGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGT

TGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTAGTCAACAAGGGGCTGAAGGATGCCCA

GAAGGTACCCCATTGTATGGGAATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAA

AAAAGCTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAAGCTTGCCACAACCC

CGGGGCCACCATGGGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGGC

CATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGGT

CCTCACCTGTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGAGGTCTTAGGCTCTGG

CAAAACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGGAGGCGAGGTTCT

AAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGACCAGAAAGA

ACCCAAAAATAAGACCTTTCTAAGATGCGAGGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGAC

AATCAGTACTGATTTGACATTCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGC

TGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAG

TGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAA

CTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGCTGAAGCCATTAAA

GAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGTACTCCACATTCCTACTTCTCCCTGAC

ATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCAC

GGTCATCTGCCGCAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGCGAATG

GGCATCTGTGCCCTGCAGTGTTCCTGGAGTAGGGGTACCTGGGGTGGGCGCCAGAAACCTCCCCGTGGCCACTCC

AGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGC

CAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGATTGATCATGAAGATATCACAAAAGATAAAACCAG

CACAGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCAT

AACTAATGGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGA

CTCGAAGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCT

AGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAA

ATCCTCCCTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAGAATTCG

GGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCTAAAAAGCGAGGTCCCTCCAAACCGTTGTCAT

TTTTATAAAACTTTGAAATGAGGAAACTTTGATAGGATGTGGATTAAGAACTAGGGAGGGGGAAAGAAGGATGGG

ACTATTACATCCACATGATACCTCTGATCAAGTATTTTTGACATTTACTGTGGATAAATTGTTTTTAAGTTTTCA

TGAATGAATTGCTAAGAAGGGGGGAATTCTTTTGCTTTTTACCCTCGACTAGCGGCCGCTTCCCTTTAGTGAGGG

TTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAA

TGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAAC

AACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTAC

AAATGTGGTAAAATCCGATAAGGATCGATCCGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCC

AACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTAC

GCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCAC

GTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCT

CGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTT

GACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTA

TTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAA

CGCGAATTTTAACAAAATATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATT

TCACACCGCATACGCGGATCTGCGCAGCACCATGGCCTGAAATAACCTCTGAAAGAGGAACTTGGTTAGGTACCT

TCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCA

GAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAA

GTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTC

CGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGG

CTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGATTCTTCTGA

CACAACAGTCTCGAACTTAAGGCTAGAGCCACCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCT

TGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTG

TCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCA

GCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGG

GACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCC

ATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACAT

CGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGG

CTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGC

GATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTG

GCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGC

TTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTC

TGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGATGGCCGCAATAAAATA

TCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGATAGCGATAAGGATCCGCGTATGGTGCACT

CTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGA

CGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTT

TCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATA

ATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAA

ATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT

ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCA

GAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAAC

AGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGT

GGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG

GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCGATA

AGCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTG

CACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAAGCGGAGCTGAATGAAGCCATACCAAACGACGAG

CGTGACACCACGATGCCTGTAGCAATGGGAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT

TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCT

GGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGAT

GGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATC

GCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGAT

TTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAA

CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTG

CGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCA

ACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTA

GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCC

AGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGA

ACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTA

TGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG

CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAG

CGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTC

CTGGCCTTTTGCTGGCCTTTTGCTCACATGGCTCGACAGATCT

Claims (2)

1. hepatitis B DNA vaccine, contain hepatitis b virus hbv envelope protein gene preS1-preS2-HBsAg, it is characterized in that also containing HBV core protein gene HBc, human interleukin-12 gene hIL-12, HBV envelope protein gene and core protein gene are formed a fusion gene preS1-preS2-HBsAg-HBc, 5 ' the end of fusion gene preS1-preS2-HBsAg-HBc has been introduced the nucleotide sequence ATGGGAATGCAGGTGCAGATCCAGAGCCTGTTTCTGCTCCTCCTGTGGGTGCCCGG GTCCAGAGGAGCCAAGTTCGTGGCTGCCTGGACCCTGAAGGCTGCCGCTGGATCC of one section coded signal peptide and universal helper T lymphocyte epi-position, and the nucleotide sequence of dna vaccination is as follows:

TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTA

TCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTA

ATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCC

TGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA

TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCC

TATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTA

CATCTACGTATTAGTCATCGCTATACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCA

CGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGT

CGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTC

GTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTG

ACACAACAGTCTCGAACTTAAGCTGCAGTGACTCTCTTAAGGTAGCCTTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTA

AGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTTTGCGTTTCTGAT

AGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAG

GCTAGAGTACTTAATACGACTCACTATAGGCTAGCCTCGAGAATTCACGCGTGGTACCTCTAGAGTCGACCCGGGAAGCTT

GCCGCCACCATGGGAATGCAGGTGCAGATCCAGAGCCTGTTTCTGCTCCTCCTGTGGGTGCCCGGGTCCAGAGGAGCCAAG

TTCGTGGCTGCCTGGACCCTGAAGGCTGCCGCTGGATCCACCATGGGAGGTTGGTCTTCCAAACCTCGACAAGGCATGGGG

ACGAATCTTTCTGTTCCCAATCCTCTGGGATTCTTTCCCGATCACCAGTTGGACCCTGCGTTCGGAGCCAACTCAAACAAT

CCAGATTGGGACTTCAACCCCAACAAGGATCACTGGCCAGAGGCAAATCAGGTAGGAGCGGGAGCATTCGGGCCAGGGTTC

ACCCCACCACACGGCGGTCTTTTGGGGTGGAGCCCTCAGGCTCAGGGCATATTGACAACAGTGCCAGTAGCACCTCCTCCT

GCCTCCACCAATCGGCAGTCAGGAAGACAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTCATCCTCAGGCCATGCAG

TGGAACTCCACAACATTCCACCAAGCTCTGCTAGACCCCAGAGTGAGGGGCCTATACTTTCCTGCTGGTGGCTCCAGTTCC

GGAACAGTAAACCCTGTTCCGACTACTGCCTCACCCATATCGTCAATCTTCTCGAGGACTGGGGACCCTGCACCGAACATG

GAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATA

CCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCACCCACGTGTCTTGGCCAAAATTCGCAGTCC

CCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGTTATCGTTGGATGTGTCTGCGGCGTTTTATCATA

TTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTTGCCCGTTTGTCCTCTA

CTTCCAAGAACATCAACTACCAGCACGGGACCATGCAAGACCTGCACGATTCCTGCTCAAGGAACCTCTATGTTTCCCTCT

TGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAGATTCCTATGG

GAGTGGGCCTCAGTCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTT

TGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTTTACCTCTATTACCA

ATTTTCTTTTGTCTTTGGGTATACATTGGATCCACCATGATGGACATTGACCCGTATAAAGAATTTGGAGCTTCTGTGGAG

TTACTCTCTTTTTTGCCTTCTGACTTCTTTCCTTCTATTCGAGATCTCCTTGACACCGCCTCAGCTCTGTATCGGGAGGCC

TTAGAGTCTCCGGAACATTGCTCACCTCACCATACCGCACTCAGGCAAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTG

GCCACCTGGGTGGGAAGTAATTTGGAAGACCCAGCATCCAGGGAATTAGTAGTCAGCTATGTCAATGTTAATATGGGCCTA

AAAATCAGACAACTACTGTGGTTTCACATTTCCTGTCTTACTTTTGGAAGAGAAACTGTTCTTGAGTATTTGGTGTCTTTT

GGAGTGTGGATTCGCACTCCTCCTGCTTACAGACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTT

AGACGACGAGGACGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCT

CAATCTCGGGAATCTCAATGTTAGGAATTCAGTGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTAATTCGCTGTCT

GCGAGGGCCGGCTGTTGGGGTGAGTACTCCCTCTCAAAAGCGGGCATGACTTCTGCGCTAAGATTGTCAGTTTCCAAAAAC

GAGGAGGATTTGATATTCACCTGGCCCGCGGTGATGCCTTTGAGGGTGGCCGCGTCCATCTGGTCAGAAAAGACAATCTTT

TTGTTGTCAAGCTTGAGGTGTGGCAGGCTTGAGATCTGGCCATACACTTGAGTGACAATGACATCCACTTTGCCTTTCTCT

CCACAGGTGTCCACTCCCAGGTCCAACTGCAGGTCGATCGAGCATGCATCTAGGGCGGCCAATTCGCCCCTCTCCCTCCCC

CCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGTGTTTGTCTATATGTGATTTTCCACCATATTGCC

GTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAA

AGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGAC

CCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAA

GGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTAGTCAACAA

GGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGAATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTT

AGTCGAGGTTAAAAAAGCTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAAGCTTGCCA

CAACCCCGGGGCCACCATGGGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGGC

CATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGGTCCTCAC

CTGTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGAGGTCTTAGGCTCTGGCAAAACCCTGAC

CATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCT

GCTTCACAAAAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAG

ATGCGAGGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAGTACTGATTTGACATTCAGTGTCAA

AAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAA

CAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCAT

GGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACC

CAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGTACTCC

ACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGTCTTCACGGA

CAAGACCTCAGCCACGGTCATCTGCCGCAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTG

GAGCGAATGGGCATCTGTGCCCTGCAGTGTTCCTGGAGTAGGGGTACCTGGGGTGGGCGCCAGAAACCTCCCCGTGGCCAC

TCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCCAG

ACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGATTGATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGA

GGCCTGTTTACCATTGGAATTAACCAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTG

CCTGGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTCGAAGATGTACCAGGTGGA

GTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTTATTGA

TGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCGGATTTTTATAAAAC

TAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGC

TTCCTAAAAAGCGAGGTCCCTCCAAACCGTTGTCATTTTTATAAAACTTTGAAATGAGGAAACTTTGATAGGATGTGGATT

AAGAACTAGGGAGGGGGAAAGAAGGATGGGACTATTACATCCACATGATACCTCTGATCAAGTATTTTTGACATTTACTGT

GGATAAATTGTTTTTAAGTTTTCATGAATGAATTGCTAAGAAGGGGGGAATTCTTTTGCTTTTTACCCTCGACTAGCGGCC

GCTTCCCTTTAGTGAGGGTTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATG

CAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT

AACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTAC

AAATGTGGTAAAATCCGATAAGGATCGATCCGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGT

TGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGAC

CGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCG

TCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGG

TGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGG

ACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGC

CTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCTGATG

CGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGCGGATCTGCGCAGCACCATGGCCTGAAATAACCT

CTGAAAGAGGAACTTGGTTAGGTACCTTCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGT

CCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCT

CCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGC

CCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCT

CGGCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGATTCTTCTGACAC

AACAGTCTCGAACTTAAGGCTAGAGCCACCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAG

AGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGC

CCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCC

ACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCG

GGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACG

CTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTT

GTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCC

GACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTC

ATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGC

GGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTT

GACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGATGGCCGCAAT

AAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGATAGCGATAAGGATCCGCGTATGGTGCACT

CTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCT

TGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCA

CCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACG

TCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTC

ATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTT

ATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAG

TTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT

CCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGC

CGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA

GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAG

CTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCA

AACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTA

GCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC

TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCC

TCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC

TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT

AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCA

GACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCA

CCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAG

ATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT

CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCG

GATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGA

TACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTC

GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGA

CTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTC

CTGGCCTTTTGCTGGCCTTTTGCTCACATGGCTCGACAGATCT。

2. the application of the described hepatitis B DNA vaccine of claim 1 in preparation treatment hepatitis B medicament.

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