CN104592390B - A kind of double special restructuring AntiHBsAg antibodies, preparation method and the usage - Google Patents

Abstract

The invention belongs to biological technical field, and the invention discloses a kind of and hepatitis B surface antibody（HBsAg）The bispecific antibody of different epitopes specific binding.Present invention also offers the amino acid sequence of this antibody-like, encode the DNA sequence dna and the expression vector containing this DNA sequence dna and the host cell containing this expression vector of this antibody-like.The invention also discloses the preparation method of above-mentioned antibody, and the purposes in prevention is hepatitis b virus infected.

Description

A kind of double special restructuring AntiHBsAg antibodies, preparation method and the usage

Technical field

The invention belongs to biological technical field, to resist more specifically, the invention discloses a kind of double special restructuring anti-HBsAgs Body, its preparation method and its prevent, treat it is hepatitis b virus infected in application.

Background technology

Hepatitis type B virus（Hepatitis B virus, HBV）It is double-stranded DNA virus, causes chronic hepatitis B, and Cause the generation of hepatic failure, hepatic sclerosis and liver cancer.Global about 300,015,000 HBV infection persons, and this also turns into world's public affairs Health problem altogether.Nearest decades, hepatitis B vaccine are succeeded in developing, and the prevention to hepatitis B brings very big hope.However, really There is a minority's HB vaccination but to excite immune system to produce anti-hepatitis B antibody, this part population once touches The high risk factor of hepatitis B, Human Hepatitis B Immune Globulin can only be injected to prevent.But human immunoglobulin(HIg) composition is relative complex, use Hazards it is relatively uncertain.Therefore the biological products relatively controllable there is an urgent need to develop a kind of people source, hazards Substitute Human Hepatitis B Immune Globulin.

With the development of biotechnology, the development of genetic engineering antibody is maked rapid progress.Human monoclonal antibody, which produces, to be included EBV immortalizes bone-marrow-derived lymphocyte, human B lymphocyte hybridoma technology, the humanization of other kind antibody, based on phage library exhibition The human antibody technology shown, human antibody technology and Single cell RT-PCR monoclonal antibody skills based on humanization mouse Art.Due to EBV immortalize bone-marrow-derived lymphocyte conversion ratio it is low, seriously limit this technology popularization [Guan Y, Sajadi MM, Kamin-Lewis R, et al. Discordant memory B cell and circulating anti-Env antibody responses in HIV-1 infection. Proc Natl Acad Sci U S A 2009;106: 3952-7].Human B lymphocyte and myeloma cell's fusion efficiencies are extremely low, cause human B lymphocyte hybridoma technology seldom by with In preparing human monoclonal antibody.Phage display library human antibody technology is to be based on human immunoglobulin(HIg) light chain and heavy chain gene Random pair, do not undergo double Sexual behavior modes of somatic mutation and immune system, the antibody for causing its screening to obtain is generally affine Power is low（KD ＞ 10^-7）, dissociation rate is fast（V ＜ 60min）, antibody Tm values are low（65 DEG C of ＜）, heat endurance is poor, potential allergy With autoimmunity risk [Mohapatra S, Juan HS. Designer monoclonal antibodies as drugs: the state of the art. Expert Rev Clin Immunol 2008;4:305-7].Due to the people of most potential quality The patent of source antibody technique-humanization mouse is only rested in a small number of international pharmacy giant hands, and development cost is high, serious system The about popularization of this technology.

The B that Single cell RT-PCR monoclonal antibodies technologies separate the antibody containing purpose directly from human peripheral is thin Born of the same parents, with single-cell RT-PCR technology Direct Cloning antibody light chain and heavy chain, antibody is prepared by vitro recombination.Prepared Antibody sources are natural, and hazards are controllable.Because monoclonal antibody is cloned from the bone-marrow-derived lymphocyte of human peripheral, The double Sexual behavior modes, long-term body fluid circulatory and somatic mutation of human immune system are experienced, therefore, show its fabulous parent And power（KD ＜ 10^-9）, dissociation rate is slow（V ＞ 6hours）, heat endurance is good, and potential allergy and autoimmunity risk is low [Smith K, Garman L, Wrammert J, et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 2009;4: 372-84].Single cell RT-PCR monoclonal antibody technological development costs are cheap, can more be shown especially for exotic antigen Its superiority.

Due to the multifactor property of disease, single target treatment can not meet the needs of clinical.In clinical test There is the report of two kinds of monoclonal antibody preclinical laboratories of conjunctive use, but this use in conjunction monoclonal antibody there are many limitations Property [Kou G, Shi J, Chen L, et al. A bispecific antibody effectively inhibits tumor growth and metastasis by simultaneous blocking vascular endothelial growth factor A and osteopontin. Cancer Lett 2010;299:130-6].For example, use in conjunction two The security and effect of kind monoclonal antibody need to go to evaluate respectively, resulting long period, high development cost limit Its development process is made, bispecific antibody solves this bottleneck [Wu C, Ying H, Grinnell C, et al. just Simultaneous targeting of multiple disease mediators by a dual-variable- domain immunoglobulin. Nat Biotechnol 2007;25:1290-7].In addition, bispecific antibody sometimes can also Obtain some novel characteristics not available for its parental monoclonal antibody：As often having the dissociation lower than former parental antibody [Lu D, Kotanides H, Jimenez X, the et al. Acquired such as speed, stronger sealing process antagonistic activity of a bispecific diabody directed against two different epitopes on vascular endothelial growth factor receptor 2. J Immunol Methods 1999;230:159-71]。

The content of the invention

For problem above, present invention design is had based on one plant of Single Cell RT-PCR monoclonal antibodies technological development The Anti-HBsAg bispecific antibodies of fabulous neutralization HBV viruses.The present invention uses ultrahigh speed streaming separation system from inoculation second Special memory B cells (the HBsAg of HBsAg are separated in healthy volunteer's peripheral blood of liver vaccine⁺IgG⁺CD19⁺), and be prepared into Unicellular sample.The gene of the antibody correlation in each unicellular sample is cloned using Single Cell RT-PCR technologies, And it is cloned into mammal efficient expression vector, the corresponding antibody of in-vitro recombination expression.

The invention discloses：

1. a kind of bispecific antibody, it contains two different weight chain variable districts, light chain variable districts, can be respectively in connection with Functional epitopes different HBsAg.

A kind of bispecific antibody described in 2., it is characterized in that weight chain variable district has SEQ ID NO：Amino shown in 2 Acid sequence, light chain variable district have SEQ ID NO：Amino acid sequence shown in 4.

3. a kind of nucleic acid molecule of separation, encodes the amino acid sequence, there is SEQ ID NO：Nucleotides shown in 1 Sequence and SEQ ID NO：Nucleotide sequence shown in 3.

4. a kind of carrier, containing described nucleic acid molecule, the table being connected with the series of operations of the nucleic acid molecule Up to regulating and controlling sequence, wherein carrier can be pDR1, pcDNA3.1（+）, pcDNA3.1/ZEO（+）One of, pDHFR.

5. carrier described in is pcDNA3.1（+）.

6. a kind of host cell, it is eukaryotic containing the carrier.

7. host cell described in, it is mammalian cell.

8. host cell described in, it is Freestyle 293F cells.

9. a kind of method for preparing described bispecific antibody, this method include：

A) it is special using HBsAg in volunteer's peripheral blood of ultra high rate airflow classification system separation HB vaccination Memory B cells, and it is prepared into unicellular sample；

B) each sample moderate resistance body phase correlation gene, in-vitro recombination expression phase are cloned using Single Cell RT-PCR technologies Answer antibody；

C) pass through functional epitope measure, affinity measure and neutralize HBV determinations of activity, it is strong, active high to filter out affinity Combination difference in functionality epitope two anti-HBsAgs monoclonal antibody C4G4 and D2H2；

D) heavy chain of the two monoclonal antibodies of C4G4, D2H2, light chain variable district are passed through respectively using biotechnology Heavy chain linker, light chain linker connections are crossed, is prepared into difunctional antibody；

E) under expression condition, any described host cell is cultivated, expresses bispecific antibody C4D2-BsAb, separation Purifying.

10. a kind of composition, contain described bispecific antibody, and pharmaceutically acceptable carrier.

Application of the bispecific antibody in anti HBV infecting is prepared described in 11..

Application of the composition in anti HBV infecting is prepared described in 12..

13. any described purposes, in addition to and other anti HBV infecting Drug combinations.

In the present invention, 42 plants of brand-new Anti-HBsAg monoclonal antibodies of successful clone, reflected using ELISA method Its functional epitope, affinity and neutralization HBV activity are determined.Through a series of test experiences, 2 plants are have selected therefrom and neutralizes HBV work Property best Anti-HBsAg monoclonal antibody C4G4 and D2H2, using biotechnology prepared by 2 strain antibodies in pairs special Property antibody C4D2-BsAb.

In HepaRG cell models, C4D2-BsAb shows the work that stronger neutralization HBV is combined than C4G4 and D2H2 Property.In addition, research shows that FcRn mediates release of the C4D2-BsAb endocytosis to suppression HBsAg and virion in liver cell.

Brief description of the drawings

Fig. 1, streaming CD19⁺IgG⁺HBsAg⁺B cell are analyzed.

Fig. 2, PCR expand antibody variable gene figure.

SDS-PAGE analyses restructuring human monoclonal antibodies electrophoretogram under Fig. 3, reducing condition.

Fig. 4, bispecific antibody structural representation.

Under Fig. 5, reducing condition, the bispecific antibody electrophoretogram of SDS-PAGE analysis purifying.

The feature of Fig. 6, anti-HBsAg monoclonal antibody, position of the synthetic peptide on HBsAg.

Fig. 7, HepaRG cell assess monoclonal antibody in and HBV activity.

Fig. 8, monoclonal antibody C4G4 and D2H2 coordinate repression.

Fig. 9, bispecific antibody C4D2-BsAb and D2C4-BsAb combination synthesis polypeptide P2, P3 activity analysis.

Figure 10, bispecific antibody C4D2-BsAb and D2C4-BsAb combination HBsAg activity analysis.

In Figure 11, bispecific antibody C4D2-BsAb and HBV activity analysis.

Embodiment

With reference to embodiments, further the present invention will be described for experimental example, and these embodiments, experimental example should not be understood For limitation of the present invention.

Embodiment 1：The preparation of the single B cell of HBsAg specificity

The lymphocyte of the peripheral blood of hepatitis B vaccine volunteer was injected by separation, with CD19-PE/Cy5, IgG-PE, HBsAg-biotin, Streptavidin-FITC fluorescence antibody separate HBsAg⁺CD19⁺IgG⁺ B cell.As shown in Figure 1, it is seen that Cell positive a small set of HBsAg（Account for whole B cell group 0.01-0.02%）.MoFlo XDP hypervelocity fluidic cell sortings system Unite each HBsAg sorted⁺CD19⁺IgG⁺ In B cell injection 96-well Single cell PCR plates, it is ensured that each A cell in PCR holes.Then add lysate, after liquid nitrogen flash freezer, be stored in standby in -80 DEG C of ice.

Embodiment 2：RT-PCR and Chao Shi PCR clonal antibodies variable region genes and gene expression

Take and freeze the 96-well Single cell PCR plates in -80 DEG C of refrigerators, make slowly to melt on ice, add Reverse transcriptase synthesizes cDNA.Because the cDNA of individual cells synthesis is especially rare, it is necessary to expand it using Chao Shi PCR method and contain Some antibody genes.Visible obvious PCR bands amplification after two-wheeled PCR（Size about 400bp）.The paired PCR bands of light and weight chain It is sequenced after recovery.As shown in Fig. 2 analyzing specific PCR after its sequence expands antibody variable region band, and introduce restricted digestion Site.Amplification antibody variable region PCR primer inserts expression vector pcDNA3.1 (+) after being connected with antibody constant region.

Embodiment 3：Monoclonal antibody gene is expressed

Paired antibody light chain and heavy chain plasmid transiently transfect the Freestyle 293F cells of serum free medium culture, Transfection collects cell conditioned medium after 7 days, IgG antibody in Protein A purifying cells supernatants.Anti-hbs monoclonal antibodies are obtained altogether 42 plants.SDS-PAGE electroresis appraisals are as shown in figure 3, the IgG antibody of purifying shows two bands in 10% SDS-PAGE, wherein one Bar is 50KD or so（Heavy chain）, one is 25KD or so in addition（Light chain）.

Embodiment 4：Anti-hbs monoclonal antibodies HBsAg affinity determines

Certain density monoclonal antibody is taken to be mixed with the antigen HBsAg of serial doubling dilution, 4 DEG C overnight, so as to ensure Reaction reaches balance.Antigen antibody complex is transferred in coating HBsAg ELISA Plates, and 37 DEG C are incubated 1 hour.Added after washing The sheep anti-human kappa chain antibody of HRP marks is incubated 1 hour.TMB colour developing 450nm wavelength determines the absorptivity in each hole after washing, calculates anti- The affinity of body, as a result as shown in table 1.

Embodiment 5：Antibody epitope is identified

Knot of the hepatitis B surface antigen small peptide measure anti-HBsAg monoclonal antibodies of synthesizing biotinylated mark on HBsAg Close region.As shown in fig. 6, four biotin labeling hepatitis B surface antigen small peptide P1 of synthesis（aa:104-120）, P2（aa: 121-137）, P3（aa:139-148）, P4（aa:149-163）, and antigen small peptide P1, P2, P3 are marked on HBsAg ideographs, The structure in the region corresponding to P4；Wherein antigen small peptide P1 and P4 is linear structure, and P2 and P3 are cyclic structure.[Jin A, Ozawa T, Tajiri K, et al. A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood. Nat Med 2009;15:1088-92]。

	Amino acid sequence number	Amino acid sequence	Structure
				P1	104-120	LPVCPLLPGTSTTSTGP	Linear peptides
P2	123-137	TCTIPAQGTSMFPSC	Cyclic peptide
				P3	135-144	CTKPSDGNCT	Cyclic peptide
P4	149-163	CIPIPSSWAFARFLW	Linear peptides

The epitope of the combination of 42 plants of Anti-HBsAg monoclonal antibodies is analyzed using ELISA method.Such as the institute of table 1 Show, what as a result display was incorporated in P1 accounts for 7.1% (3/42), and be incorporated in P2 accounts for 38.1% (16/42), is incorporated in accounting for for P3 31.0% (13/42) and be incorporated in P4 account for 9.5% (4/42), but have the monoclonal antibody of 14.3% (6/42) not with four Individual synthetic peptide combines.

Embodiment 6：Anti-hbs monoclonal antibodies neutralize the identification of HBV activity in vitro

HepaRG cell lines are the currently the only HBV Infection in Vitro models being widely recognized as.Therefore, we use HepaRG The ability of the neutralization HBV infection HepaRG cells of 42 plants of Anti-HBsAg monoclonal antibodies of raji cell assay Raji.Use Anti-HBsAg Monoclonal antibody（1μg）And HBV（1×10⁶copies）Preincubate, is then added to DMSO and hydrocortisone induces differentiation HepaRG cells, determine it and neutralize the ability of HBV activity.Infect the 7th day we determine containing for HBsAg in cells and supernatant HBV-DNA copy numbers in amount and cell.As shown in Figure 8 A, infect the 7th day, HBsAg goes out in most cells cells and supernatant Now it is decreased obviously (*P＜ 0.05).It is also shown in FIG. 7, infect the 7th day, HBV-DNA copies count in most cells cell Now it is decreased obviously (*P＜ 0.05).As shown in table 1, the comprehensive multifactor neutralization for carrying out 42 plants of Anti-HBsAg monoclonal antibodies HBV infection HepaRG cell abilities are analyzed, the preferable two monoclonals C4G4 and D2H2 of final choice effect.

Embodiment 7：Anti-hbs monoclonal antibodies collaboration neutralizes HBV determinations of activity

Two plants of selection neutralizes the best monoclonal antibody of HBV activity（C4G4 and D2H2）Detect its cooperative effect.

HBV virions (1 × 10⁶ Copies) respectively with 1 μ g monoclonal antibody C4G4, D2H2 and Control human IgG, preincubate 1 hour, it is added in the HepaRG cell lines that induction has been broken up（Contain 4%PEG8000 in culture medium）Training overnight Support, next day washes PBS three times, renews fresh culture medium.In HBV-DNA copy numbers and culture supernatant in the 6th day detection cell HBsAg contents.HBV-DNA detections use HBV-DNA PCR kit for fluorescence quantitative（China of Shanghai section biology）.HBsAg measure is collected Culture supernatant, with hepatitis B surface antigen quantification kit（Electrochemiluminescince）（The U.S. biology of Beijing section）Detection.As shown in figure 8, C4G4 and D2H2 combinations can be substantially reduced in cell conditioned medium in HBsAg expression and cell compared with C4G4 and D2H2 are alone HBV-DNA copy numbers（*P＜ 0.05）.

Embodiment 8：The structure of bispecific antibody and identification

Selection neutralizes two best strain antibody C4G4 and D2H2 of HBV activity, designs bispecific antibody, antibody structure such as Fig. 4. The weight chain variable district of one monoclonal antibody and light chain variable district connect peptide by heavy chain respectively and light chain connect peptide be fused to it is another The weight chain variable district of individual monoclonal antibody and 5 ' ends of light chain variable district.It is ASTKGPSVFPLAP that heavy chain, which connects peptide amino acid sequence, It is TVAAPSVFIFPP that light chain, which connects peptide amino acid sequence,.By correct bispecific antibody heavy chain, chain variable region gene difference gram It is grand enter IgG1-AbVec plasmids and Ig κ-AbVec plasmids in.Bispecific antibody is expressed in Freestyle 293F cell lines C4D2-BsAb and D2C4-BsAb.SDS-PAGE electroresis appraisal bispecific antibody C4D2-BsAb and D2C4-BsAb, as a result such as Fig. 5.

Experimental example 1：The affinity measure of bispecific antibody

Elisa plate is coated with synthetic peptide P2 and P3 respectively, the bispecific of two kinds of different structures is detected using ELISA method The same parental monoclonal antibody of antibody（C4G4 and D2H2）Between affinity difference.As Fig. 9 is shown：When C4G4 variable regions are placed on When before D2H2 variable regions, the C4D2-BsAb of composition maintains to synthetic antigen small peptide P2 and P3 to be resisted with parental monoclonal Body（C4G4 and D2H2）Similar affinity；And when D2C4-BsAb is formed before D2H2 variable regions are put in into C4G4, D2C4- BsAb and synthetic antigen small peptide P2 binding activity decline substantially compared with C4G4；D2C4-BsAb's and synthetic antigen small peptide P3 Binding activity is with D2H2 without significant difference.

Equally two bispecific antibody C4D2-BsAb and D2C4-BsAb and HBsAg of measure affinity.Such as Figure 10 institutes Show, C4D2-BsAb and D2C4-BsAb can be combined with HBsAg well.

Experimental example 2：In bispecific antibody C4D2-BsAb and HBV determinations of activity

HBV virions (1 × 10⁶ Copies) C4G4, D2H2, C4G4+D2H2 and C4D2- with various concentrations respectively BsAb preincubates 1 hour, it is added in the HepaRG cell lines that induction has been broken up（Contain 4%PEG8000 in culture medium）Training overnight Support, next day washes PBS three times, renews fresh culture medium.In HBV-DNA copy numbers and culture supernatant in the 6th day detection cell HBsAg contents.HBV-DNA detections use HBV-DNA PCR kit for fluorescence quantitative（China of Shanghai section biology）.HBsAg measure is collected Culture supernatant, with hepatitis B surface antigen quantification kit（Electrochemiluminescince）（The U.S. biology of Beijing section）Detection.Such as Figure 11, as a result Display bispecific antibody C4D2-BsAb shows fabulous neutralization HBV ability, has exceeded effect associated with C4G4 and D2H2 Fruit.

Characterization of anti-HBsAg antibodies.

Antibody

Affinity KD(M) a

Epito peb

Neutraliz ation activityc

Antibody

Affinity KD (M) a

Epit opeb

Neutraliza tion activityc

A3E3

5.67×10−8

3

++

D12H12

2.04×10−7

2

++

A5E5

3.75×10−9

4

-/+

2A1E1

2.39×10−9

4

-/+

A6E6

3.24×10−8

ND

+

2A2E2

3.72×10−8

3

++

A7E7

6.64×10−9

1

-/+

2A9E9

4.35×10−8

ND

-/+

A10E10

2.12×10−8

2

+

2B4F4

1.71×10−9

2

++

A11E11

5.63×10−8

1

-/+

2B5F5

6.63×10−8

2

+

B1F1

2.95×10−7

2

++

2B6F6

5.11×10−8

3

++

B6F6

4.82×10−8

2

ND

2B10F10

8.35×10−9

4

++

B7F7

6.55×10−9

3

-/+

2B11F11

6.28×10−8

ND

-/+

B9F9

2.86×10−9

4

++

2C2G2

1.77×10−7

2

+

B12F12

1.33×10−8

3

+

2C3G3

3.66×10−8

ND

-/+

C1G1

6.74×10−7

3

ND

2C5G5

3.28×10−9

2

+

C2G2

1.85×10−9

2

++

2C6G6

1.11×10−8

3

++

C4G4

3.66×10−9

2

++

2C7G7

8.6×10−8

3

+

C8G8

3.03×10−7

2

-/+

2C8G8

8.28×10−7

3

++

C9G9

3.81×10−8

ND

+

2D1H1

5.04×10−8

1

++

D2H2

5.82×10−9

3

++

2D4H4

3.48×10−9

2

++

D3H3

4.14×10−8

2

ND

2D5H5

2.73×10−7

2

ND

D5H5

1.88×10−9

2

++

2D8H8

2.12×10−8

3

++

D6H6

4.86×10−7

3

++

2D9H9

5.29×10−8

3

+

D10H10

1.58×10−8

2

+

C4D2-BsAb

3.88×10−9

2,3

++

D11H11

8.92×10−9

ND

-/+

D2C4-BsAb

4.95×10−9

2,3

^aAffinities were analyzed with ELISA.

^bEpitopes were analyzed by ELISA using synthesized peptides (P1, amino acids 104–120; P2, amino acids 121–137; P3, amino acids 139–148; P4, amino acids 149–163) covering extracellular domain of HBsAg.

^cHBV-neutralization activity : ++, more than 60% inhibition; +, 40%– 60% inhibition; +/–, 20-40% inhibition at day 7 post-infection from that with control IgG.

SEQUENCE LISTING

<110>Shanghai Hai Sitaike pharmaceutcal corporation, Ltds of Shanghai Zhangjiang Biological Technology Co

<120>A kind of double special restructuring AntiHBsAg antibodies, preparation method and the usage

<130> 2013

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1842

<212> DNA

<213>Artificial sequence

<400> 1

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt gcacagccag 60

gtgcagctgg tggaaagcgg cggaggagtg gtgagacccg gaagaagcct gagactgagc 120

tgcgccgcta gcggctttgc cttcagcgac tacagcatca actgggtgag gcaagctccc 180

ggcaagggac tggaatgggt ggccatcatc agctacgacg gcaggatcac ctactacagg 240

gacagcgtga agggcaggtt caccatctcc agggacgaca gcaagaacac cctgtacctg 300

cagatgaaca gcctgaggac cgaggacacc gccgtgtatt actgcgccag gcagtactac 360

gacttctgga gcggctcctc cgtgggcagg aactacgacg gaatggacgt gtggggcctg 420

ggcaccacag tcaccgtgag ctcagctagc accaagggac cttctgtgtt ccctctggcc 480

cctcaggtgc agctggtgga aagcggagga ggcgtggtgc aacccggagg aagcctgagg 540

ctgagctgcg ctcctagcgg cttcgtgttc aggagctacg gcatgcactg ggtgaggcag 600

acacccggca aaggcctgga gtgggtgagc ctgatctggc acgacggcag caacaggttc 660

tacgccgaca gcgtgaaggg caggttcacc atcagcaggg acaacagcaa gaacaccctg 720

tacctgcaga tgaacagcct gagggctgag gacaccgcca tgtacttctg cgccagggag 780

aggctgattg ccgctcctgc cgctttcgac ctgtggggac agggcaccct ggtgaccgtc 840

agctcggcgt cgaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 900

tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 960

gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 1020

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 1080

cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 1140

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 1200

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 1260

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1320

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1380

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1440

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1500

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1560

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1620

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1680

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1740

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1800

tacacgcaga agagcctctc cctgtctccg ggtaaatgat ga 1842

<210> 2

<211> 612

<212> PRT

<213>Artificial sequence

<400> 2

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe

35 40 45

Ser Asp Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ile Ile Ser Tyr Asp Gly Arg Ile Thr Tyr Tyr Arg

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gln Tyr Tyr Asp Phe Trp Ser Gly Ser Ser Val

115 120 125

Gly Arg Asn Tyr Asp Gly Met Asp Val Trp Gly Leu Gly Thr Thr Val

130 135 140

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

145 150 155 160

Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly

165 170 175

Gly Ser Leu Arg Leu Ser Cys Ala Pro Ser Gly Phe Val Phe Arg Ser

180 185 190

Tyr Gly Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp

195 200 205

Val Ser Leu Ile Trp His Asp Gly Ser Asn Arg Phe Tyr Ala Asp Ser

210 215 220

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

225 230 235 240

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Phe

245 250 255

Cys Ala Arg Glu Arg Leu Ile Ala Ala Pro Ala Ala Phe Asp Leu Trp

260 265 270

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

275 280 285

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

290 295 300

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

305 310 315 320

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

325 330 335

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

340 345 350

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

355 360 365

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

370 375 380

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

385 390 395 400

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

405 410 415

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

420 425 430

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

435 440 445

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

450 455 460

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

465 470 475 480

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

485 490 495

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

500 505 510

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

515 520 525

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

530 535 540

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

545 550 555 560

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

565 570 575

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

580 585 590

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

595 600 605

Ser Pro Gly Lys

610

<210> 3

<211> 1080

<212> DNA

<213>Artificial sequence

<400> 3

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt gcacggcgac 60

atcgtcatga cccagagccc tctcagcctg tccgtgaccc ctggagaacc cgccagcatc 120

tcctgcagga gcagccagag cctgctgcac aggagcggca acaactacct ggactggtac 180

ctgcagaagc ccggccatag cccccagctg ctgatctacg tgggcagcaa cagagcttcc 240

ggcgtgcccg acagattcag cggaagcgga tccggcaccg agtacaccct gaagatcagc 300

agagtggagg ccgaggacgt gggcgtgtac tactgcatgc aggccctgca gacccccagg 360

acattcggcc agggcaccaa gctggagatc aagcgtaccg tggctgcccc tagcgtgttc 420

atcttccctc ctgagctggt gatgacccag agcccttcca gcctgagcgc tagcgtggga 480

gacagggtga ccatcacctg cagggccagc cagggcatct acaacagcat cgcctggtac 540

cagcagaagc ccggcaaggc tcccaagctg ctgctgtaca gcaccagcac actgctgagc 600

ggcgtgccca gcagattcag cggaagcggc agcggcaccg attacaccct gaccatcacc 660

aacctgcagc ctgaggactt cgccacctac tactgccagc agtacttcgt gacccccgag 720

accttcggac agggcaccaa ggtggagatc aagcgtacgg tggctgcacc atctgtcttc 780

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 840

aataacttct accccagaga agccaaagtg cagtggaagg tggacaacgc cctgcagagc 900

ggaaacagcc aggaaagcgt gacagagcag gattccaagg attccacata cagcctgagc 960

agcacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 1020

acacaccagg gactgtcctc ccctgtgaca aagagcttca acagaggaga atgctgatga 1080

<210> 4

<211> 358

<212> PRT

<213>Artificial sequence

<400> 4

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

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

20 25 30

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

35 40 45

Leu His Arg Ser Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro

50 55 60

Gly His Ser Pro Gln Leu Leu Ile Tyr Val Gly Ser Asn Arg Ala Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

100 105 110

Met Gln Ala Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

145 150 155 160

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Asn Ser

165 170 175

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

180 185 190

Tyr Ser Thr Ser Thr Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly

195 200 205

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

210 215 220

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Val Thr Pro Glu

225 230 235 240

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

245 250 255

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

260 265 270

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

275 280 285

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

290 295 300

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

305 310 315 320

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

325 330 335

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

340 345 350

Phe Asn Arg Gly Glu Cys

355

Claims (11)

1. A kind of 1. bispecific antibody, it is characterized in that the amino acid sequence of weight chain variable district is SEQ ID NO：Amino shown in 2 Acid sequence, the amino acid sequence of light chain variable district is SEQ ID NO：Amino acid sequence shown in 4.
2. 2. a kind of nucleic acid molecule of separation, amino acid sequence described in claim 1 is encoded, is SEQ ID NO：Core shown in 1 Nucleotide sequence and SEQ ID NO：Nucleotide sequence shown in 3.
3. 3. a kind of carrier, containing the nucleic acid molecule described in claim 2, it is connected with the series of operations of the nucleic acid molecule Expression regulation sequence, wherein carrier is pDR1, pcDNA3.1 (+), one of pcDNA3.1/ZEO (+), pDHFR.
4. A kind of 4. carrier described in claim 3, it is characterized in that described carrier is pcDNA3.1 (+).
5. 5. a kind of host cell, it is eukaryotic containing carrier described in claim 4.
6. 6. host cell described in claim 5, it is mammalian cell.
7. 7. host cell described in claim 6, it is Freestyle293F cells.
8. 8. a kind of composition, contain the bispecific antibody described in claim 1, and pharmaceutically acceptable carrier.
9. 9. application of the bispecific antibody in anti HBV infecting medicine is prepared described in claim 1.
10. 10. application of the composition in anti HBV infecting medicine is prepared described in claim 8.
11. 11. any described purposes of claim 9,10, in addition to and other anti HBV infecting Drug combinations.