CN102233136B - Recombinant plasmid vaccine for treating hepatitis B and composition thereof - Google Patents

Abstract

The invention relates to a recombinant plasmid DNA (deoxyribonucleic acid) vaccine which simultaneously carries a hepatitis B surface antigen protein-coding gene and a hepatitis B core antigen protein-coding gene. The constructed recombinant plasmid DNA vaccine comprises two target protein expression cassettes, wherein one target protein expression cassette is an optimized target protein expression cassette, and the two genes are separated by one target protein expression cassette. The invention also relates to a double plasmid DNA vaccine which comprises the recombinant plasmid DNA vaccine provided by the invention and a recombinant interleukin 12 adjuvant plasmid. The recombinant plasmid DNA vaccine and the composition thereof constructed by the invention can be used for preparing medicaments for preventing and treating hepatitis B.

Description

A kind of recombiant plasmid vaccine and compositions thereof that is used for the treatment of hepatitis B

Technical field

The present invention relates to a kind of recombinant plasmid DNA vaccine and compositions thereof for the treatment of hepatitis B diseases, belong to biomedical sector.

Background technology

Hepatitis B serious harm human health, there is no the specific treatment means at present, and the main cause of hepatitis B chronicity is that after hepatitis B virus (HBV) infects, body lacks lasting specific cellular immunity and humoral immunization.

The HBV coat protein is that large protein LS (S1S2S antigen is arranged; by S2-S gene code before front S1-), in albumen MS (S2S antigen; by front S2-S gene code), small protein S (S antigen; by the S gene code) three kinds of molecular compositions; wherein, the preS 2 antigen molecular weight is little, and antigenicity is strong; relate to the absorption of hepatitis B virus to host cell, thereby should be more effective for inoculator's protective effect containing the vaccine of preS 2 antigen.

HBcAg is by hepatitis B virogene C district gene code, from the 2nd the initial translation of ATG, containing 183-185 amino acid polypeptide, molecular weight is about 21KD, its C end is rich in arginine and multiple protein enzyme action site, the ability in conjunction with RNA is arranged, and be assembled into the hepatitis B core granule with it closely related.The HBcAg granule consists of a plurality of core protein subunits, the granular structure that is the regular dodecahedron symmetry, single core heart protein subunit (comprising 183-185 amino acid polypeptide chain) first forms homodimer (Homodimer), and dimer is further assembled and formed the HBcAg granule.

Recombinant plasmid DNA vaccine be exogenous gene cloning to carrier for expression of eukaryon, then recombiant plasmid is injected directly in animal body, exogenous gene is in vivo expressed, produce the immune system of antigenic activation body, cause immunoreation.With traditional vaccine, compare, recombinant plasmid DNA vaccine can not only effectively be induced humoral immunization, can also the inducing cell immunity, can doublely do prevention and therapeutic vaccine, and the DNA plasmid vaccine is demonstrating application prospect widely aspect the treatment of numerous disease and prevention.

Mancini (Mancini M, Hadchouel M, Davis HL, et al.DNA-mediated immunizationin a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.ProNatl Acad Sci USA.1996, the recombiant plasmid immunity HBV transgenic mice of application coding preS2/S HBV envelope protein such as 93:12496-12501), just can remove the HBsAg in the transgenic mice circulation through an intramuscular injection, and the virus replication in can the long term inhibition hepatocyte, the potential advantages of DNA vaccination at the treatment HBV chronic carrier have been confirmed.(the Mancini-Bourgine M such as Mancini-Bourgine M, Fontaine H, Scott-Algara D, et al.Induction or expansion of T-cell responsese by a hepatitis B DanAvaccine administered to chronic HBV carriers.Hepatology.2004,40:874-882) the recombiant plasmid immunity 10 routine HBV chronic carriers of application coding preS2/S HBV envelope protein, this 10 routine carrier was not accepted any antiviral therapy.Found that, vaccine can tolerate well, the T lymphproliferation response that the PBMC generation antigenic specificity of 2 routine carriers is arranged, and the T cell number of HBV specific secretion IFN-γ has raising by a relatively large margin after immunity, have the serum HBV DNA level of 5 routine carriers to descend, but this decline is only temporary.This time research has also confirmed the safety of HBV DNA vaccination and can in part HBV chronic carrier body, induce special T cellullar immunologic response.

HBcAg is as dominant antigen, in the research of Hepatitis B virus vaccine, also be widely applied, (the Veremeiko TA such as Veremeiko TA, Lebedev LR, Chikaev NA, et al.Humoral immune response ofBalb/c mice immunized with chimer HBcAg proteins carrying the epitopes of surfacehepatic B virus protein.Vopr virusol, 2007, 52:40-45) HBV coat protein epi-position is inserted into to HBcAg, successfully built the HBcAg-HBsAg fusion rotein, and with this fusion protein immunization Balb/cxiaoshi, produced high titre for inserting epi-position HBsAg and carrier protein HBcAg antibody.Zhang Wei etc. (Zhang Wei, understand living good fortune, Sun Shuhui, etc. hepatitis B virus core antigen DNA vaccination inducing mouse antigenic specificity CD8 ⁺t kinetics of cells and DYNAMIC DISTRIBUTION. Chinese Journal of Immunology .2004,20 (6): 379-383) studied the immunization of rHBcAg eukaryon expression plasmid pHBc144 to the C57BL/6 mice, found that, antigenic specificity CD8 after the pHBc144 face ⁺the T cell increases gradually, at initial immunity, first immunne response peak, after this antigenic specificity CD8 within the 14th day, occur ⁺the T cell quantity is set up to descend and is entered the immunological memory phase and maintained maintenance level in 1 year.

Thymosin, GSF, IL-2, IL-4, IL-12 or IFN-γ etc. are Chang Zuowei immunological adjuvant or reinforcing agent, wherein using thymosin, GSF, IL-2 or IFN-γ as adjuvant, with the HBV vaccine, combine and use existing research.IL-12 has multiple biological activity, is the strongest N K cell-stimulating factor, can promote CD4 ⁺the Th0 cell differentiation is the Th1 cell, and the inferior suitable dosage IL-2 of tunable induces LAK (activated killer cells) cytoactive.As important immune modulatory molecules, IL-12 is the topmost cytokine of inducing Th1 type immunne response known to so far, and substance is as the research of the DNA vaccination of antibacterial, parasite, fungus and viral infection associated diseases and tumor.

Chinese patent CN1316518A discloses a kind of eukaryotic expression recombinant plasmid pS2S, contain cell in plasmid and shoot down stimulating factor, and the recombiant vaccine plasmid is combined to use with the adjuvant plasmid of reforming containing IL-2 and IFN-γ, find that restructuring adjuvant plasmid can improve immunity and the therapeutical effect of recombiant vaccine plasmid.This project has entered the clinical research stage at present.

During by hepatitis B surface antigen and hepatitis B virus core antigen joint mapping recombiant vaccine plasmid, present stage normally utilizes HBcAg can be assembled into the characteristics of the nutty structure that diameter is 27nm, the HBcAg of directly usining is fused to HBcAg as expression vector by hepatitis B virus surface antigen, structure obtains amalgamation protein vaccine, perhaps increase HBV S and C gene and be spliced into SC and merge fragment, will merge fragment and insert the recombinant dna plasmid that builds amalgamation and expression HBsAg/HBcAg in carrier for expression of eukaryon.But this mode builds the recombinant dna plasmid obtained, when obtaining wherein a kind of antibody titer, the antibody titer that another kind of antigen produces is very weak, descends obviously.

Although the preliminary clinical test results of DNA vaccination is satisfactory, but its immune effect does not also reach desirable level, therefore how to strengthen immune effect and become the key of DNA vaccination research, aspect the immune effect that especially how guarantees how to make DNA vaccination can simultaneously obtain stronger HBsAg and HBcAg.

Summary of the invention

The object of the present invention is to provide a kind of recombinant plasmid DNA vaccine through optimizing that is used for the treatment of hepatitis B, described recombinant plasmid DNA vaccine carries hepatitis b surface antigen protein encoding gene and hepatitis B virus core antigen protein coding gene simultaneously.

In the present invention, hepatitis b surface antigen protein encoding gene in described recombinant plasmid DNA vaccine and hepatitis B virus core antigen protein coding gene are not to form the form of fusion rotein encoding gene by direct connection, but by optimizing plasmid, two destination protein expression cassettes of design on recombiant plasmid, two encoding genes are spaced.

In the present invention, HBV just face antigen protein encoding gene can be S1S2S protein coding gene, the white encoding gene of S2S albumen or S protein coding gene, in preferred hepatitis-B virus cytomembrane, just, described hepatitis B virus core antigen protein coding gene is the encoding gene of coding hepatitis B virus core Core albumen to Protein S 2S protein coding gene.

In the present invention, described hepatitis B virus S2S protein coding gene, its nucleotide sequence and Seq ID No.5 have at least 70% homology, preferred at least 80% homology, at least 90% homology more preferably, most preferably be with natural hepatitis B virus in Protein S 2S encoding gene nucleotide sequence identical.

In the present invention, described hepatitis B virus Core protein coding gene, its nucleotide sequence and Seq ID No.11 have at least 70% homology, preferred at least 80% homology, more preferably at least 90% homology, most preferably be with natural hepatitis B virus core Core protein coding gene nucleotide sequence identical.

In the present invention, the recombinant plasmid DNA vaccine of structure also comprises escherichia coli replication origin ColE1, blocks that resistant gene sequence Kan and two destination protein expression cassettes; Wherein the expression cassette in upstream position is for optimizing the destination protein expression cassette, and it comprises enhancer element Enhancer, transcriptional regulatory element intron Intron1 and the exon Exon1 that derives from SV40, the posttranscriptional regulatory element HPRE that derives from HBV.

In the present invention, the recombinant plasmid DNA vaccine built can be to express the recombiant vaccine plasmid pS2S-C of hepatitis B core Core albumen (C) in optimization expression HBV peplos when Protein S 2S, and described recombiant vaccine plasmid pS2S-C has the nucleotide sequence as shown in Seq ID No.1; Or expressing the recombiant vaccine plasmid pC-S2S of the S2S of albumen in hepatitis B peplos when optimization expression hepatitis B core Core albumen, described recombiant vaccine plasmid pC-S2S has the nucleotide sequence as shown in Seq ID No.2.

In the present invention, core Core albumen can be natural total length core Core albumen, can be also the core Core albumen after truncate, and the Core albumen after its truncate, containing the 144aa of N-terminal, is preferably the core Core albumen of total length.

A further object of the present invention is to provide a kind of compositions that contains recombinant plasmid DNA vaccine of the present invention, described compositions is except containing recombinant plasmid DNA vaccine of the present invention, the medicine that can also contain some other treatments or there is adjunctive therapeutic, medicine as ucleosides treatment hepatitis B, cytokine, improve the Chinese medicine ingredients of immunization etc.

A further object of the present invention is to provide a kind of two plasmid DNA vaccine combinations that are used for the treatment of hepatitis B that build by gene recombination technology, of the present invention pair of plasmid DNA vaccine combination, comprise HBV DNA recombiant vaccine plasmid and IL-12 restructuring adjuvant plasmid pIL12.

In the present invention, the two plasmid DNA vaccine combinations that provide, wherein constructed recombiant vaccine plasmid is for carrying the recombiant vaccine plasmid of Protein S 2S encoding gene and core Core (C) protein coding gene in HBV, in described recombiant vaccine plasmid, the expression sequencing of antigen can be first to express in HBV to express HBV core Core albumen after Protein S 2S, the recombiant plasmid built is pS2S-C (the plasmid structural representation is shown in Fig. 1), has the nucleotide sequence as shown in Seq ID No.1; Can be also to express Protein S 2S in HBV after first expressing HBV core Core albumen, the recombiant plasmid built be pC-S2S (the plasmid structural representation is shown in Fig. 2), has the nucleotide sequence as shown in Seq ID No.2.

In the present invention, described recombinant interleukin 12 adjuvant plasmids (pIL12), wherein interleukin 12 can be the interleukin 12 that derives from Mus, described Mus interleukin 12 can be the interleukin 12 of rat, mice or other Mus, constructed restructuring adjuvant plasmid pmIL12 (having the nucleotide sequence as shown in Seq ID No.4); Can be also the interleukin 12 that derives from the people, build restructuring adjuvant plasmid phIL12 (thering is the nucleotide sequence as shown in Seq ID No.3).

In the present invention; described recombinant interleukin 12 adjuvant plasmids also comprise escherichia coli replication origin ColE1, block that resistant gene sequence Kan and two destination protein expression cassettes, and the elements such as enhancer element Enhancer, immunomodulating sequence C pG that derive from SV40.

In the present invention, described recombiant vaccine plasmid and restructuring adjuvant plasmid also portability have multiple clone site district MCS, and described multiple clone site district comprises the restriction enzyme sites such as SwaI, KpnI, SalI, EcoRV, BgIII.

The present invention also aims to provide the construction method of a kind of pair of plasmid DNA vaccine combination.

1, recombiant plasmid HBV DNA vaccination plasmid pS2S-C (pRec2.0-preS2S-C)

(1) first construction of recombinant plasmid vector skeleton pOE-EKS:

Take pDRVISV1.0 as template, take Primer1 and Primer2 as primer, the primer that wherein primer 2 is 5 ' terminal phosphate, amplification obtains the replicon zone (Ori) that size is 748bp, introduces EcoRI, KpnI and SwaI restriction enzyme site simultaneously;

Primer1：5’-GGAATTCGGGGTACCATTTAAATTTGAACGTTCGCAAtATGTGAGCAAAAGGCCAGC-3’

Primer2：5’-CGGCGCGCGCCGAAAACGACGATTGCGAACGTTCAACCCGTAGAAAAGATCAAAGG-3’

Take pDRVISV1.0 as template, take Primer3 and Primer4 as primer, the primer that wherein primer 3 is 5 ' terminal phosphate, amplification obtains that resistant maker gene of card (Kan) that size is 1056bp, introduces the EcoRI restriction enzyme site simultaneously;

Primer3：5’-tcgtcgttttcggcgcgcgccgTTGAACGTTCGCAAtTCAAGTCAGCGTAATGCTC-3’

Primer4：5’-GGAATTCGGCGCGCGCCGAAAACGACGATGCGAACGTTCAAGAAAGCCACGTTGTGTCT-3’

Above-mentioned two fragments are carried out to single endonuclease digestion with EcoRI respectively, after connecting, build recombinant clone pOK-EKS;

(2) construction recombination plasmid DNA vaccination pRec2.0-PreS2.S

Synthetic gene PreS2S is connected with the carrier pHPRE of SalI+XbaI double digestion by the XhoI+XbaI double digestion, builds and obtain recombinant clone pHPRE-PreS2S;

Synthetic gene PreS2S used entrusts thunder silent bio tech ltd in Beijing synthetic, and its nucleotides sequence is classified as has the nucleotide sequence shown in Seq ID No.5:

Take pDRVISV1.0 as template, take Primer5 and Primer6 as primer, it is the BGHpolyA district of 271bp that amplification obtains size, introduces the BamHI restriction enzyme site in upstream simultaneously, and the BglII-EcoRV-SalI-KpnI restriction enzyme site is introduced in downstream; Enter by BamHI+KpnI double digestion rear clone in the pHPRE-PreS2S carrier of BglII+KpnI double digestion, obtain recombiant plasmid pHPRE '-PreS2S;

Primer5：5’-CGGGATCCgctgtgccttctagttgccag-3’

Primer6：5’-GGGGTACCGTCGACAACGTTGATATCAACGTTAGATCTCATAGAGCCCACCGCATCC-3’

By EcoRI+KpnI, the PreS2S expression cassette of recombiant plasmid pHPRE '-PreS2S is cloned in carrier pOK-EKS to construction recombination plasmid pRec2.0-PreS2S.

(3) construction recombination plasmid pRec2.0-C

Synthetic gene CPreS1 is connected with the carrier pHPRE of SalI+XbaI double digestion by the XhoI+XbaI double digestion, builds and obtain recombinant clone pHPRE-CPreS1;

The nucleotides sequence of synthetic gene CPreS1 is classified the nucleotide sequence shown in Seq ID No.6 as.

Take pDRVISV1.0 as template, take Primer5 and Primer6 as primer, it is the BGHpolyA district of 271bp that amplification obtains size, introduces the BamHI restriction enzyme site in upstream simultaneously, and the BglII-EcoRV-SalI-KpnI restriction enzyme site is introduced in downstream; Enter by BamHI+KpnI double digestion rear clone in the pHPRE-CPreS1 carrier of BglII+KpnI double digestion, obtain recombiant plasmid pHPRE '-CPreS1;

By EcoRI+KpnI, the CPreS1 expression cassette of recombiant plasmid pHPRE '-CPreS1 is cloned in carrier pOK-EKS to construction recombination plasmid pRec2.0-CPreS1;

Take pRec2.0-CPreS1 as template, take Primer7 and Primer8 as primer, amplification obtains the Core gene (C) that size is 596bp, and by PstI+XbaI double digestion rear clone, enters in the pRec2.0-CPreS1 carrier of PstI+XbaI double digestion, obtains recombiant plasmid pRec2.0-C.

Primer7：5’-gtcttttctgcagtcaccgtcgTCGAG-3’

Primer8：5’-GCTCTAGAATCAACACTGGGATTCGCGGCTCTG-3’

(4) construction recombination plasmid pRec2.0-PreS2S-C (pS2S-C)

Take pRec2.0-C as template, take Primer9 and Primer8 as primer, amplification obtains the Core gene (C) that size is 579bp, by the HindIII+XbaI double digestion, with the carrier pcDNA3.1 of HindIII+XbaI double digestion, is connected, and builds and obtains pcDNA3.1-C;

Primer9：5’-CCCAAGCTTGCCGCCACCATGGATATTG-3’

Primer8：5’-GCTCTAGAATCAACACTGGGATTCGCGGCTCTG-3’

Cut out the C expression cassette by the BglII+PvuII double digestion from plasmid pcDNA3.1-C, be cloned in the pRec2.0-PreS2S of BglII+EcoRV double digestion, build and obtain recombiant plasmid pRec2.0-PreS2S-C.

2 restructuring HBV DNA vaccination plasmid pC-S2S (being pRec2.0-C-preS2S)

The pRec2.0-PreS2S that the middle structure of the above-mentioned steps (2) of take obtains is template, take Primer11 and Primer12 as primer, it is the PreS2S gene of 873bp that amplification obtains size, be connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, build and obtain pcDNA3.1-PreS2S;

Primer11：5’-CCCAAGCTTGCCGCCACCATGCAGTGGAACTC-3’

Primer12：5’-GCTCTAGAATCAGATGTAAACCCAC-3’

Cut out the PreS2S expression cassette by the BglII+PvuII double digestion from plasmid pcDNA3.1-PreS2S, be cloned in the pRec2.0-C of BglII+EcoRV double digestion, build and obtain recombiant plasmid pRec2.0-C-PreS2S (pC-S2S).

The structure of 3 restructuring adjuvant plasmid pmIL12

(1) synthetic gene mP35 (having the nucleotide sequence shown in Seq ID No.7) is connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, builds and obtain recombiant plasmid pcDNA3.1-mP35;

(2) cut out the mP35 expression casette by the BglII+PvuII double digestion from plasmid pcDNA3.1-mP35, be cloned in the plasmid pRec2.0-PreS2S of BglII+EcoRV double digestion, obtain recombiant plasmid pRec2.0-PreS2S-mP35;

(3) synthetic gene mP40 (having the nucleotide sequence shown in Seq ID No.8) is connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, builds and obtain recombiant plasmid pcDNA3.1-mP40;

(4) go out the mP40 expression casette by the SalI+PvuII double digestion, be cloned in the plasmid pRec2.0-PreS2S-mP35 of SalI+SwaI double digestion, obtain recombiant plasmid pRec2.0-PreS2S-mIL12;

(5) by NdeI digested plasmid pRec2.0-PreS2S-mIL12, cut out altogether 3 bands (1870bp+3448bp+3855bp), reclaim 1870bp fragment and 3855bp fragment, after the 3855bp fragment of recovery is carried out to the CIP dephosphorylation, be connected screening forward clone's recombiant plasmid pmIL12 with the 1870bp fragment.

The structure of 4 restructuring adjuvant plasmid phIL12

(1) synthetic gene hP35 (having the nucleotide sequence shown in Seq ID No.9) is connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, builds and obtain recombiant plasmid pcDNA3.1-hP35;

(2) cut out the hP35 expression casette by the BglII+PvuII double digestion from plasmid pcDNA3.1-hP35, be cloned in the plasmid pRec2.0-PreS2S of BglII+EcoRV double digestion, obtain recombiant plasmid pRec2.0-PreS2S-hP35;

(3) synthetic gene hP40 (having the nucleotide sequence shown in Seq ID No.10) is connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, builds and obtain recombiant plasmid pcDNA3.1-hP40;

(4) go out the hP40 expression casette by the SalI+PvuII double digestion, be cloned in the plasmid pRec2.0-PreS2S-hP35 of SalI+SwaI double digestion, obtain recombiant plasmid pRec2.0-PreS2S-hIL12;

(5) by NdeI digested plasmid pRec2.0-PreS2S-hIL12, cut out altogether 3 bands (1882bp+3446bp+3812bp), reclaim 1882bp fragment and 3812bp fragment, after the 3812bp fragment of recovery is carried out to the CIP dephosphorylation, be connected screening forward clone's recombiant plasmid phIL12 with the 1882bp fragment.

A further object of the present invention is to provide a kind of preparation method of vaccine, the preparation method of each plasmid (pS2S-C, pC-S2S and pIL12) is identical, but separately produce, recombiant plasmid pS2S-C, pC-S2S, pmIL12, phIL12 are transformed respectively to bacillus coli DH 5 alpha, screening positive clone, contain the positive monoclonal of recombiant plasmid through fermentation culture, amplification, then obtain the recombiant plasmid of purifying after cracking and column purification, be specifically as follows:

Fermentation: take out the bacillus coli DH 5 alpha that contains plasmid in-70 ℃, pick a small amount of bacterium liquid with inoculating loop, on plate, the picking monoclonal is placed in 5-10ml and contains corresponding antibiotic LB fluid medium, 37 ℃, 200rpm are cultivated 10-12h, with the ratio of 1: 1000, add 2.5L to contain corresponding antibiotic LB culture medium culture fluid, 37 ℃, 200rpm carry out centrifugal treating by culture after cultivating 12-16h, collect thalline, obtain the fermentation culture medium containing plasmid.

Cracking: thalline is added to Buffer liquid, and soft puts upside down centrifuge tube for several times, and room temperature is placed, and makes its cracking abundant.

Column chromatography: the thalline after cracking is by the DNA preparative column, and eluting separates, and collects plasmid and uses organic solvent deposit, by the physiological saline solution plasmid DNA and get final product.

In order convenient and efficient ground more to express recombiant vaccine plasmid of the present invention and restructuring adjuvant plasmid, the present invention has also built the escherichia coli that contain recombiant plasmid of the present invention, apply recombination bacillus coli of the present invention, can prepare more quickly each plasmid in the present invention.

Coli strain DH5 α/pmIL12, contain restructuring adjuvant plasmid pmIL12, by prior construction recombination plasmid pmIL12, screen and obtain in the LB plate containing that resistance of card after transforming e.colistraindh5α, be preserved in (address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, China Microbial Culture Preservation Commission's common micro-organisms center, postcode: 100101), preservation date: on 04 13rd, 2010, the classification of suggestion colon bacillus Esherichia coli by name, deposit number is: CGMCC No.3726.

Coli strain DH5 α/phIL12, contain restructuring adjuvant plasmid phIL12, by prior construction recombination plasmid phIL12, screen and obtain in the LB plate containing that resistance of card after transforming e.colistraindh5α, be preserved in (address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, China Microbial Culture Preservation Commission's common micro-organisms center, postcode: 100101), preservation date: on 04 13rd, 2010, the classification of suggestion colon bacillus Esherichia coli by name, deposit number is: CGMCC No.3727.

Coli strain DH5 α/pRec2.0-S2S-C, contain restructuring adjuvant plasmid pS2S-C, by prior construction recombination plasmid pS2S-C, screen and obtain in the LB plate containing that resistance of card after transforming e.colistraindh5α, be preserved in (address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, China Microbial Culture Preservation Commission's common micro-organisms center, postcode: 100101), preservation date: on 04 13rd, 2010, the classification of suggestion colon bacillus Esherichia coli by name, deposit number is: CGMCC No.3728.

Coli strain DH5 α/pRec2.0-C-S2S, contain restructuring adjuvant plasmid pC-S2S, by prior construction recombination plasmid pC-S2S, screen and obtain in the LB plate containing that resistance of card after transforming e.colistraindh5α, be preserved in (address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, China Microbial Culture Preservation Commission's common micro-organisms center, postcode: 100101), preservation date: on 04 13rd, 2010, the classification of suggestion colon bacillus Esherichia coli by name, deposit number is: CGMCC No.3729.

A further object of the present invention also is to provide recombinant plasmid DNA vaccine of the present invention and two plasmid DNA vaccine to have the application in prevention and treatment hepatitis B medicine in preparation.

An also purpose of the present invention is to provide a kind of recombinant plasmid DNA vaccine of the present invention or the immune administering mode of two plasmid vaccine when prevention and treatment hepatitis B, its administering mode can be subcutaneous injection, intramuscular injection or by the electroporation mode, preferred modes for by the electroporation mode administration carry out immunity or treatment.

The external evaluation that the present invention is used for the treatment of two plasmid DNA vaccines of hepatitis B adopts in-vitro transfection 293-T and COS-7 cell, the high level expression of destination protein (HBsAg, HBcAg, IL12) detected.Two plasmid DNA vaccines can induce body to produce high-caliber hepatitis B surface antibody (HBsAb) and core antibody (HBcAb) level and specific cell immune response.

Even more noteworthy, after carrying out face by means of electroporation technology, the two plasmid DNA vaccines of hepatitis B can induce the immune effect significantly improved in the present invention.

The accompanying drawing explanation

Fig. 1 is the structural representation of restructuring vaccine plasmid pRec2.0-S2S-C (pS2S-C).

Fig. 2 is the structural representation of restructuring vaccine plasmid pRec2.0-C-S2S (pC-S2S).

Fig. 3 is the structural representation of restructuring adjuvant plasmid phIL12.

Fig. 4 is the structural representation of restructuring adjuvant plasmid pmIL12.

Fig. 5 is the restriction enzyme digestion and electrophoresis collection of illustrative plates of restructuring vaccine plasmid pS2S-C and pC-S2S, wherein:

Swimming lane 1 through the collection of illustrative plates that XbaI is mono-after cutting, can cut out two bands for restructuring vaccine plasmid pS2S-C, is respectively 2300bp and 4570bp;

Swimming lane 2 for restructuring vaccine plasmid pS2S-C through EcoRI is mono-cut after, cut out the general band that size is about 6870bp;

Swimming lane 3 is not for carrying out the recombiant vaccine plasmid pS2S-C of enzyme action;

Swimming lane 4 after the NdeI+SalI double digestion, cuts out three general bands that size is about 1300bp, 2100bp, 3400bp for restructuring vaccine plasmid pS2S-C;

Swimming lane 5 after the PvuII+XbaI double digestion, cuts out three general bands that size is about 1000bp, 2200bp, 3600bp for restructuring vaccine plasmid pS2S-C;

Swimming lane 6 is marker DL2000;

Swimming lane 7 is marker DL15000;

Swimming lane 8 is for restructuring vaccine plasmid pC-S2S through the collection of illustrative plates that XbaI is mono-after cutting, and two bands that cut out are respectively 2550bp and 4320bp;

Swimming lane 9, cuts out size and is about 6870 general band through the collection of illustrative plates that XbaI is mono-after cutting for recombiant plasmid pC-S2S;

Swimming lane 10 is not for carrying out the recombiant vaccine plasmid pS2S-C of enzyme action;

Swimming lane 11 after the NdeI+SalI double digestion, cuts out three general bands that size is about 1500bp, 2300bp, 3100bp for restructuring vaccine plasmid pC-S2S;

Swimming lane 12 after the PvuII+XbaI double digestion, cuts out four general bands that size is about 250bp, 500bp, 2500bp, 3600bp for restructuring vaccine plasmid pC-S2S;

Fig. 6 is restructuring adjuvant plasmid pmIL12 restriction enzyme digestion and electrophoresis collection of illustrative plates, wherein:

Swimming lane 1 is after the PstI single endonuclease digestion, three general bands that cut out, and size is 280bp, 2100bp, 3300bp, and wherein 280bp size is general is with fuzzyly, and all the other two are entirely true;

The 2 bar general bands of swimming lane 2 for cutting out after the NdeI single endonuclease digestion, size is respectively 1900bp, 3800bp, and two bands are entirely true;

The two bar general bands of swimming lane 3 for cutting out after the EcoRI+BglII double digestion, size is respectively 1700bp, 4000bp, and two bands are entirely true;

Swimming lane 4,5 is respectively DL15000, DL2000marker;

Swimming lane 6 is the linear fragment through EcoRI is mono-after cutting, and size is about 5700bp;

Swimming lane 7 is not for carrying out the restructuring adjuvant plasmid pmIL12 of enzyme action.

Fig. 7 is restructuring adjuvant plasmid phIL12 restriction enzyme digestion and electrophoresis collection of illustrative plates, wherein:

Swimming lane 1,2 is respectively DL2000, DL15000marker.

Swimming lane 3 is after the HindIII single endonuclease digestion, three general bands that cut out, and size is respectively 1496bp, 1882bp, 2343bp, and reality is entirely true;

Swimming lane 4 is after the EcoRI+BglII double digestion, two general bands that cut out, and size is respectively 1752,3942bp, and two band reality are entirely true;

Swimming lane 5 is the linear fragment after the EcoRI single endonuclease digestion, and size is about 5700bp;

Swimming lane 6 is not for carrying out the restructuring adjuvant plasmid phIL12 of enzyme action.

Specific embodiment

The present invention further illustrates by following embodiment, but any embodiment or its combination not should be understood to the restriction to scope of the present invention or embodiment.Scope of the present invention is limited by appended claims, and in conjunction with the general general knowledge of this description and this area, those of ordinary skills can clearly understand claims limited range.Under prerequisite without departing from the spirit and scope of the present invention, those skilled in the art can carry out any modification or change to technical scheme of the present invention, and this modification and change are also contained in scope of the present invention.

Agents useful for same and material: bacillus coli DH 5 alpha is purchased from Invitrogen company; The DNA vaccination plasmid pInH, the plasmid pDRVISV1.0 that carry posttranscriptional regulatory element (HPRE) are so kind as to give (building process of this plasmid and particulars be full disclosure in Chinese patent CN1560259A) by Venereal Disease AIDS Preventing Controlling Center China Disease Preventing Con's virus immunity chamber; Eukaryon expression plasmid pcDNA3.1 is purchased from Invitrogen company; Carrier for expression of eukaryon pHPRE is built by Venereal Disease AIDS Preventing Controlling Center China Disease Preventing Con's virus immunity chamber; Carry the double-cistron expressing plasmid pIRESneo of internal ribosome entry site (IRES) purchased from Clontech company; 293T, COS-7 cell are purchased from ATCC; Mouse boosting cell is cultivated (10% hyclone, Invitrogen company) with 1640 complete mediums; Various restricted enzyme, pfu polymerase, DNA connect reagent Sol I all purchased from NEB company; The plasmid extraction purification kit is purchased from TIANGEN company; Hepatitis B surface antigen and surface antibody quantitative ELISA detection kit (chemoluminescence method) are purchased from source, Beijing moral bio tech ltd.The ELISPOT detection kit of mice IFN-γ is purchased from BD company.The antigenic stimulus peptide is synthetic by match Parkson, Beijing Bioisystech Co., Ltd.The synthetic high bio tech ltd of gene sequencing working delegation Beijing English that reaches of gene carries out.

The structure of embodiment 1 recombiant plasmid pRec2.0-preS2S-C (being abbreviated as pS2S-C)

(1) first construction of recombinant plasmid vector skeleton pOE-EKS, its nucleotides sequence is classified the nucleotide sequence that SeqNo.1 indicates as:

Take pDRVISV1.0 as template, take Primer1 and Primer2 as primer, the primer that wherein primer 2 is 5 ' terminal phosphate, amplification obtains the replicon zone (Ori) that size is 748bp, introduces EcoRI, KpnI and SwaI restriction enzyme site simultaneously;

Primer1：5’-GGAATTCGGGGTACCATTTAAATTTGAACGTTCGCAAtATGTGAGCAAAAGGCCAGC-3’

Primer2：5’-CGGCGCGCGCCGAAAACGACGATTGCGAACGTTCAACCCGTAGAAAAGATCAAAGG-3’

Take pDRVISV1.0 as template, take Primer3 and Primer4 as primer, the primer that wherein primer 3 is 5 ' terminal phosphate, amplification obtains that resistant maker gene of card (Kan) that size is 1056bp, introduces the EcoRI restriction enzyme site simultaneously;

Primer3：5’-tcgtcgttttcggcgcgcgccgTTGAACGTTCGCAAtTCAAGTCAGCGTAATGCTC-3’

Primer4：5’-GGAATTCGGCGCGCGCCGAAAACGACGATGCGAACGTTCAAGAAAGCCACGTTGTGTCT-3’

Above-mentioned two fragments are carried out to single endonuclease digestion with EcoRI respectively, after connecting, build recombinant clone pOK-EKS;

(2) construction recombination plasmid DNA vaccination pRec2.0-PreS2.S

Synthetic gene PreS2S is connected with the carrier pHPRE of SalI+XbaI double digestion by the XhoI+XbaI double digestion, builds and obtain recombinant clone pHPRE-PreS2S;

Take pDRVISV1.0 as template, take Primer5 and Primer6 as primer, it is the BGHpolyA district of 271bp that amplification obtains size, introduces the BamHI restriction enzyme site in upstream simultaneously, and the BglII-EcoRV-SalI-KpnI restriction enzyme site is introduced in downstream; Enter by BamHI+KpnI double digestion rear clone in the pHPRE-PreS2S carrier of BglII+KpnI double digestion, obtain recombiant plasmid pHPRE '-PreS2S;

Primer5：5’-CGGGATCCgctgtgccttctagttgccag-3’

Primer6：5’-GGGGTACCGTCGACAACGTTGATATCAACGTTAGATCTCATAGAGCCCACCGCATCC-3’

By EcoRI+KpnI, the PreS2S expression cassette of recombiant plasmid pHPRE '-PreS2S is cloned in carrier pOK-EKS, construction recombination plasmid pRec2.0-PreS2S, its nucleotides sequence is classified the nucleotide sequence that Seq No.2 indicates as.

(3) construction recombination plasmid pRec2.0-C

Synthetic gene CPreS1 is connected with the carrier pHPRE of SalI+XbaI double digestion by the XhoI+XbaI double digestion, builds and obtain recombinant clone pHPRE-CPreS1;

Take pDRVISV1.0 as template, take Primer5 and Primer6 as primer, it is the BGHpolyA district of 271bp that amplification obtains size, introduces the BamHI restriction enzyme site in upstream simultaneously, and the BglII-EcoRV-SalI-KpnI restriction enzyme site is introduced in downstream; Enter by BamHI+KpnI double digestion rear clone in the pHPRE-CPreS1 carrier of BglII+KpnI double digestion, obtain recombiant plasmid pHPRE '-CPreS1;

By EcoRI+KpnI, the CPreS1 expression cassette of recombiant plasmid pHPRE '-CPreS1 is cloned in carrier pOK-EKS to construction recombination plasmid pRec2.0-CPreS1;

Take pRec2.0-CPreS1 as template, take Primer7 and Primer8 as primer, amplification obtains the Core gene (C) that size is 596bp, and by PstI+XbaI double digestion rear clone, enters in the pRec2.0-CPreS1 carrier of PstI+XbaI double digestion, obtains recombiant plasmid pRec2.0-C.

Primer7：5’-gtcttttctgcagtcaccgtcgTCGAG-3’

Primer8：5’-GCTCTAGAATCAACACTGGGATTCGCGGCTCTG-3’

(4) construction recombination plasmid pRec2.0-PreS2S-C

Take pRec2.0-C as template, take Primer9 and Primer8 as primer, amplification obtains the Core gene (C) that size is 579bp, by the HindIII+XbaI double digestion, with the carrier pcDNA3.1 of HindIII+XbaI double digestion, is connected, and builds and obtains pcDNA3.1-C;

Primer9：5’-CCCAAGCTTGCCGCCACCATGGATATTG-3’

Primer8：5’-GCTCTAGAATCAACACTGGGATTCGCGGCTCTG-3’

Cut out the Core expression cassette by the BglII+PvuII double digestion from plasmid pcDNA3.1-C, be cloned in the pRec2.0-preS2S of BglII+EcoRV double digestion, build and obtain recombiant plasmid pRec2.0-PreS2S-C.

The structure of embodiment 2 recombiant plasmid pRec2.0-C-preS2S (pC-S2S)

Take that to build the pRec2.0-PreS2S obtained in embodiment 1 be template, take Primer11 and Primer12 as primer, it is the PreS2S gene of 873bp that amplification obtains size, be connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, build and obtain pcDNA3.1-PreS2S;

Primer11：5’-CCCAAGCTTGCCGCCACCATGCAGTGGAACTC-3’

Primer 12：5’-GCTCTAGAATCAGATGTAAACCCAC-3’

Cut out the PreS2S expression cassette by the BglII+PvuII double digestion from plasmid pcDNA3.1-PreS2S, be cloned in the pRec2.0-C of BglII+EcoRV double digestion, build and obtain recombiant plasmid pRec2.0-C-PreS2S (pC-S2S).

The structure of embodiment 3 restructuring adjuvant plasmid pmIL12

(1) synthetic gene mP35 is connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, builds and obtain recombiant plasmid pcDNA3.1-mP35;

(2) cut out the mP35 expression casette by the BglII+PvuII double digestion from plasmid pcDNA3.1-mP35, be cloned in the plasmid pRec2.0-PreS2S of BglII+EcoRV double digestion, obtain recombiant plasmid pRec2.0-PreS2S-mP35;

(3) synthetic gene mP40 is connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, builds and obtain recombiant plasmid pcDNA3.1-mP40;

(4) go out the mP40 expression casette by the SalI+PvuII double digestion, be cloned in the plasmid pRec2.0-PreS2S-mP35 of SalI+SwaI double digestion, obtain recombiant plasmid pRec2.0-PreS2S-mIL12;

(5) by NdeI digested plasmid pRec2.0-PreS2S-mIL12, cut out altogether 3 bands (1870bp+3448bp+3855bp), reclaim 1870bp fragment and 3855bp fragment, after the 3855bp fragment of recovery is carried out to the CIP dephosphorylation, be connected screening forward clone's recombiant plasmid pRec2.0-mIL12 (pmIL12) with the 1870bp fragment.

The structure of embodiment 4 restructuring adjuvant plasmid phIL12

(1) synthetic gene hP35 is connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, builds and obtain recombiant plasmid pcDNA3.1-hP35;

(2) cut out the hP35 expression casette by the BglII+PvuII double digestion from plasmid pcDNA3.1-hP35, be cloned in the plasmid pRec2.0-PreS2S of BglII+EcoRV double digestion, obtain recombiant plasmid pRec2.0-PreS2S-hP35;

(3) synthetic gene hP40 is connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, builds and obtain recombiant plasmid pcDNA3.1-hP40;

(4) go out the hP40 expression casette by the SalI+PvuII double digestion, be cloned in the plasmid pRec2.0-PreS2S-hP35 of SalI+SwaI double digestion, obtain recombiant plasmid pRec2.0-PreS2S-hIL12;

(5) by NdeI digested plasmid pRec2.0-PreS2S-hIL12, cut out altogether 3 bands (1882bp+3446bp+3812bp), reclaim 1882bp fragment and 3812bp fragment, after the 3812bp fragment of recovery is carried out to the CIP dephosphorylation, be connected screening forward clone's recombiant plasmid pRec2.0-hIL 12 with the 1882bp fragment.

The recombiant plasmid pS2S-C of the recombiant plasmid pcDNA3.1-SC that embodiment 5 amalgamation modes build and the present invention's design or the pC-S2S detection to the mice serum specific antibody

The structure of recombiant plasmid pcDNA3.1-SC is pressed literature method (Chen Min, etc. the little tree immunne response that the recombiant plasmid of amalgamation and expression HBsAg/HBcAg is induced for Zhou Taoyou, Zhao Liansan) and is built and obtain.

6-8 Balb/c female mice in age in week is purchased from Chinese Academy of Medical Sciences's animal reproduction center, clean level is raised, 30 mices are divided into 3 groups at random, every group 10, gastrocnemius is injected recombiant plasmid pcDNA3.1-SC (100 μ g), recombiant plasmid pS2S-C (50 μ g), recombiant plasmid pC-S2S (50 μ g) respectively, after the 2nd, 4 weeks, each booster shot is 1 time, gathers mice serum.

Mice serum detection of specific antibody: respectively at the 3rd, 5,8,12 weeks collection mice socket of the eye venous blood after primary vaccination, serum separates rearmounted-80 ℃ of preservations, the mice serum specimen was diluted by 1: 5,1: 10,1: 25,1: 50,1: 100,1: 250,1: 500, adopt the anti-HBs of hepatitis B virus and-anti-HBc detection kit (ELISA method), above-mentioned specimen is unified to detect.

The anti-HBs of mice serum specific antibody check result: start inoculation latter 3,5,8,12 weeks, the anti-HBs sun of pcDNA3.1-SC group mice serum rate of rotation is respectively 60%, 80%, 100% and 70%, the anti-HBs titre of serum raises gradually, reached peak value at the 8th week, and its anti-HBc value is lower, only the part mice can detect, but its positive rate of rotation is all lower than 30%.And for the recombiant plasmid pS2S-C group of optimizing, after starting to inoculate 3,5,8,12 weeks, the anti-HBs sun of its serum rate of rotation is respectively 70%, 100%, 100% and 80%, antibody titer can reach peak value at the 5th week, meanwhile, the anti-HBc sun of serum rate of rotation is respectively 30%, 40%, 70% and 50%, antibody titer can reach the highest at the 3rd week, with recombiant plasmid pcDNA3.1-SC group, compare, obtain the anti-HBc sun of serum rate of rotation significantly excellent, difference has significance (P<0.01); For the recombiant plasmid pC-S2S optimized, after starting to inoculate 3,5,8,12 weeks, the anti-HBs sun of its serum rate of rotation is respectively 0,20%, 30% and 10%, anti-HBs sun rate of rotation is lower, all lower than 30%, but its for the anti-HBc of serum sun rate of rotation be respectively 80%, 100%, 100% and 90%, the titre of the anti-HBc of serum obviously will be higher than the pcDNA3.1-SC group.

The immunogenicity that embodiment 6 expresses multi-form HBcAg nucleic acid vaccine compares

Relatively truncate, body fluid and the cell immunogenicity of 2 kinds of multi-form hepatitis B virus core antigens of total length (HBcAg) nucleic acid vaccine in Mice Body, obtain the immunogen design of optimizing; The multi-form hepatitis B virus HBcAg nucleic acid vaccine that structure is optimized through codon is respectively 1. pRec2.0-Ct: express the Core albumen that is truncated to 144aa; 2. pRec2.0-C expresses the Core albumen of total length 183aa; By above-mentioned vaccine plasmid transfection 293T cell, WestemBlot testing goal antigen gene expression.After by intramuscular injection (each plasmid intramuscular injection 50 μ g), the Balb/c mice being carried out to immunoprophylaxis, the ELISA method detects the antibody horizontal of the anti-HBc of mice serum, and IFN-γ ELISPOT method detects HBcAg Specific T cell immunity in Mice Body and replys.Result: except blank group mice, the mice of respectively organizing after vaccine immunity has all produced HBc antigen-specific antibodies and t cell response, wherein pRec2.0-C organizes anti-HBc antibody (110.86 ± 87.03U/ml) and t cell immune response level (471.3 ± 88.0spots) all is significantly higher than pRec2.0-Ct group (anti-HBc:38.60 ± 41.87U/ml, IFN-γ: 203.5 ± 126.7spots) (P<0.05).Show that HBcAg is truncated to 144 aminoacid maybe can reduce its body fluid and cell immunogenicity.

The expression of HBsAg, IL12 destination protein after 7 pairs of plasmid cell in vitro transient transfections of embodiment

Cultivate respectively according to a conventional method COS-7 cell strain, 293T cell strain in containing RPMI1640 (DMEM) culture medium of 10% newborn calf serum (FBS), before transfection, 24h uses trypsinization note parietal cell, be resuspended in containing calf serum RPMI1640 culture fluid, by 3 * 10 ⁵the cells/well transferred species in 6 well culture plates, 37 ℃, 5%C0 ₂cultivate, treat that the growth of note parietal cell reaches 90% degrees of fusion, get recombiant vaccine plasmid pS2S-C, pCS2S, restructuring adjuvant plasmid pmIL12, phIL12, (method is shown in Lipofectamine2000protocol to carry out cell transfecting with the Lipofectamine2000 transfection reagent, Invitrogen company), the plasmid consumption is 5 μ g/ holes, 37 ℃, 5%CO ₂condition is carried out cell culture, continue to cultivate and after transfection 48h collect supernatant, the expression of testing goal gene, the quantitative assay of HBsAg and IL12 is undertaken by the description of test kit separately, the results are shown in Table 1 and table 2.

The vitro detection of HBsAg, HBcAg after table 1 recombiant vaccine plasmid in-vitro transfection COS-7,293T cell

From the result of table 1 and table 2, can find out, recombiant vaccine plasmid pS2S-C and pC-S2S all can carry out the expression of genes of interest HBsAg at COS-7,293-T cell, and the expression that detects HBsAg is respectively 93.2,15.1 and 67.9,8.4ng/ml; Restructuring adjuvant plasmid pmIL12 and phIL12 also all can carry out the expression of genes of interest IL12 at COS-7,293-T cell, and the expression that detects IL12 is respectively 358.7,564.1 and 336.9,381.3ng/ml.

The preparation of 8 pairs of plasmid vaccines of embodiment

Vaccine plasmid (pS2S-C, pC-S2S) is identical with the preparation method of adjuvant plasmid (pmIL12, phIL12), and each plasmid is separately produced, and concrete steps are as follows:

1) on plate, the picking monoclonal is placed in 5-10ml containing corresponding antibiotic LB fluid medium, and 37 ℃, 200rpm cultivates 10-12h;

2) with the ratio of 1: 1000, add 2.5L to contain in corresponding antibiotic LB culture medium culture fluid, 37 ℃, 200rpm cultivates 12-16h;

3) in 4 ℃, the centrifugal 15min of 6000rpm, supernatant discarded, be resuspended in precipitation in the STE solution of 500ml ice pre-cooling, and 4 ℃, the centrifugal 15min of 6000rpm, supernatant discarded;

4) bacterial sediment is resuspended in the Buffer P1 of 125ml, and vibration, to occurring without the precipitation agglomerate, adds the Buffer P2 of 125ml, soft puts upside down centrifuge tube for several times, and standing 5min under room temperature adds the Buffer P3 of 125ml after cracking fully, put upside down centrifuge tube for several times, fully mix;

5) pyrolysis product after mixing is poured in filter (Mega-Giga Cartridge), under room temperature more than standing 20min, open vacuum pump pressurization sucking filtration, after treating the liquid sucking filtration, the Buffer FWB2 that adds 50ml, stir gently the precipitation agglomerate with aseptic Glass rod, proceed sucking filtration;

6) add the Buffer ER of 30ml in lysate, put upside down and mix, ice bath 30min adds the good preparative column of Buffer QBT balance of using in advance 75ml after the solution clarification;

7), after lysate passes through the DNA preparative column fully, the Buffer QC eluting impurity of use 600ml, after the whole posts excessively of liquid, with the eluent Buffer QN eluted dna of 100ml;

8) collect the DNA eluent, add the isopropyl alcohol of 70ml, after fully mixing, in 12 ℃, the centrifugal 50min of 10000rpm;

9) carefully remove supernatant, by 10ml 70% washing with alcohol precipitation, go the ethanol washing liquid, centrifuge tube is upside down in natural drying on napkin;

10) add the physiological saline solution plasmid DNA of 5ml-10ml;

11) concentration of the prepared plasmid DNA of spectrophotometric determination, diluted accordingly according to the plasmid concentration of measuring, and dilutes for final concentration is 1 μ g/ μ l (or 0.4 μ g/ μ l, 40ng/ μ l)-20 ℃ of preservations.Embodiment 9 is containing hepatitis B core Core protein gene and containing the Core protein gene, body fluid and the cellular immunization to healthy Balb/c mice compares

6-8 Balb/c female mice in age in week is purchased from Chinese Academy of Medical Sciences's animal reproduction center, and clean level is raised.25 mices are divided into 5 groups, for giving respectively recombinant plasmid DNA vaccine pRec2.0-S2S group, control plasmid pcDNA3.1-S2S, recombinant plasmid DNA vaccine pRec2.0-S2S-C (pS2S-C), each 50 μ g of recombinant plasmid DNA vaccine pRec2.0-C-S2S (pC-S2S), pRec2.0 empty carrier group, 4 immune group carry out immunity, every mice is by each group dosage injection plasmid DNA, volume is 100 μ l, and every mice two hind leg tibialis anteriors are injected 50 μ l.Immune programme for children be each immunity in 0,2,4 week once, within the 6th week, kill Mus and get serum and separate lymphocyte.

1, humoral immunization detects: get mice serum strictly by the test kit operation instructions method operation of source, Beijing moral biotechnology company.

2, cellular immunization detects: the mouse lymphocyte preparation: the cervical vertebra dislocation method is put to death mice, put in the beaker that fills 75% ethanol, make mice chaeta complete wetting approximately more than 3 minutes, then cut mouse peritoneal, take out spleen, the spleen of separation is put on 200 eye mesh screens of the aseptic beaker of underlying (the cultivation drop is arranged), twist gently the spleen tissue into pieces, splenocyte is extruded in grinding, get 5ml serum-free RPMI-1640 and slowly sweep away cell to beaker, 5ml splenocyte suspension is slowly added in the 15ml centrifuge tube that the 2.5ml lymphocyte separation medium is housed, the centrifugal 20min of 2000-2500r/min, after collecting the separation lymphocyte, with twice, IC centrifuge washing cell, each centrifugal 6min of 1500r/min, with the appropriate splenocyte containing the resuspended separation of 5%FCS-1640 complete culture solution, platform expects that every mice of blue dyeing counting separates lymphocyte living cells sum, adjust cell density to 3 * 10 ⁵/ ml, cultivate and detect for ELISPOT in 37 ℃ of 5%CO2 incubators.

3, ELISPOT detects: strictly press the test kit operation instructions method operation of BD company, think that meeting following two persons regards as the ELISPOT detection HBsAg specific cell positive: under (1) certain cell density condition, in the ELISPOT check-out console, same sample HBsAg and non-HBsAg stimulate the ratio of each 3 multiple hole speckle means (SFC) should be not less than 2; (2) immunization experiment group sample lymphocyte density is 3 * 10 ⁵/ ml hole is detected, and HBsAg and non-HBsAg stimulate the difference of each 3 multiple hole SFC means to be not less than 5.

As the positive evaluation criterion of mice specific cellular immunity, the serum-HBs of HBV DNA vaccination immune group >=10mIU is considered to body protective valid density, the results are shown in 3.

Table 3 is containing hepatitis B core Core protein gene and containing the Core protein gene, body fluid and the cellular immunization to healthy Balb/c mice compares

From the table result can find out, the HBsAg of empty carrier plasmid, HBcAg cellular immunization and humoral immunization all are negative, and for control plasmid pcDNA3.1-S2S, the humoral immunization value of its HBsAg is (46.25 ± 20.31) mIU/ml, cellular immunization value (188.67 ± 91.81) SFU/ 1,000,000 cells, and for the recombiant plasmid pRec2.0-S2S through optimizing, its humoral immunization value is (97.49 ± 40.21) mIU/ml, cellular immunization value (379.15 ± 129.25) SFU/ 1,000,000 cells, obviously be better than control plasmid (difference has significance, P<0.01).Express the recombiant plasmid pS2S-C of HBV core Core albumen for Protein S 2S in optimization expression HBV peplos simultaneously, the humoral immunization value of its HBsAg is a little more than not containing the recombiant plasmid pRec2.0-S2S of Core albumen, the cellular immunization value will be lower than not containing the recombiant plasmid pRec2.0-S2S of Core albumen, its value is respectively (106.34 ± 35.29) mIU/ml, (348.21 ± 59.05) SFU/ 1,000,000 cells, mutual no significant difference, but recombiant plasmid also can obtain the humoral immunity level of HBcAg, its value is (46.41 ± 19.14) U/ml, but the cellular immunization value of HBcAg does not detect, be negative.Express the recombiant plasmid pC-S2S of albumen in HBV peplos for optimization expression HBV core Core albumen simultaneously, the cellular immunization value of its HBsAg will be lower than pRec2.0-S2S, for (113.40 ± 43.37) SFU/ 1,000,000 cells, the humoral immunization value does not detect, be negative, but the humoral immunization of HBcAg and cellular immunization value are higher, be respectively (275.49 ± 84.07) U/ml, (146.30 ± 58.15) SFU/ 1,000,000 cells.Containing the recombiant plasmid of albumen in HBV peplos and HBV core Core albumen, with the recombiant plasmid that does not contain Core albumen, compare when building, when obtaining the HBsAg immune level, also can obtain the level of HBcAg, play more fully immunization.

Body fluid and the cellular immunization of 10 pairs of plasmid DNA vaccines of embodiment to healthy Balb/c mice

6-8 Balb/c female mice in age in week is purchased from Chinese Academy of Medical Sciences's animal reproduction center, and clean level is raised.35 mices are divided into 7 groups, 6 immune group are injected respectively plasmid vaccine pS2S-C+pRec2.0 (50+50) μ g (group II), pS2S-C+pmIL12 (50+50) μ g (group III), pS2S-C+phIL12 (50+50) μ g (group IV), pC-S2S+pRec2.0 (50+50) μ g (group V), pC-S2S+pmIL12 (50+50) μ g (group VI), pC-S2S+phI L12 (50+50) μ g (group VII), with a blank group (injection pRec2.0 empty carrier 50 μ g, group I), every mice is by each group dosage injection plasmid DNA, volume is 100 μ l, every mice two hind leg tibialis anteriors are injected 50 μ l.Immune programme for children be each immunity in 0,2,4 week once, within the 6th week, kill Mus and get serum and separate lymphocyte.

Humoral immunization detection, cellular immunization detection and ELISPOT detection method are with the method in embodiment 8.The results are shown in Table 4

The immune level of the different vaccine plasmids of table 4 to normal BaLb/c mice HBsAg, HBcAg

Compare * P<0.05, * * P<0.01 with (pS2S-C+pRec2.0) group; Compare #P<0.05 with (pC-S2S+pRec2.0) group.

From the table result known, after pRec2.0 empty carrier inoculation normal mouse almost the anti-HBs of serum-free and the anti-HBc concentration of serum produce, the anti-HBs of its mice and anti-HBc number positive are 0.By comparison, pS2S-C, pS2S-C+pmIL12, pS2S-C+phIL12 all can induce anti-HBs to produce, (pS2S-C+pRec2.0) group is (348.21 ± 94.05) SFU/ 1,000,000 cells to the cellular immunization value of healthy Balb/c mice HBsAg, the humoral immunization value is (106.34 ± 35.29) mIU/ml, the cellular immunization of HBcAg is negative, the humoral immunization value is (46.41 ± 19.14) U/ml, and inject (pS2S-C+pmIL12), to the cellular immunization value of healthy Balb/c mice HBsAg, be (468.42 ± 87.29) SFU/ 1,000,000 cells, the humoral immunization value is (318.53 ± 59.67) mIU/ml, the cellular immunization of HBcAg is negative, the humoral immunization value is (278.49 ± 62.53) U/ml, injection (pS2S-C+phIL12) is (406.13 ± 82.40) SFU/ 1,000,000 cells to the cellular immunization value of healthy Balb/c mice HBsAg, the humoral immunization value is (339.62 ± 73.44) mIU/ml, the cellular immunization of HBcAg is negative, the humoral immunization value is (231 ± 66) U/ml, can find out, recombiant vaccine plasmid pS2S-C is with after recombinant interleukin 12 adjuvant plasmids are combined use, recombinant interleukin 12 adjuvant plasmids can obviously improve the cellular immunization of HBsAg of recombiant vaccine plasmid pS2S-C and the humoral immunization value of humoral immunization value and HBcAg, difference has significance.Equally, after recombiant vaccine plasmid pC-S2S and recombinant interleukin adjuvant plasmid are share, recombinant interleukin 12 can obviously improve cellular immunization and the humoral immunization value of the HBcAg of recombiant vaccine plasmid pC-S2S, and the cellular immunization value of HBsAg, and difference has significance.Therefore, after union and recombination interleukin 12 adjuvant plasmid, its immunization to healthy Balb/c mice obviously is better than independent injection recombiant vaccine plasmid pS2S-C or pC-S2S.

Embodiment 11 different dosing regimes compare the immune effect of healthy Balb/c Female Rats

6-8 Balb/c female mice in age in week is purchased from Chinese Academy of Medical Sciences's animal reproduction center, 24, be divided at random four groups, be respectively (pS2S-C+pmIL12) group (administered intramuscular, two plasmid amounts are 50 μ g), (pS2S-C+pmIL12) group (by the immunity of electroporation mode), (pC-S2S+pmIL12) group (administered intramuscular, two plasmid amounts are 50 μ g), (pC-S2S+pmIL12) group (by the immunity of electroporation mode), immune programme for children be each immunity in 0,2,4 week once, within the 6th week, kill Mus and get serum and separate lymphocyte.Humoral immunization detection, cellular immunization detection and ELISPOT detection method are with the method in embodiment 8, and experimental result is in Table 5

Table 5 different dosing regimes compares the immune effect of healthy Balb/c Female Rats

With the intramuscular injection immunization ways, compare, ^*p<0.05, ^*p<0.01

From the table result can find out, for (pS2S-C+pmIL12) group, the obtained immune effect of two dosage groups that carries out immune scheme by the electroporation mode all is better than the intramuscular injection group, difference has significance (P<0.05, P<0.01), especially (10+10) μ g group, the cellular immunization value of its HBsAg is (762.42 ± 77.25) SFU/ 1,000,000 cells, the humoral immunization value is (573.63 ± 81.07) mIU/ml, the humoral immunization value of HBcAg is (384.47 ± 68.39) U/ml, its value will be carried out immune group apparently higher than muscle injection mode, corresponding value is respectively (318.53 ± 59.67) SFU/ 1,000,000 cells, (468.42 ± 37.29) mIU/ml, (278.49 ± 62.53) U/ml, difference has extremely significance (p<0.01), for (pC-S2S+pmIL12) group, apply two electroporation mediated dosage, carry out the obtained effect of immunity and all be better than the obtained immune effect of intramuscular injection group, difference has significance, the immunity of its (10+10) μ g dosage group to healthy Balb/c mice, the cellular immunization value of its HBsAg is (293.37 ± 47.69) SFU/ 1,000,000 cells, the cellular immunization value of HBcAg is (347.25 ± 65.53) SFU/ 1,000,000 cells, the humoral immunization value is (415.38 ± 77.26) U/ml, be better than the muscle injection mode immune group, its value is respectively (185.57 ± 67.24) SFU/ 1,000,000 cells, (332.46 ± 97.06) U/ml, (257.39 ± 63.17) SFU/ 1,000,000 cells, difference has significance (p<0.05).

The nucleotides sequence list

<110 > Beijing KaiYin Bioisystech Co., Ltd, Beijing KaiYin Science Co., Ltd

<120 > a kind of recombiant plasmid vaccine and compositions thereof that is used for the treatment of hepatitis B

<160>11

<210>1

<211>6848

<212>DNA

<213 > synthetic

<400>1

GAATTCTGGT TGCTGACTAA TTGAGATGCA TGCTTTGCAT ACTTCTGCCT GCTGGGGAGC 60

CTGGGGACTT TCCACACCCA TTACCGCCAT GTTGACATTG ATTATTGACT AGTTATTAAT 120

AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC 180

TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA 240

TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA TGGGTGGAGT 300

ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA AGTACGCCCC 360

CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT 420

GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC 480

GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC 540

TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG GACTTTCCAA 600

AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA CGGTGGGAGG 660

TCTATATAAG CAGAGCTCGT TTAGTGAACC GTCAGATCGC CTGGAGACGC CATCCACGCT 720

GTTTTGACCT CCATAGAAGA CACCGGGACC GATCCAGCCT CCGCGGCCGG GAACGGTGCA 780

TTGGAACGCG GATTCCCCGT GCCAAGAGTG ACGTAAGTAC CGCCTATAGA CTCTATAGGC 840

ACACCCCTTT GGCTCTTATG CATGCTATAC TGTTTTTGGC TTGGGGCCTA TACACCCCCG 900

CTTCCTTATG CTATAGGTGA TGGTATAGCT TAGCCTATAG GTGTGGGTTA TTGACCATTA 960

TTGACCACTC CCCTATTGGT GACGATACTT TCCATTACTA ATCCATAACA TGGCTCTTTG 1020

CCACAACTAT CTCTATTGGC TATATGCCAA TACTCTGTCC TTCAGAGACT GACACGGACT 1080

CTGTATTTTT ACAGGATGGG GTCCCATTTA TTATTTACAA ATTCACATAT ACAACAACGC 1140

CGTCCCCCGT GCCCGCAGTT TTTATTAAAC ATAGCGTGGG ATCTCCACGC GAATCTCGGG 1200

TACGTGTTCC GGACATGGGC TCTTCTCCGG TAGCGGCGGA GCTTCCACAT CCGAGCCCTG 1260

GTCCCATGCC TCCAGCGGCT CATGGTCGCT CGGCAGCTCC TTGCTCCTAA CAGTGGAGGC 1320

CAGACTTAGG CACAGCACAA TGCCCACCAC CACCAGTGTG CCGCACAAGG CCGTGGCGGT 1380

AGGGTATGTG TCTGAAAATG AGCGTGGAGA TTGGGCTCGC ACGGCTGACG CAGATGGAAG 1440

ACTTAAGGCA GCGGCAGAAG AAGATGCAGG CAGCTGAGTT GTTGTATTCT GATAAGAGTC 1500

AGAGGTAACT CCCGTTGCGG TGCTGTTAAC GGTGGAGGGC AGTGTAGTCT GAGCAGTACT 1560

CGTTGCTGCC GCGCGCGCCA CCAGACATAA TAGCTGACAG ACTAACAGAC TGTTCCTTTC 1620

CATGGGTCTT TTCTGCAGTC ACCGTCGTCG AGCCGCCACC ATGCAGTGGA ACTCTACCAC 1680

ATTCCATCAG GCTCTGCTGG ACCCTAGAGT GAGGGGACTC TATTTCCCCG CCGGAGGAAG 1740

CTCCTCTGGC ACTGTGAACC CAGTGCCCAC AACCGCAAGC CCTATTTCCT CTATCTTTAG 1800

CCGGACCGGA GACCCCGCTC CTAATATGGA AAACACTACC TCCGGCTTTC TGGGTCCACT 1860

GCTCGTCCTG CAGGCCGGCT TCTTCCTGCT CACCAGAATC CTGACAATCC CCCAGAGCCT 1920

GGACTCTTGG TGGACATCCC TGAACTTCCT CGGAGGCGCA CCCACTTGTC CTGGTCAGAA 1980

TAGCCAGTCC CCAACCAGCA ACCACTCCCC TACATCTTGT CCCCCAATTT GCCCTGGCTA 2040

CAGGTGGATG TGTCTGAGAC GCTTTATCAT TTTCCTGTTT ATCCTGCTCC TGTGCCTGAT 2100

TTTCCTGCTC GTGCTGCTGG ACTACCAGGG AATGCTCCCC GTGTGTCCAC TGCTCCCCGG 2160

AACCAGCACC ACTTCCACCG GTCCTTGTAA GACATGCACA ATCCCCGCTC AGGGCACCAG 2220

CATGTTTCCA TCCTGTTGCT GTACCAAACC CAGCGACGGC AACTGCACTT GTATTCCCAT 2280

CCCTTCTTCC TGGGCCTTCG CCAGATTCCT GTGGGAATGG GCAAGCGTCA GGTTCTCCTG 2340

GCTGAGCCTG CTCGTGCCCT TTGTGCAGTG GTTTGTCGGC CTGTCTCCCA CAGTGTGGCT 2400

GAGCGTGATT TGGATGATGT GGTATTGGGG ACCATCCCTG TATAATATCC TCTCTCCCTT 2460

CCTGCCTCTG CTCCCAATTT TCTTTTGCCT GTGGGTTTAC ATCTGATTCT AGAGCATGCG 2520

TGGAACCTTT GTGGCTCCTC TGCGATCCAT ACTGCGGAAC TCCTAGCCGC TTGTTTTGCT 2580

CGCAGCCGGT CTGGAGCAAA GCTCATCGGA ACTGACAATT CTGTCGTCCT CTCGCGGAAA 2640

TATACATCGT TTCCATGGCT GCTAGGCTGT ACTGCCAACT GGATCCTTCG CGGGACGTCC 2700

TTTGTTTACG TCCCGTCGGC GCTGAATCCC GCGGACGACC CCTCTCGGGG CCGCTTGGGA 2760

CTCTCTCGTC CCCTCTCCGT CTGCCGTTCC AGCCGACCAC GGGGCGCACC TCTCTTTACG 2820

CCGGTCTCCC CGTCTGTGCC TTCTCATCTG CCGGTCCGTG TGCACTTCGC TTCACCTCTG 2880

CACGTTGCAT GGAGACCACC GTGAACGCCC ATCAGATCCT GCCCAAGGTC TTACATAAGA 2940

GGACTCTTGG ACTCCCAGCA ATGTCAACGA CCGACCTTGA GGCCTACTTC AAAGACTGTG 3000

TGTTTAAGGA CTGGGAGGAG CTGGGGGAGG AGATTAGGTT AAAGGTCTTT GTATTAGGAG 3060

GCTGTAGGCA CAAATTGGTC TGCGCAAGAT CCGCTGTGCC TTCTAGTTGC CAGCCATCTG 3120

TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG TGCCACTCCC ACTGTCCTTT 3180

CCTAATAAAA TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG 3240

GTGGGGTGGG GCAGGACAGC AAGGGGGAGG ATTGGGAAGA CAATAGCAGG CATGCTGGGG 3300

ATGCGGTGGG CTTATGAGAT CTCCCGATCC CCTATGGTGC ACTCTCAGTA CAATCTGCTC 3360

TGATGCCGCA TAGTTAAGCC AGTATCTGCT CCCTGCTTGT GTGTTGGAGG TCGCTGAGTA 3420

GTGCGCGAGC AAAATTTAAG CTACAACAAG GCAAGGCTTG ACCGACAATT GCATGAAGAA 3480

TCTGCTTAGG GTTAGGCGTT TTGCGCTGCT TCGCGATGTA CGGGCCAGAT ATACGCGTTG 3540

ACATTGATTA TTGACTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC 3600

ATATATGGAG TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA 3660

CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC CAATAGGGAC 3720

TTTCCATTGA CGTCAATGGG TGGAGTATTT ACGGTAAACT GCCCACTTGG CAGTACATCA 3780

AGTGTATCAT ATGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGGTAAAT GGCCCGCCTG 3840

GCATTATGCC CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT 3900

AGTCATCGCT ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG 3960

GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA GTTTGTTTTG 4020

GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT TGACGCAAAT 4080

GGGCGGTAGG CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTCTCTGGC TAACTAGAGA 4140

ACCCACTGCT TACTGGCTTA TCGAAATTAA TACGACTCAC TATAGGGAGA CCCAAGCTGG 4200

CTAGCGTTTA AACTTAAGCT TGCCGCCACC ATGGATATTG ATCCTTACAA AGAGTTTGGC 4260

GCCTCTGTGG AACTGCTCAG CTTCCTGCCC TCTGACTTCT TTCCATCCAT TAGAGACCTG 4320

CTCGATACCG CCAGCGCACT GTATCGCGAG GCCCTGGAAT CCCCTGAGCA CTGCAGCCCT 4380

CACCATACAG CCCTGAGACA GGCTATCCTC TGTTGGGGAG AACTGATGAA CCTGGCAACC 4440

TGGGTCGGCT CCAACCTGGA GGACCCAGCC TCTAGGGAAC TCGTGGTGAG CTACGTCAAT 4500

GTGAACATGG GCCTGAAGAT TAGACAGCTC CTGTGGTTCC ACATCTCCTG CCTCACCTTC 4560

GGTCGGGAGA CTGTGCTGGA ATATCTGGTG AGCTTTGGCG TTTGGATTAG AACTCCTCCT 4620

GCTTACAGGC CTCCAAATGC CCCTATCCTG TCCACCCTCC CTGAGACCAC CGTGGTCAGA 4680

CGCAGAGGCA GGTCTCCAAG ACGGAGGACA CCCAGCCCTA GACGCAGGAG ATCACAGTCC 4740

CCTAGACGGA GAAGGTCTCA GAGCCGCGAA TCCCAGTGTT GATTCTAGAG GGCCCGTTTA 4800

AACCCGCTGA TCAGCCTCGA CTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTC 4860

CCCCGTGCCT TCCTTGACCC TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGA 4920

GGAAATTGCA TCGCATTGTC TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCA 4980

GGACAGCAAG GGGGAGGATT GGGAAGACAA TAGCAGGCAT GCTGGGGATG CGGTGGGCTC 5040

TATGGCTTCT GAGGCGGAAA GAACCAGATC AACGTTGTCG ACGGTACCAT TTAAATTTGA 5100

ACGTTCGCAA TATGTGAGCA AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AAGGCCGCGT 5160

TGCTGGCGTT TTTCCATAGG CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA 5220

GTCAGAGGTG GCGAAACCCG ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT 5280

CCCTCGTGCG CTCTCCTGTT CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC 5340

CTTCGGGAAG CGTGGCGCTT TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG 5400

TCGTTCGCTC CAAGCTGGGC TGTGTGCACG AACCCCCCGT TCAGCCCGAC CGCTGCGCCT 5460

TATCCGGTAA CTATCGTCTT GAGTCCAACC CGGTAAGACA CGACTTATCG CCACTGGCAG 5520

CAGCCACTGG TAACAGGATT AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA 5580

AGTGGTGGCC TAACTACGGC TACACTAGAA GAACAGTATT TGGTATCTGC GCTCTGCTGA 5640

AGCCAGTTAC CTTCGGAAAA AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG 5700

GTAGCGGTGG TTTTTTTGTT TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG 5760

AAGATCCTTT GATCTTTTCT ACGGGTTGAA CGTTCGCAAT CGTCGTTTTC GGCGCGCGCC 5820

GTTGAACGTT CGCAATTCAA GTCAGCGTAA TGCTCTGCCA GTGTTACAAC CAATTAACCA 5880

ATTCTGATTA GAAAAACTCA TCGAGCATCA AATGAAACTG CAATTTATTC ATATCAGGAT 5940

TATCAATACC ATATTTTTGA AAAAGCCGTT TCTGTAATGA AGGAGAAAAC TCACCGAGGC 6000

AGTTCCATAG GATGGCAAGA TCCTGGTATC GGTCTGCGAT TCCGACTCGT CCAACATCAA 6060

TACAACCTAT TAATTTCCCC TCGTCAAAAA TAAGGTTATC AAGTGAGAAA TCACCATGAG 6120

TGACGACTGA ATCCGGTGAG AATGGCAAAA GCTTATGCAT TTCTTTCCAG ACTTGTTCAA 6180

CAGGCCAGCC ATTACGCTCG TCATCAAAAT CACTCGCATC AACCAAACCG TTATTCATTC 6240

GTGATTGCGC CTGAGCGAGA CGAAATACGC GATCGCTGTT AAAAGGACAA TTACAAACAG 6300

GAATCGAATG CAACCGGCGC AGGAACACTG CCAGCGCATC AACAATATTT TCACCTGAAT 6360

CAGGATATTC TTCTAATACC TGGAATGCTG TTTTCCCGGG GATCGCAGTG GTGAGTAACC 6420

ATGCATCATC AGGAGTACGG ATAAAATGCT TGATGGTCGG AAGAGGCATA AATTCCGTCA 6480

GCCAGTTTAG TCTGACCATC TCATCTGTAA CATCATTGGC AACGCTACCT TTGCCATGTT 6540

TCAGAAACAA CTCTGGCGCA TCGGGCTTCC CATACAATCG ATAGATTGTC GCACCTGATT 6600

GCCCGACATT ATCGCGAGCC CATTTATACC CATATAAATC AGCATCCATG TTGGAATTTA 6660

ATCGCGGCCT CGAGCAAGAC GTTTCCCGTT GAATATGGCT CATAACACCC CTTGTATTAC 6720

TGTTTATGTA AGCAGACAGT TTTATTGTTC ATGATGATAT ATTTTTATCT TGTGCAATGT 6780

AACATCAGAG ATTTTGAGAC ACAACGTGGC TTTCTTGAAC GTTCGCATCG TCGTTTTCGG 6840

CGCGCGCC 6848

<210>2

<211>6849

<212>DNA

<213 > synthetic

<400>2

GAATTCTGGT TGCTGACTAA TTGAGATGCA TGCTTTGCAT ACTTCTGCCT GCTGGGGAGC 60

CTGGGGACTT TCCACACCCA TTACCGCCAT GTTGACATTG ATTATTGACT AGTTATTAAT 120

AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC 180

TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA 240

TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA TGGGTGGAGT 300

ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA AGTACGCCCC 360

CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT 420

GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC 480

GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC 540

TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG GACTTTCCAA 600

AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA CGGTGGGAGG 660

TCTATATAAG CAGAGCTCGT TTAGTGAACC GTCAGATCGC CTGGAGACGC CATCCACGCT 720

GTTTTGACCT CCATAGAAGA CACCGGGACC GATCCAGCCT CCGCGGCCGG GAACGGTGCA 780

TTGGAACGCG GATTCCCCGT GCCAAGAGTG ACGTAAGTAC CGCCTATAGA CTCTATAGGC 840

ACACCCCTTT GGCTCTTATG CATGCTATAC TGTTTTTGGC TTGGGGCCTA TACACCCCCG 900

CTTCCTTATG CTATAGGTGA TGGTATAGCT TAGCCTATAG GTGTGGGTTA TTGACCATTA 960

TTGACCACTC CCCTATTGGT GACGATACTT TCCATTACTA ATCCATAACA TGGCTCTTTG 1020

CCACAACTAT CTCTATTGGC TATATGCCAA TACTCTGTCC TTCAGAGACT GACACGGACT 1080

CTGTATTTTT ACAGGATGGG GTCCCATTTA TTATTTACAA ATTCACATAT ACAACAACGC 1140

CGTCCCCCGT GCCCGCAGTT TTTATTAAAC ATAGCGTGGG ATCTCCACGC GAATCTCGGG 1200

TACGTGTTCC GGACATGGGC TCTTCTCCGG TAGCGGCGGA GCTTCCACAT CCGAGCCCTG 1260

GTCCCATGCC TCCAGCGGCT CATGGTCGCT CGGCAGCTCC TTGCTCCTAA CAGTGGAGGC 1320

CAGACTTAGG CACAGCACAA TGCCCACCAC CACCAGTGTG CCGCACAAGG CCGTGGCGGT 1380

AGGGTATGTG TCTGAAAATG AGCGTGGAGA TTGGGCTCGC ACGGCTGACG CAGATGGAAG 1440

ACTTAAGGCA GCGGCAGAAG AAGATGCAGG CAGCTGAGTT GTTGTATTCT GATAAGAGTC 1500

AGAGGTAACT CCCGTTGCGG TGCTGTTAAC GGTGGAGGGC AGTGTAGTCT GAGCAGTACT 1560

CGTTGCTGCC GCGCGCGCCA CCAGACATAA TAGCTGACAG ACTAACAGAC TGTTCCTTTC 1620

CATGGGTCTT TTCTGCAGTC ACCGTCGTCG AGCCGCCACC ATGGATATTG ATCCTTACAA 1680

AGAGTTTGGC GCCTCTGTGG AACTGCTCAG CTTCCTGCCC TCTGACTTCT TTCCATCCAT 1740

TAGAGACCTG CTCGATACCG CCAGCGCACT GTATCGCGAG GCCCTGGAAT CCCCTGAGCA 1800

CTGTAGCCCT CACCATACAG CCCTGAGACA GGCTATCCTC TGTTGGGGAG AACTGATGAA 1860

CCTGGCAACC TGGGTCGGCT CCAACCTGGA GGACCCAGCC TCTAGGGAAC TCGTGGTGAG 1920

CTACGTCAAT GTGAACATGG GCCTGAAGAT TAGACAGCTG CTGTGGTTCC ACATCTCCTG 1980

CCTCACCTTC GGTCGGGAGA CTGTGCTGGA ATATCTGGTG AGCTTTGGCG TTTGGATTAG 2040

AACTCCTCCT GCTTACAGGC CTCCAAATGC CCCTATCCTG TCCACCCTCC CTGAGACCAC 2100

CGTGGTCAGA CGCAGAGGCA GGTCTCCAAG ACGGAGGACA CCCAGCCCTA GACGCAGGAG 2160

ATCACAGTCC CCTAGACGGA GAAGGTCTCA GAGCCGCGAA TCCCAGTGTT GATTCTAGAG 2220

CATGCGTGGA ACCTTTGTGG CTCCTCTGCG ATCCATACTG CGGAACTCCT AGCCGCTTGT 2280

TTTGCTCGCA GCCGGTCTGG AGCAAAGCTC ATCGGAACTG ACAATTCTGT CGTCCTCTCG 2340

CGGAAATATA CATCGTTTCC ATGGCTGCTA GGCTGTACTG CCAACTGGAT CCTTCGCGGG 2400

ACGTCCTTTG TTTACGTCCC GTCGGCGCTG AATCCCGCGG ACGACCCCTC TCGGGGCCGC 2460

TTGGGACTCT CTCGTCCCCT CTCCGTCTGC CGTTCCAGCC GACCACGGGG CGCACCTCTC 2520

TTTACGCCGG TCTCCCCGTC TGTGCCTTCT CATCTGCCGG TCCGTGTGCA CTTCGCTTCA 2580

CCTCTGCACG TTGCATGGAG ACCACCGTGA ACGCCCATCA GATCCTGCCC AAGGTCTTAC 2640

ATAAGAGGAC TCTTGGACTC CCAGCAATGT CAACGACCGA CCTTGAGGCC TACTTCAAAG 2700

ACTGTGTGTT TAAGGACTGG GAGGAGCTGG GGGAGGAGAT TAGGTTAAAG GTCTTTGTAT 2760

TAGGAGGCTG TAGGCACAAA TTGGTCTGCG CAAGATCCGC TGTGCCTTCT AGTTGCCAGC 2820

CATCTGTTGT TTGCCCCTCC CCCGTGCCTT CCTTGACCCT GGAAGGTGCC ACTCCCACTG 2880

TCCTTTCCTA ATAAAATGAG GAAATTGCAT CGCATTGTCT GAGTAGGTGT CATTCTATTC 2940

TGGGGGGTGG GGTGGGGCAG GACAGCAAGG GGGAGGATTG GGAAGACAAT AGCAGGCATG 3000

CTGGGGATGC GGTGGGCTCT ATGAGATCTC CCGATCCCCT ATGGTGCACT CTCAGTACAA 3060

TCTGCTCTGA TGCCGCATAG TTAAGCCAGT ATCTGCTCCC TGCTTGTGTG TTGGAGGTCG 3120

CTGAGTAGTG CGCGAGCAAA ATTTAAGCTA CAACAAGGCA AGGCTTGACC GACAATTGCA 3180

TGAAGAATCT GCTTAGGGTT AGGCGTTTTG CGCTGCTTCG CGATGTACGG GCCAGATATA 3240

CGCGTTGACA TTGATTATTG ACTAGTTATT AATAGTAATC AATTACGGGG TCATTAGTTC 3300

ATAGCCCATA TATGGAGTTC CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC 3360

CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA 3420

TAGGGACTTT CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG 3480

TACATCAAGT GTATCATATG CCAAGTACGC CCCCTATTGA CGTCAATGAC GGTAAATGGC 3540

CCGCCTGGCA TTATGCCCAG TACATGACCT TATGGGACTT TCCTACTTGG CAGTACATCT 3600

ACGTATTAGT CATCGCTATT ACCATGGTGA TGCGGTTTTG GCAGTACATC AATGGGCGTG 3660

GATAGCGGTT TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT 3720

TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAACAACTCC GCCCCATTGA 3780

CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT CTCTGGCTAA 3840

CTAGAGAACC CACTGCTTAC TGGCTTATCG AAATTAATAC GACTCACTAT AGGGAGACCC 3900

AAGCTGGCTA GCGTTTAAAC TTAAGCTTGC CGCCACCATG CAGTGGAACT CTACCACATT 3960

CCATCAGGCT CTGCTGGACC CTAGAGTGAG GGGACTCTAT TTCCCCGCCG GAGGAAGCTC 4020

CTCTGGCACT GTGAACCCAG TGCCCACAAC CGCAAGCCCT ATTTCCTCTA TCTTTAGCCG 4080

GACCGGAGAC CCCGCTCCTA ATATGGAAAA CACTACCTCC GGCTTTCTGG GTCCACTGCT 4140

CGTCCTGCAG GCCGGCTTCT TCCTGCTCAC CAGAATCCTG ACAATCCCCC AGAGCCTGGA 4200

CTCTTGGTGG ACATCCCTGA ACTTCCTCGG AGGCGCACCC ACTTGTCCTG GTCAGAATAG 4260

CCAGTCCCCA ACCAGCAACC ACTCCCCTAC ATCTTGTCCC CCAATTTGCC CTGGCTACAG 4320

GTGGATGTGT CTGAGACGCT TTATCATTTT CCTGTTTATC CTGCTCCTGT GCCTGATTTT 4380

CCTGCTCGTG CTGCTGGACT ACCAGGGAAT GCTCCCCGTG TGTCCACTGC TCCCCGGAAC 4440

CAGCACCACT TCCACCGGTC CTTGTAAGAC ATGCACAATC CCCGCTCAGG GCACCAGCAT 4500

GTTTCCATCC TGTTGCTGTA CCAAACCCAG CGACGGCAAC TGCACTTGTA TTCCCATCCC 4560

TTCTTCCTGG GCCTTCGCCA GATTCCTGTG GGAATGGGCA AGCGTCAGGT TCTCCTGGCT 4620

GAGCCTGCTC GTGCCCTTTG TGCAGTGGTT TGTCGGCCTG TCTCCCACAG TGTGGCTGAG 4680

CGTGATTTGG ATGATGTGGT ATTGGGGACC ATCCCTGTAT AATATCCTCT CTCCCTTCCT 4740

GCCTCTGCTC CCAATTTTCT TTTGCCTGTG GGTTTACATC TGATTCTAGA GGGCCCGTTT 4800

AAACCCGCTG ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT GTTTGCCCCT 4860

CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC TAATAAAATG 4920

AGGAAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT GGGGTGGGGC 4980

AGGACAGCAA GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT GCGGTGGGCT 5040

CTATGGCTTC TGAGGCGGAA AGAACCAGAT CAACGTTGTC GACGGTACCA TTTAAATTTG 5100

AACGTTCGCA ATATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA AAAGGCCGCG 5160

TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA TCGACGCTCA 5220

AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC 5280

TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC 5340

CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG TTCGGTGTAG 5400

GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA CCGCTGCGCC 5460

TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC GCCACTGGCA 5520

GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC AGAGTTCTTG 5580

AAGTGGTGGC CTAACTACGG CTACACTAGA AGAACAGTAT TTGGTATCTG CGCTCTGCTG 5640

AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA AACCACCGCT 5700

GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC GCAGAAAAAA AGGATCTCAA 5760

GAAGATCCTT TGATCTTTTC TACGGGTTGA ACGTTCGCAA TCGTCGTTTT CGGCGCGCGC 5820

CGTTGAACGT TCGCAATTCA AGTCAGCGTA ATGCTCTGCC AGTGTTACAA CCAATTAACC 5880

AATTCTGATT AGAAAAACTC ATCGAGCATC AAATGAAACT GCAATTTATT CATATCAGGA 5940

TTATCAATAC CATATTTTTG AAAAAGCCGT TTCTGTAATG AAGGAGAAAA CTCACCGAGG 6000

CAGTTCCATA GGATGGCAAG ATCCTGGTAT CGGTCTGCGA TTCCGACTCG TCCAACATCA 6060

ATACAACCTA TTAATTTCCC CTCGTCAAAA ATAAGGTTAT CAAGTGAGAA ATCACCATGA 6120

GTGACGACTG AATCCGGTGA GAATGGCAAA AGCTTATGCA TTTCTTTCCA GACTTGTTCA 6180

ACAGGCCAGC CATTACGCTC GTCATCAAAA TCACTCGCAT CAACCAAACC GTTATTCATT 6240

CGTGATTGCG CCTGAGCGAG ACGAAATACG CGATCGCTGT TAAAAGGACA ATTACAAACA 6300

GGAATCGAAT GCAACCGGCG CAGGAACACT GCCAGCGCAT CAACAATATT TTCACCTGAA 6360

TCAGGATATT CTTCTAATAC CTGGAATGCT GTTTTCCCGG GGATCGCAGT GGTGAGTAAC 6420

CATGCATCAT CAGGAGTACG GATAAAATGC TTGATGGTCG GAAGAGGCAT AAATTCCGTC 6480

AGCCAGTTTA GTCTGACCAT CTCATCTGTA ACATCATTGG CAACGCTACC TTTGCCATGT 6540

TTCAGAAACA ACTCTGGCGC ATCGGGCTTC CCATACAATC GATAGATTGT CGCACCTGAT 6600

TGCCCGACAT TATCGCGAGC CCATTTATAC CCATATAAAT CAGCATCCAT GTTGGAATTT 6660

AATCGCGGCC TCGAGCAAGA CGTTTCCCGT TGAATATGGC TCATAACACC CCTTGTATTA 6720

CTGTTTATGT AAGCAGACAG TTTTATTGTT CATGATGATA TATTTTTATC TTGTGCAATG 6780

TAACATCAGA GATTTTGAGA CACAACGTGG CTTTCTTGAA CGTTCGCATC GTCGTTTTCG 6840

GCGCGCGCC 6849

<210>3

<211>5693

<212>DNA

<213 > synthetic

<400>3

GAATTCTGGT TGCTGACTAA TTGAGATGCA TGCTTTGCAT ACTTCTGCCT GCTGGGGAGC 60

CTGGGGACTT TCCACACCCA TTACCGCCAT GTTGACATTG ATTATTGACT AGTTATTAAT 120

AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC 180

TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA 240

TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA TGGGTGGAGT 300

ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA AGTACGCCCC 360

CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT 420

GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC 480

GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC 540

TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG GACTTTCCAA 600

AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA CGGTGGGAGG 660

TCTATATAAG CAGAGCTCTC TGGCTAACTA GAGAACCCAC TGCTTACTGG CTTATCGAAA 720

TTAATACGAC TCACTATAGG GAGACCCAAG CTGGCTAGCG TTTAAACTTA AGCTTGCCGC 780

CACCATGTGT CCTGCTAGAT CACTGCTTCT GGTTGCCACA CTCGTGCTGC TGGACCACCT 840

TAGCCTGGCA AGGAACCTGC CAGTGGCTAC TCCCGATCCT GGCATGTTCC CATGCCTCCA 900

CCATTCCCAG AATCTGCTGA GAGCCGTGAG CAACATGCTT CAGAAGGCCA GGCAGACCCT 960

GGAGTTCTAC CCATGTACAT CTGAGGAAAT CGACCACGAG GACATCACCA AGGACAAAAC 1020

TTCCACCGTC GAAGCATGCC TGCCTCTGGA GCTCACAAAG AATGAGAGCT GTCTGAACTC 1080

CAGAGAAACT TCCTTCATTA CCAATGGAAG CTGCCTGGCC TCTCGCAAAA CATCCTTTAT 1140

GATGGCTCTG TGTCTTAGCT CTATCTATGA GGATCTGAAG ATGTACCAGG TGGAGTTCAA 1200

GACTATGAAC GCCAAACTGC TCATGGACCC AAAGAGGCAG ATTTTCCTGG ACCAGAATAT 1260

GCTGGCAGTG ATTGACGAGC TGATGCAGGC CCTGAACTTC AACTCCGAGA CCGTGCCCCA 1320

GAAAAGCTCT CTTGAAGAGC CTGATTTCTA CAAGACTAAG ATCAAACTGT GCATTCTGCT 1380

CCATGCTTTC AGAATCAGGG CCGTCACAAT CGACCGGGTG ATGTCCTACC TGAATGCCAG 1440

TTGATTCTAG AGGGCCCGTT TAAACCCGCT GATCAGCCTC GACTGTGCCT TCTAGTTGCC 1500

AGCCATCTGT TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA 1560

CTGTCCTTTC CTAATAAAAT GAGGAAATTG CATCGCATTG TCTGAGTAGG TGTCATTCTA 1620

TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA TTGGGAAGAC AATAGCAGGC 1680

ATGCTGGGGA TGCGGTGGGC TCTATGGCTT CTGAGGCGGA AAGAACCAGA TCAACGTTGT 1740

CGACGGATCG GGAGATCTCC CGATCCCCTA TGGTGCACTC TCAGTACAAT CTGCTCTGAT 1800

GCCGCATAGT TAAGCCAGTA TCTGCTCCCT GCTTGTGTGT TGGAGGTCGC TGAGTAGTGC 1860

GCGAGCAAAA TTTAAGCTAC AACAAGGCAA GGCTTGACCG ACAATTGCAT GAAGAATCTG 1920

CTTAGGGTTA GGCGTTTTGC GCTGCTTCGC GATGTACGGG CCAGATATAC GCGTTGACAT 1980

TGATTATTGA CTAGTTATTA ATAGTAATCA ATTACGGGGT CATTAGTTCA TAGCCCATAT 2040

ATGGAGTTCC GCGTTACATA ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC 2100

CCCCGCCCAT TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC 2160

CATTGACGTC AATGGGTGGA GTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG 2220

TATCATATGC CAAGTACGCC CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT 2280

TATGCCCAGT ACATGACCTT ATGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC 2340

ATCGCTATTA CCATGGTGAT GCGGTTTTGG CAGTACATCA ATGGGCGTGG ATAGCGGTTT 2400

GACTCACGGG GATTTCCAAG TCTCCACCCC ATTGACGTCA ATGGGAGTTT GTTTTGGCAC 2460

CAAAATCAAC GGGACTTTCC AAAATGTCGT AACAACTCCG CCCCATTGAC GCAAATGGGC 2520

GGTAGGCGTG TACGGTGGGA GGTCTATATA AGCAGAGCTC TCTGGCTAAC TAGAGAACCC 2580

ACTGCTTACT GGCTTATCGA AATTAATACG ACTCACTATA GGGAGACCCA AGCTGGCTAG 2640

CGTTTAAACT TAAGCTTGCC GCCACCATGT GTCATCAGCA GCTTGTTATC TCTTGGTTTA 2700

GCCTGGTGTT CCTGGCCTCT CCCCTGGTGG CAATCTGGGA GCTCAAGAAG GACGTGTACG 2760

TCGTGGAGCT GGATTGGTAT CCAGACGCTC CTGGCGAAAT GGTGGTTCTG ACCTGCGATA 2820

CACCCGAGGA AGACGGCATT ACTTGGACCC TGGATCAGAG CTCCGAGGTG CTTGGCTCCG 2880

GAAAGACACT GACTATCCAG GTGAAGGAGT TTGGCGACGC CGGCCAGTAC ACCTGTCACA 2940

AAGGAGGCGA AGTGCTGAGC CACTCTCTGC TCCTGCTTCA TAAGAAGGAG GATGGCATCT 3000

GGTCCACAGA CATTCTGAAA GATCAGAAGG AACCAAAGAA CAAGACTTTC CTGAGGTGCG 3060

AGGCCAAGAA TTATAGCGGA AGATTTACCT GTTGGTGGCT GACAACTATC TCTACCGACC 3120

TCACATTCTC CGTGAAAAGC TCTCGCGGCT CCAGCGATCC TCAGGGCGTC ACTTGCGGAG 3180

CAGCCACCCT GTCTGCTGAG AGGGTGAGAG GCGACAACAA GGAATACGAG TATTCCGTGG 3240

AATGTCAGGA GGATAGCGCA TGTCCCGCCG CTGAGGAATC TCTGCCAATC GAGGTGATGG 3300

TGGACGCCGT GCACAAACTG AAGTACGAAA ATTATACATC CAGCTTCTTC ATTCGGGACA 3360

TCATTAAGCC TGACCCACCT AAGAACCTGC AGCTCAAGCC TCTTAAGAAT AGCAGGCAGG 3420

TGGAGGTGTC CTGGGAGTAC CCCGATACTT GGAGCACACC ACACTCTTAT TTCAGCCTGA 3480

CCTTCTGCGT TCAGGTGCAG GGCAAGTCTA AGCGCGAGAA GAAGGACCGC GTGTTTACTG 3540

ATAAAACATC TGCAACCGTG ATCTGCAGGA AGAACGCTTC CATCAGCGTC AGAGCCCAGG 3600

ACCGGTACTA TTCTAGCTCC TGGTCTGAGT GGGCCTCCGT GCCTTGCAGT TGATTCTAGA 3660

GGGCCCGTTT AAACCCGCTG ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT 3720

GTTTGCCCCT CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC 3780

TAATAAAATG AGGAAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT 3840

GGGGTGGGGC AGGACAGCAA GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT 3900

GCGGTGGGCT CTATGGCTTC TGAGGCGGAA AGAACCAGAA ATTTGAACGT TCGCAATATG 3960

TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC 4020

CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA 4080

AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT 4140

CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG 4200

GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG 4260

CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT 4320

CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC 4380

AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC 4440

TACGGCTACA CTAGAAGAAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC 4500

GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT 4560

TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC 4620

TTTTCTACGG GTTGAACGTT CGCAATCGTC GTTTTCGGCG CGCGCCGTTG AACGTTCGCA 4680

ATTCAAGTCA GCGTAATGCT CTGCCAGTGT TACAACCAAT TAACCAATTC TGATTAGAAA 4740

AACTCATCGA GCATCAAATG AAACTGCAAT TTATTCATAT CAGGATTATC AATACCATAT 4800

TTTTGAAAAA GCCGTTTCTG TAATGAAGGA GAAAACTCAC CGAGGCAGTT CCATAGGATG 4860

GCAAGATCCT GGTATCGGTC TGCGATTCCG ACTCGTCCAA CATCAATACA ACCTATTAAT 4920

TTCCCCTCGT CAAAAATAAG GTTATCAAGT GAGAAATCAC CATGAGTGAC GACTGAATCC 4980

GGTGAGAATG GCAAAAGCTT ATGCATTTCT TTCCAGACTT GTTCAACAGG CCAGCCATTA 5040

CGCTCGTCAT CAAAATCACT CGCATCAACC AAACCGTTAT TCATTCGTGA TTGCGCCTGA 5100

GCGAGACGAA ATACGCGATC GCTGTTAAAA GGACAATTAC AAACAGGAAT CGAATGCAAC 5160

CGGCGCAGGA ACACTGCCAG CGCATCAACA ATATTTTCAC CTGAATCAGG ATATTCTTCT 5220

AATACCTGGA ATGCTGTTTT CCCGGGGATC GCAGTGGTGA GTAACCATGC ATCATCAGGA 5280

GTACGGATAA AATGCTTGAT GGTCGGAAGA GGCATAAATT CCGTCAGCCA GTTTAGTCTG 5340

ACCATCTCAT CTGTAACATC ATTGGCAACG CTACCTTTGC CATGTTTCAG AAACAACTCT 5400

GGCGCATCGG GCTTCCCATA CAATCGATAG ATTGTCGCAC CTGATTGCCC GACATTATCG 5460

CGAGCCCATT TATACCCATA TAAATCAGCA TCCATGTTGG AATTTAATCG CGGCCTCGAG 5520

CAAGACGTTT CCCGTTGAAT ATGGCTCATA ACACCCCTTG TATTACTGTT TATGTAAGCA 5580

GACAGTTTTA TTGTTCATGA TGATATATTT TTATCTTGTG CAATGTAACA TCAGAGATTT 5640

TGAGACACAA CGTGGCTTTC TTGAACGTTC GCATCGTCGT TTTCGGCGCG CGC 5693

<210>4

<211>5703

<212>DNA

<213 > synthetic

<400>4

GAATTCTGGT TGCTGACTAA TTGAGATGCA TGCTTTGCAT ACTTCTGCCT GCTGGGGAGC 60

CTGGGGACTT TCCACACCCA TTACCGCCAT GTTGACATTG ATTATTGACT AGTTATTAAT 120

AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC 180

TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA 240

TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA TGGGTGGAGT 300

ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA AGTACGCCCC 360

CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT 420

GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC 480

GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC 540

TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG GACTTTCCAA 600

AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA CGGTGGGAGG 660

TCTATATAAG CAGAGCTCTC TGGCTAACTA GAGAACCCAC TGCTTACTGG CTTATCGAAA 720

TTAATACGAC TCACTATAGG GAGACCCAAG CTGGCTAGCG TTTAAACTTA AGCTTGCCGC 780

CACCATGTGT CAGAGCAGGT ATCTGCTCTT TCTGGCTACC CTGGCCCTTC TGAATCACCT 840

GTCCCTCGCA AGGGTGATCC CCGTGTCTGG CCCTGCCCGG TGCCTGAGCC AGTCCAGGAA 900

CCTGCTTAAG ACAACTGACG ATATGGTGAA GACCGCTAGA GAGAAACTGA AGCACTACTC 960

TTGTACAGCC GAAGACATCG ATCATGAGGA CATTACTAGA GATCAGACCA GCACACTGAA 1020

AACTTGCCTG CCACTGGAAC TCCACAAGAA TGAGTCCTGT CTGGCAACCA GGGAGACATC 1080

TAGCACTACC AGAGGCTCCT GCCTGCCACC TCAGAAGACA TCTCTCATGA TGACTCTGTG 1140

TCTGGGAAGC ATCTATGAAG ACCTCAAAAT GTACCAGACC GAGTTCCAGG CCATCAACGC 1200

TGCCCTGCAG AATCATAACC ACCAGCAGAT TATCCTGGAT AAGGGCATGC TGGTCGCAAT 1260

CGACGAGCTT ATGCAGTCCC TGAATCACAA CGGCGAAACA CTGCGGCAGA AACCACCCGT 1320

GGGAGAAGCC GATCCTTATA GGGTGAAGAT GAAACTGTGC ATTCTCCTGC ATGCTTTTTC 1380

TACTAGAGTC GTGACCATCA ATCGCGTGAT GGGCTACCTG AGCTCCGCCT GATTCTAGAG 1440

GGCCCGTTTA AACCCGCTGA TCAGCCTCGA CTGTGCCTTC TAGTTGCCAG CCATCTGTTG 1500

TTTGCCCCTC CCCCGTGCCT TCCTTGACCC TGGAAGGTGC CACTCCCACT GTCCTTTCCT 1560

AATAAAATGA GGAAATTGCA TCGCATTGTC TGAGTAGGTG TCATTCTATT CTGGGGGGTG 1620

GGGTGGGGCA GGACAGCAAG GGGGAGGATT GGGAAGACAA TAGCAGGCAT GCTGGGGATG 1680

CGGTGGGCTC TATGGCTTCT GAGGCGGAAA GAACCAGATC AACGTTGTCG ACGGATCGGG 1740

AGATCTCCCG ATCCCCTATG GTGCACTCTC AGTACAATCT GCTCTGATGC CGCATAGTTA 1800

AGCCAGTATC TGCTCCCTGC TTGTGTGTTG GAGGTCGCTG AGTAGTGCGC GAGCAAAATT 1860

TAAGCTACAA CAAGGCAAGG CTTGACCGAC AATTGCATGA AGAATCTGCT TAGGGTTAGG 1920

CGTTTTGCGC TGCTTCGCGA TGTACGGGCC AGATATACGC GTTGACATTG ATTATTGACT 1980

AGTTATTAAT AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC 2040

GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG 2100

ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA 2160

TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA 2220

AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC 2280

ATGACCTTAT GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC 2340

ATGGTGATGC GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA 2400

TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG 2460

GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA 2520

CGGTGGGAGG TCTATATAAG CAGAGCTCTC TGGCTAACTA GAGAACCCAC TGCTTACTGG 2580

CTTATCGAAA TTAATACGAC TCACTATAGG GAGACCCAAG CTGGCTAGCG TTTAAACTTA 2640

AGCTTGCCGC CACCATGTGT CCACAGAAGC TCACAATCTC CTGGTTTGCA ATCGTGCTGC 2700

TGGTGAGCCC CCTGATGGCC ATGTGGGAGC TTGAGAAGGA CGTGTACGTC GTGGAAGTGG 2760

ATTGGACCCC TGACGCTCCA GGCGAGACTG TGAACCTGAC CTGCGATACA CCCGAAGAGG 2820

ACGATATTAC TTGGACCTCT GACCAGAGGC ACGGCGTTAT CGGAAGCGGC AAAACACTGA 2880

CTATCACCGT GAAGGAGTTC CTGGATGCCG GCCAGTATAC ATGTCACAAA GGAGGCGAAA 2940

CTCTCTCCCA TTCTCACCTG CTCCTGCATA AGAAAGAGAA TGGCATTTGG AGCACCGAAA 3000

TCCTGAAGAA CTTTAAAAAT AAGACATTCC TGAAATGCGA GGCACCTAAC TACTCCGGAA 3060

GATTTACTTG TTCTTGGCTG GTGCAGCGGA ATATGGACCT TAAGTTCAAC ATCAAGAGCT 3120

CCTCTAGCTC CCCAGATTCT AGGGCCGTGA CCTGCGGCAT GGCTAGCCTG TCCGCCGAGA 3180

AAGTCACACT GGACCAGAGA GATTATGAGA AGTACTCTGT GAGCTGTCAG GAAGACGTGA 3240

CTTGCCCCAC CGCAGAGGAA ACACTGCCTA TTGAGCTCGC CCTGGAGGCT CGCCAGCAGA 3300

ATAAGTATGA AAACTACTCC ACTTCTTTCT TTATCAGGGA CATCATTAAA CCAGACCCTC 3360

CCAAGAATCT GCAGATGAAA CCACTGAAGA ACAGCCAGGT GGAGGTCTCC TGGGAATATC 3420

CTGATTCTTG GAGCACCCCC CACTCCTACT TTTCTCTCAA GTTCTTTGTG AGAATCCAGC 3480

GGAAGAAAGA GAAGATGAAA GAGACAGAAG AGGGCTGTAA TCAGAAGGGA GCCTTCCTGG 3540

TGGAAAAGAC TAGCACCGAG GTTCAGTGCA AAGGCGGAAA CGTGTGTGTG CAGGCACAGG 3600

ACAGGTATTA CAATTCCTCT TGCAGCAAGT GGGCCTGTGT GCCCTGCAGA GTCAGGTCCT 3660

GATTCTAGAG GGCCCGTTTA AACCCGCTGA TCAGCCTCGA CTGTGCCTTC TAGTTGCCAG 3720

CCATCTGTTG TTTGCCCCTC CCCCGTGCCT TCCTTGACCC TGGAAGGTGC CACTCCCACT 3780

GTCCTTTCCT AATAAAATGA GGAAATTGCA TCGCATTGTC TGAGTAGGTG TCATTCTATT 3840

CTGGGGGGTG GGGTGGGGCA GGACAGCAAG GGGGAGGATT GGGAAGACAA TAGCAGGCAT 3900

GCTGGGGATG CGGTGGGCTC TATGGCTTCT GAGGCGGAAA GAACCAGAAA TTTGAACGTT 3960

CGCAATATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG 4020

GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG 4080

AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC 4140

GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG 4200

GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT 4260

CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC 4320

GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC 4380

ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG 4440

TGGCCTAACT ACGGCTACAC TAGAAGAACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA 4500

GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC 4560

GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT 4620

CCTTTGATCT TTTCTACGGG TTGAACGTTC GCAATCGTCG TTTTCGGCGC GCGCCGTTGA 4680

ACGTTCGCAA TTCAAGTCAG CGTAATGCTC TGCCAGTGTT ACAACCAATT AACCAATTCT 4720

GATTAGAAAA ACTCATCGAG CATCAAATGA AACTGCAATT TATTCATATC AGGATTATCA 4800

ATACCATATT TTTGAAAAAG CCGTTTCTGT AATGAAGGAG AAAACTCACC GAGGCAGTTC 4860

CATAGGATGG CAAGATCCTG GTATCGGTCT GCGATTCCGA CTCGTCCAAC ATCAATACAA 4920

CCTATTAATT TCCCCTCGTC AAAAATAAGG TTATCAAGTG AGAAATCACC ATGAGTGACG 4980

ACTGAATCCG GTGAGAATGG CAAAAGCTTA TGCATTTCTT TCCAGACTTG TTCAACAGGC 5040

CAGCCATTAC GCTCGTCATC AAAATCACTC GCATCAACCA AACCGTTATT CATTCGTGAT 5100

TGCGCCTGAG CGAGACGAAA TACGCGATCG CTGTTAAAAG GACAATTACA AACAGGAATC 5160

GAATGCAACC GGCGCAGGAA CACTGCCAGC GCATCAACAA TATTTTCACC TGAATCAGGA 5220

TATTCTTCTA ATACCTGGAA TGCTGTTTTC CCGGGGATCG CAGTGGTGAG TAACCATGCA 5280

TCATCAGGAG TACGGATAAA ATGCTTGATG GTCGGAAGAG GCATAAATTC CGTCAGCCAG 5340

TTTAGTCTGA CCATCTCATC TGTAACATCA TTGGCAACGC TACCTTTGCC ATGTTTCAGA 5400

AACAACTCTG GCGCATCGGG CTTCCCATAC AATCGATAGA TTGTCGCACC TGATTGCCCG 5460

ACATTATCGC GAGCCCATTT ATACCCATAT AAATCAGCAT CCATGTTGGA ATTTAATCGC 5520

GGCCTCGAGC AAGACGTTTC CCGTTGAATA TGGCTCATAA CACCCCTTGT ATTACTGTTT 5580

ATGTAAGCAG ACAGTTTTAT TGTTCATGAT GATATATTTT TATCTTGTGC AATGTAACAT 5640

CAGAGATTTT GAGACACAAC GTGGCTTTCT TGAACGTTCG CATCGTCGTT TTCGGCGCGC 5700

GCC 5703

<210>5

<211>872

<212>DNA

<213 > synthetic

<400>5

CCGCTCGAGC CGCCACCATG CAGTGGAACT CTACCACATT CCATCAGGCT CTGCTGGACC 60

CTAGAGTGAG GGGACTCTAT TTCCCCGCCG GAGGAAGCTC CTCTGGCACT GTGAACCCAG 120

TGCCCACAAC CGCAAGCCCT ATTTCCTCTA TCTTTAGCCG GACCGGAGAC CCCGCTCCTA 180

ATATGGAAAA CACTACCTCC GGCTTTCTGG GTCCACTGCT CGTCCTGCAG GCCGGCTTCT 240

TCCTGCTCAC CAGAATCCTG ACAATCCCCC AGAGCCTGGA CTCTTGGTGG ACATCCCTGA 300

ACTTCCTCGG AGGCGCACCC ACTTGTCCTG GTCAGAATAG CCAGTCCCCA ACCAGCAACC 360

ACTCCCCTAC ATCTTGTCCC CCAATTTGCC CTGGCTACAG GTGGATGTGT CTGAGACGCT 420

TTATCATTTT CCTGTTTATC CTGCTCCTGT GCCTGATTTT CCTGCTCGTG CTGCTGGACT 480

ACCAGGGAAT GCTCCCCGTG TGTCCACTGC TCCCCGGAAC CAGCACCACT TCCACCGGTC 540

CTTGTAAGAC ATGCACAATC CCCGCTCAGG GCACCAGCAT GTTTCCATCC TGTTGCTGTA 600

CCAAACCCAG CGACGGCAAC TGCACTTGTA TTCCCATCCC TTCTTCCTGG GCCTTCGCCA 660

GATTCCTGTG GGAATGGGCA AGCGTCAGGT TCTCCTGGCT GAGCCTGCTC GTGCCCTTTG 720

TGCAGTGGTT TGTCGGCCTG TCTCCCACAG TGTGGCTGAG CGTGATTTGG ATGATGTGGT 780

ATTGGGGACC ATCCCTGTAT AATATCCTCT CTCCCTTCCT GCCTCTGCTC CCAATTTTCT 840

TTTGCCTGTG GGTTTACATC TGATTCTAGA GC 872

<210>6

<211>950

<212>DNA

<213 > synthetic

<400>6

CCGCTCGAGC CGCCACCATG GATATTGATC CTTACAAAGA GTTTGGCGCC TCTGTGGAAC 60

TGCTCAGCTT CCTGCCCTCT GACTTCTTTC CATCCATTAG AGACCTGCTC GATACCGCCA 120

GCGCACTGTA TCGCGAGGCC CTGGAATCCC CTGAGCACTG TAGCCCTCAC CATACAGCCC 180

TGAGACAGGC TATCCTCTGT TGGGGAGAAC TGATGAACCT GGCAACCTGG GTCGGCTCCA 240

ACCTGGAGGA CCCAGCCTCT AGGGAACTCG TGGTGAGCTA CGTCAATGTG AACATGGGCC 300

TGAAGATTAG ACAGCTCCTG TGGTTCCACA TCTCCTGCCT CACCTTCGGT CGGGAGACTG 360

TGCTGGAATA TCTGGTGAGC TTTGGCGTTT GGATTAGAAC TCCTCCTGCT TACAGGCCTC 420

CAAATGCCCC TATCCTGTCC ACCCTCCCTG AGACCACCGT GGTCAGACGC AGAGGCAGGT 480

CTCCAAGACG GAGGACACCC AGCCCTAGAC GCAGGAGATC ACAGTCCCCT AGACGGAGAA 540

GGTCTCAGAG CCGCGAATCC CAGTGTGGCG GAGGCGGTAG CATGGGCGGA TGGTCTTCCA 600

AACCAAGACA GGGCATGGGC ACCAACCTGA GCGTGCCTAA CCCTCTGGGC TTCTTCCCAG 660

ATCACCAGCT CGACCCTGCA TTTGGCGCCA ATTCCAATAA CCCAGACTGG GATTTCAACC 720

CTAACAAGGA CCATTGGCCT GAGGCCAATC AGGTGGGTGC TGGCGCCTTC GGACCCGGAT 780

TTACACCACC CCACGGTGGC CTGCTGGGTT GGAGCCCTCA GGCCCAGGGC ATTCTCACCA 840

CTGTTCCCGC AGCTCCACCT CCTGCATCTA CCAACAGGCA GTCCGGAAGA CAGCCAACTC 900

CCATCAGCCC TCCTCTGCGG GATAGCCACC CTCAGGCTTG ATTCTAGAGC 950

<210>7

<211>675

<212>DNA

<213 > synthetic

<400>7

CCCAAGCTTG CCGCCACCAT GTGTCAGAGC AGGTATCTGC TCTTTCTGGC TACCCTGGCC 60

CTTCTGAATC ACCTGTCCCT CGCAAGGGTG ATCCCCGTGT CTGGCCCTGC CCGGTGCCTG 120

AGCCAGTCCA GGAACCTGCT TAAGACAACT GACGATATGG TGAAGACCGC TAGAGAGAAA 180

CTGAAGCACT ACTCTTGTAC AGCCGAAGAC ATCGATCATG AGGACATTAC TAGAGATCAG 240

ACCAGCACAC TGAAAACTTG CCTGCCACTG GAACTCCACA AGAATGAGTC CTGTCTGGCA 300

ACCAGGGAGA CATCTAGCAC TACCAGAGGC TCCTGCCTGC CACCTCAGAA GACATCTCTC 360

ATGATGACTC TGTGTCTGGG AAGCATCTAT GAAGACCTCA AAATGTACCA GACCGAGTTC 420

CAGGCCATCA ACGCTGCCCT GCAGAATCAT AACCACCAGC AGATTATCCT GGATAAGGGC 480

ATGCTGGTCG CAATCGACGA GCTTATGCAG TCCCTGAATC ACAACGGCGA AACACTGCGG 540

CAGAAACCAC CCGTGGGAGA AGCCGATCCT TATAGGGTGA AGATGAAACT GTGCATTCTC 600

CTGCATGCTT TTTCTACTAG AGTCGTGACC ATCAATCGCG TGATGGGCTA CCTGAGCTCC 660

GCCTGATTCT AGAGC 675

<210>8

<211>1035

<212>DNA

<213 > synthetic

<400>8

CCCAAGCTTG CCGCCACCAT GTGTCCACAG AAGCTCACAA TCTCCTGGTT TGCAATCGTG 60

CTGCTGGTGA GCCCCCTGAT GGCCATGTGG GAGCTTGAGA AGGACGTGTA CGTCGTGGAA 120

GTGGATTGGA CCCCTGACGC TCCAGGCGAG ACTGTGAACC TGACCTGCGA TACACCCGAA 180

GAGGACGATA TTACTTGGAC CTCTGACCAG AGGCACGGCG TTATCGGAAG CGGCAAAACA 240

CTGACTATCA CCGTGAAGGA GTTCCTGGAT GCCGGCCAGT ATACATGTCA CAAAGGAGGC 300

GAAACTCTCT CCCATTCTCA CCTGCTCCTG CATAAGAAAG AGAATGGCAT TTGGAGCACC 360

GAAATCCTGA AGAACTTTAA AAATAAGACA TTCCTGAAAT GCGAGGCACC TAACTACTCC 420

GGAAGATTTA CTTGTTCTTG GCTGGTGCAG CGGAATATGG ACCTTAAGTT CAACATCAAG 480

AGCTCCTCTA GCTCCCCAGA TTCTAGGGCC GTGACCTGCG GCATGGCTAG CCTGTCCGCC 540

GAGAAAGTCA CACTGGACCA GAGAGATTAT GAGAAGTACT CTGTGAGCTG TCAGGAAGAC 600

GTGACTTGCC CCACCGCAGA GGAAACACTG CCTATTGAGC TCGCCCTGGA GGCTCGCCAG 660

CAGAATAAGT ATGAAAACTA CTCCACTTCT TTCTTTATCA GGGACATCAT TAAACCAGAC 720

CCTCCCAAGA ATCTGCAGAT GAAACCACTG AAGAACAGCC AGGTGGAGGT CTCCTGGGAA 780

TATCCTGATT CTTGGAGCAC CCCCCACTCC TACTTTTCTC TCAAGTTCTT TGTGAGAATC 840

CAGCGGAAGA AAGAGAAGAT GAAAGAGACA GAAGAGGGCT GTAATCAGAA GGGAGCCTTC 900

CTGGTGGAAA AGACTAGCAC CGAGGTTCAG TGCAAAGGCG GAAACGTGTG TGTGCAGGCA 960

CAGGACAGGT ATTACAATTC CTCTTGCAGC AAGTGGGCCT GTGTGCCCTG CAGAGTCAGG 1020

TCCTGATTCT AGAGC 1035

<210>9

<211>687

<212>DNA

<213 > synthetic

<400>9

CCCAAGCTTG CCGCCACCAT GTGTCCTGCT AGATCACTGC TTCTGGTTGC CACACTCGTG 60

CTGCTGGACC ACCTTAGCCT GGCAAGGAAC CTGCCAGTGG CTACTCCCGA TCCTGGCATG 120

TTCCCATGCC TCCACCATTC CCAGAATCTG CTGAGAGCCG TGAGCAACAT GCTTCAGAAG 180

GCCAGGCAGA CCCTGGAGTT CTACCCATGT ACATCTGAGG AAATCGACCA CGAGGACATC 240

ACCAAGGACA AAACTTCCAC CGTCGAAGCA TGCCTGCCTC TGGAGCTCAC AAAGAATGAG 300

AGCTGTCTGA ACTCCAGAGA AACTTCCTTC ATTACCAATG GAAGCTGCCT GGCCTCTCGC 360

AAAACATCCT TTATGATGGC TCTGTGTCTT AGCTCTATCT ATGAGGATCT GAAGATGTAC 420

CAGGTGGAGT TCAAGACTAT GAACGCCAAA CTGCTCATGG ACCCAAAGAG GCAGATTTTC 480

CTGGACCAGA ATATGCTGGC AGTGATTGAC GAGCTGATGC AGGCCCTGAA CTTCAACTCC 540

GAGACCGTGC CCCAGAAAAG CTCTCTTGAA GAGCCTGATT TCTACAAGAC TAAGATCAAA 600

CTGTGCATTC TGCTCCATGC TTTCAGAATC AGGGCCGTCA CAATCGACCG GGTGATGTCC 660

TACCTGAATG CCAGTTGATT CTAGAGC 687

<210>10

<211>1014

<212>DNA

<213 > synthetic

<400>10

CCCAAGCTTG CCGCCACCAT GTGTCATCAG CAGCTTGTTA TCTCTTGGTT TAGCCTGGTG 60

TTCCTGGCCT CTCCCCTGGT GGCAATCTGG GAGCTCAAGA AGGACGTGTA CGTCGTGGAG 120

CTGGATTGGT ATCCAGACGC TCCTGGCGAA ATGGTGGTTC TGACCTGCGA TACACCCGAG 180

GAAGACGGCA TTACTTGGAC CCTGGATCAG AGCTCCGAGG TGCTTGGCTC CGGAAAGACA 240

CTGACTATCC AGGTGAAGGA GTTTGGCGAC GCCGGCCAGT ACACCTGTCA CAAAGGAGGC 300

GAAGTGCTGA GCCACTCTCT GCTCCTGCTT CATAAGAAGG AGGATGGCAT CTGGTCCACA 360

GACATTCTGA AAGATCAGAA GGAACCAAAG AACAAGACTT TCCTGAGGTG CGAGGCCAAG 420

AATTATAGCG GAAGATTTAC CTGTTGGTGG CTGACAACTA TCTCTACCGA CCTCACATTC 480

TCCGTGAAAA GCTCTCGCGG CTCCAGCGAT CCTCAGGGCG TCACTTGCGG AGCAGCCACC 540

CTGTCTGCTG AGAGGGTGAG AGGCGACAAC AAGGAATACG AGTATTCCGT GGAATGTCAG 600

GAGGATAGCG CATGTCCCGC CGCTGAGGAA TCTCTGCCAA TCGAGGTGAT GGTGGACGCC 660

GTGCACAAAC TGAAGTACGA AAATTATACA TCCAGCTTCT TCATTCGGGA CATCATTAAG 720

CCTGACCCAC CTAAGAACCT GCAGCTCAAG CCTCTTAAGA ATAGCAGGCA GGTGGAGGTG 780

TCCTGGGAGT ACCCCGATAC TTGGAGCACA CCACACTCTT ATTTCAGCCT GACCTTCTGC 840

GTTCAGGTGC AGGGCAAGTC TAAGCGCGAG AAGAAGGACC GCGTGTTTAC TGATAAAACA 900

TCTGCAACCG TGATCTGCAG GAAGAACGCT TCCATCAGCG TCAGAGCCCA GGACCGGTAC 960

TATTCTAGCT CCTGGTCTGA GTGGGCCTCC GTGCCTTGCA GTTGATTCTA GAGC 1014

<210>11

<211>1014

<212>DNA

<213 > synthetic

<400>11

CCGCCACCAT GGATATTGAT CCTTACAAAG AGTTTGGCGC CTCTGTGGAA CTGCTCAGCT 60

TCCTGCCCTC TGACTTCTTT CCATCCATTA GAGACCTGCT CGATACCGCC AGCGCACTGT 120

ATCGCGAGGC CCTGGAATCC CCTGAGCACT GtAGCCCTCA CCATACAGCC CTGAGACAGG 180

CTATCCTCTG TTGGGGAGAA CTGATGAACC TGGCAACCTG GGTCGGCTCC AACCTGGAGG 240

ACCCAGCCTC TAGGGAACTC GTGGTGAGCT ACGTCAATGT GAACATGGGC CTGAAGATTA 300

GACAGCTCCT GTGGTTCCAC ATCTCCTGCC TCACCTTCGG TCGGGAGACT GTGCTGGAAT 360

ATCTGGTGAG CTTTGGCGTT TGGATTAGAA CTCCTCCTGC TTACAGGCCT CCAAATGCCC 420

CTATCCTGTC CACCCTCCCT GAGACCACCG TGGTCAGACG CAGAGGCAGG TCTCCAAGAC 480

GGAGGACACC CAGCCCTAGA CGCAGGAGAT CACAGTCCCC TAGACGGAGA AGGTCTCAGA 540

GCCGCGAATC CCAGTGT 557

Claims (15)

1. a recombinant plasmid DNA vaccine that is used for the treatment of hepatitis B, it is characterized in that described recombiant plasmid carries hepatitis B surface antigen S2S protein coding gene and hepatitis B virus core antigen Core protein coding gene, and two destination protein expression cassettes, two destination protein expression cassettes are separated from one another by two encoding genes.

2. recombinant plasmid DNA vaccine according to claim 1, is characterized in that described S2S protein coding gene, and its nucleotides sequence is classified Seq ID No.5 as, described Core protein coding gene, and its nucleotides sequence is classified Seq ID No.11 as.

3. recombinant plasmid DNA vaccine according to claim 1, is characterized in that described recombiant plasmid also comprises escherichia coli replication origin ColE1, blocks that resistant gene sequence Kan; In described two destination protein expression cassettes, expression cassette in upstream position is for optimizing the destination protein expression cassette, and it comprises the enhancer element Enhancer, the transcriptional regulatory element intron Intronl that derive from SV40 and exon Exonl and the posttranscriptional regulatory element HPRE that derives from HBV.

4. according to the described recombinant plasmid DNA vaccine of any claim of claim 1-3, it is characterized in that described recombiant plasmid is to express the recombiant vaccine plasmid pS2S-C of hepatitis B core Core albumen (C) in optimization expression HBV peplos when Protein S 2S, the nucleotide sequence of described recombiant vaccine plasmid pS2S-C is as shown in Seq IDNo.1; Or express the recombiant vaccine plasmid pC-S2S of the S2S of albumen in hepatitis B peplos when being optimization expression hepatitis B core Core albumen, the nucleotide sequence of described recombiant vaccine plasmid pC-S2S is as shown in Seq IDNo.2.

5. the two plasmid DNA vaccines that are used for the treatment of hepatitis B, is characterized in that described pair of plasmid DNA vaccine comprises the described recombinant plasmid DNA vaccine of any claim of claim 1 to 4 and restructuring adjuvant plasmid.

6. according to claim 5 pair of plasmid DNA vaccine, is characterized in that described restructuring adjuvant plasmid is for restructuring interleukin 12 (IL12) adjuvant plasmid pIL12.

7. according to claim 6 pair of plasmid DNA vaccine, it is characterized in that described recombinant interleukin 12 adjuvant plasmid behaviour recombinant interleukin 12 adjuvant plasmid phIL12 or Mus recombinant interleukin 12 adjuvant plasmid pmIL12, the nucleotides sequence of adjuvant plasmid phIL12 of wherein recombinating is classified the nucleotide sequence shown in Seq ID No.3 as, and the nucleotides sequence of restructuring adjuvant plasmid pmIL12 is classified the described nucleotide sequence of Seq ID No.4 as.

8. build the method for the described recombiant vaccine plasmid of claim 4 pS2S-C, it is characterized in that comprising the steps:

(1) first construction of recombinant plasmid vector skeleton pOE-EKS:

Take pDRVISV1.0 as template, take Primer1 and Primer2 as primer, the primer that wherein primer 2 is 5 ' terminal phosphate, amplification obtains the replicon zone (Ori) that size is 748bp, introduces EcoRI, KpnI and SwaI restriction enzyme site simultaneously;

Primer 1：5’-GGAATTCGGGGTACCATTTAAATTTGAACGTTCGCAA t ATGTGAGCAAAAGGCCAGC-3’

Primer2：5’-CGGCGCGCGCCGAAAACGACGATTGCGAACGTTCAACCCGTAGAAAAGATCAAAGG-3’

Take pDRVISV1.0 as template, take Primer3 and Primer4 as primer, the primer that wherein primer 3 is 5 ' terminal phosphate, amplification obtains that resistant maker gene of card (Kan) that size is 1056bp, introduces the EcoRI restriction enzyme site simultaneously;

Primer3：5’-tcgtcgt t t tcggcgcgcgccgTTGAACGTTCGCAAtTCAAGTCAGCGTAATGCTC-3’

Primer4：5’-GGAATTCGGCGCGCGCCGAAAACGACGATGCGAACGTTCAAGAAAGCCACGTTGTGTCT-3’

Above-mentioned two fragments are carried out to single endonuclease digestion with EcoRI respectively, after connecting, build recombinant clone pOK-EKS;

(2) construction recombination plasmid DNA vaccination pRec2.0-PreS2.S

Synthetic gene PreS2S is connected with the carrier pHPRE of SalI+XbaI double digestion by the XhoI+XbaI double digestion, builds and obtain recombinant clone pHPRE-PreS2S, wherein the nucleotide sequence of synthetic gene preS2S is as shown in Seq ID No.5;

Take pDRVISV1.0 as template, take Primer5 and Primer6 as primer, it is the BGHpolyA district of 271bp that amplification obtains size, introduces the BamHI restriction enzyme site in upstream simultaneously, and the BglII-EcoRV-SalI-KpnI restriction enzyme site is introduced in downstream; Enter by BamHI+KpnI double digestion rear clone in the pHPRE-PreS2S carrier of BglII+KpnI double digestion, obtain recombiant plasmid pHPRE '-PreS2S;

Primer5：5’-CGGGATCCgctgtgccttctagttgccag-3’

Primer6：5’-GGGGTACCGTCGACAACGTTGATATCAACGTTAGATCTCATAGAGCCCACCGCATCC-3’

By EcoRI+KpnI, the PreS2S expression cassette of recombiant plasmid pHPRE '-PreS2S is cloned in carrier pOK-EKS, construction recombination plasmid pRec2.0-PreS2S, its nucleotides sequence is classified the nucleotide sequence that Seq No.2 indicates as;

(3) construction recombination plasmid pRec2.0-C

Synthetic gene CPreS1 is connected with the carrier pHPRE of SalI+XbaI double digestion by the XhoI+XbaI double digestion, builds and obtain recombinant clone pHPRE-CPreS1, wherein the nucleotide sequence of synthetic gene CPreS1 is as shown in Seq ID No.6;

Take pDRVISV1.0 as template, take Primer5 and Primer6 as primer, it is the BGHpolyA district of 271bp that amplification obtains size, introduces the BamHI restriction enzyme site in upstream simultaneously, and the BglII-EcoRV-SalI-KpnI restriction enzyme site is introduced in downstream; Enter by BamHI+KpnI double digestion rear clone in the pHPRE-CPreS1 carrier of BglII+KpnI double digestion, obtain recombiant plasmid pHPRE '-CPreS1;

By EcoRI+KpnI, the CPreS1 expression cassette of recombiant plasmid pHPRE '-CPreS1 is cloned in carrier pOK-EKS to construction recombination plasmid pRec2.0-CPreS1;

Take pRec2.0-CPreS1 as template, take Primer7 and Primer8 as primer, amplification obtains the Core gene (C) that size is 596bp, and by PstI+XbaI double digestion rear clone, enters in the pRec2.0-CPreS1 carrier of PstI+XbaI double digestion, obtains recombiant plasmid pRec2.0-C;

Primer7：5’-gtcttttctgcagtcaccgtcgTCGAG-3’

Primer8：5’-GCTCTAGAATCAACACTGGGATTCGCGGCTCTG-3’

(4) construction recombination plasmid pRec2.0-PreS2S-C (pS2S-C)

Take pRec2.0-C as template, take Primer9 and Primer8 as primer, amplification obtains the Core gene (C) that size is 579bp, by the HindIII+XbaI double digestion, with the carrier pcDNA3.1 of HindIII+XbaI double digestion, is connected, and builds and obtains pcDNA3.1-C;

Primer9：5’-CCCAAGCTTGCCGCCACCATGGATATTG-3’

Primer8：5’-GCTCTAGAATCAACACTGGGATTCGCGGCTCTG-3’

Cut out the C expression cassette by the BglII+PvuII double digestion from plasmid pcDNA3.1-C, be cloned in the pRec2.0-PreS2S of BglII+EcoRV double digestion, build and obtain recombiant plasmid pRec2.0-PreS2S-C.

9. build the method for the described recombiant vaccine plasmid of claim 4 pC-S2S, it is characterized in that comprising the steps:

The pRec2.0-PreS2S that the middle structure of claim 8 step (2) of take obtains is template, take Primer11 and Primer12 as primer, it is the PreS2S gene of 873bp that amplification obtains size, be connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by the HindIII+XbaI double digestion, build and obtain pcDNA3.1-PreS2S;

Primer 11：5’-CCCAAGCTTGCCGCCACCATGCAGTGGAACTC-3’

Primer 12：5’-GCTCTAGAATCAGATGTAAACCCAC-3’

Cut out the PreS2S expression cassette by the BglII+PvuII double digestion from plasmid pcDNA3.1-PreS2S, be cloned in the pRec2.0-C of BglII+EcoRV double digestion, build and obtain recombiant plasmid pRec2.0-C-PreS2S (pC-S2S).

10. a method for preparing described pair of plasmid DNA vaccine of claim 7, by described recombiant vaccine plasmid pS2S-C or pC-S2S, described restructuring adjuvant plasmid phIL12 or pIL12 transform respectively bacillus coli DH 5 alpha, screening positive clone, will be containing the monoclonal of positive colony through fermentation culture, amplification, again through cracking, column purification, collect target plasmid, physiological saline solution target plasmid and get final product.

11. the colon bacillus that a strain contains recombiant vaccine plasmid pS2S-C (Esherichia coli), deposit number is: CGMCC No.3728.

12. the colon bacillus that a strain contains recombiant vaccine plasmid pC-S2S (Esherichia coli), deposit number is: CGMCC No.3729.

13. in claim 1-4, the described recombinant plasmid DNA vaccine of any claim has the application in prevention and treatment hepatitis B medicine in preparation.

14. in claim 5-7, described pair of plasmid DNA vaccine of any claim has the application in prevention and treatment hepatitis B medicine in preparation.

15. according to claim 14 pair of plasmid DNA vaccine has the application in prevention and treatment hepatitis B medicine in preparation, it is characterized in that immune administering mode is subcutaneous injection, intramuscular injection or carries out immunity by the electroporation mode.

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