CN105061591A - Anti-hepatitis B virus surface antigen completely humanized human antibody and use thereof - Google Patents
Anti-hepatitis B virus surface antigen completely humanized human antibody and use thereof Download PDFInfo
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Abstract
The invention provides an anti-hepatitis B virus surface antigen completely humanized human antibody A3D5 and a gene for encoding the antibody. Experiments show that the antibody A3D5 can specifically bind to an HBsAg protein, and has good HBV neutralization activity in order to possibly prevent HBV infection related hepatitis, hepatic cirrhosis and liver cancer. The antibody is a completely humanized human antibody obtained through cloning HBsAg specific memory B cells in the peripheral blood of hepatitis B vaccine inoculated volunteer, has lower immunogenicity than murine, chimeric and humanized antibodies, and can be used to prepare hepatitis B virus related hepatopathy prevention or treatment drugs or diagnose reagents.
Description
Technical field
The invention belongs to biological technical field, more specifically, the invention discloses the technology of preparing of anti-hepatitis b surface protein antibody and preventing the purposes in HBV infection and the relevant hepatocellular carcinoma for the treatment of HBV.
Background technology
Hepatitis B virus (HepatitisBvirus, HBV) is double-stranded DNA virus, and human body is infected easily causes chronic hepatitis B afterwards, and then causes liver cirrhosis and (or) liver cancer.About there are 3.5 hundred million HBV infection persons in the whole world in recent years according to statistics, and the liver failure about having 1,000,000 ~ 1,500,000 people to die from acute or chronic HBV infection every year to cause, liver cirrhosis and (or) liver cancer, thus prevent HBV infection to become world public health problem
[1].Hepatocellular carcinoma (Hepatocellularcarcinoma, HCC, be called for short liver cancer) in close relations with viral hepatitis, it is one of most important cause of death caused by viral hepatitis, also be one of modal malignant tumour clinically, be worldwide that the tumour occupying the 3rd is correlated with the cause of death, in annual new cases, China accounts for the whole world 42.5%, has become the deputy tumor etiology of China.
Research shows, in HBV infection associated HCC patient, the HCC incidence of HBVDNA carrying capacity height person (>105copies/ml) reaches 10.1%, and the HCC incidence of the low person of HBVDNA carrying capacity (<104copies/ml) is only 3.8%, baseline height virus load raises relevant with HCC incidence.Time high virus load (HBVDNA >=0.7mEq/ml), HCC risk of relapse ratio is 5.13, and the HCC risk of relapse ratio of the cutting edge positive is 2.14.Therefore, antiviral therapy is carried out to hepatitis B patient, contribute to delaying the progress of disease to HCC.Antiviral therapy being carried out to HBV associated HCC patient, improving survival rate by improving liver function.In recent years research display, high HBVDNA carrying capacity is relevant with high HCC recurrence rate and prognosis mala.Carrying out antiviral therapy to HBV infection associated HCC postoperative patient may be another kind of therapeutic choice except liver transplantation, and this treatment improves survival rate by improving liver function.
Mainly adopt the Antiviral Effect such as alpha-interferon and nucleotide analog to treat for HBV infection at present, but this two classes medicine life-time service curative effect is not good enough, is easy to cause virus mutation, brings extreme difficulties to treatment
[2].
Relative to the medicine of anti-virus infection, the antiviral effect of the specific immunoglobulin (Ig) of hepatitis B (HBIG) is well a lot.Give the specific immunoglobulin (Ig) of patient's hepatitis B (HBIG) treatment give by scholar's extensive concern of various countries.Such as, the research after the HBIG liver cancer of originating in hepatitis B after liver transplantation has reached good effect.HBIG is a kind of polyclone exogenous antibody of high-titer, and it screens from healthy blood donor, makes through bioconcentration technique.Accept this passive immunization preparation passively as human infection HBV, body can be made to neutralize rapidly in a short time and remove hepatitis B virus free in serum, avoiding hepatitis B virus localized infection.
But there is many non-safety factors as the source of therapeutic antibodies in current HBIG.Meanwhile, anti-hepatis B immunoglobulin complicated component, Hazard Factor are relatively uncertain, and the mode of production is limited, and these factors govern the Clinical practice of hepatitis B therapeutic antibody
[3].
Therefore Human Hepatitis B Immune Globulin is substituted in the urgent need to researching and developing a kind of safe and effective and that Hazard Factor are relatively controlled biological products, so research and development hepatitis B specificity human genetically engineered antibody more and more receives publicity.Because monoclonal antibody has high specificity, the highly sensitive and easy advantage such as scale operation, therefore research and development are for hepatitis B surface antigen (HepatitisBsurfaceAg, HBsAg) monoclonal antibody, to can safely and effectively in and HBV virus, and then the process of hepatitis, liver cirrhosis and the liver cancer that the infection of HBV may be stoped to be correlated with.
Summary of the invention
The present invention utilizes SinglecellRT-PCR monoclonal antibody technological development one strain total man's resource monoclonal antibody A of anti-hepatitis B virus surface antigen
3d
5, have employed following technical scheme:
The human antibody A of anti-hepatitis B virus surface antigen provided by the invention
3d
5, there is such feature, comprising: heavy-chain variable domains, comprise hypervariable region CDR
1, CDR
2, CDR
3; And light variable domains, comprise hypervariable region CDR
1＇, CDR
2＇, CDR
3＇, wherein, CDR
1aminoacid sequence be: Met-Tyr-Ala-Phe-Ser; CDR
2aminoacid sequence be: Gly-Ile-Ile-Pro-Ser-Phe-Asp-Thr-Thr-Asn-Tyr-Ala-Gln-Lys-Phe-Gln-Cys; CDR
3aminoacid sequence be: Ser-Val-Ile-Thr-Asp-Leu-Asp-Thr-Val-Gly-Asn-Asp-Glu-Ser-Gly-Asp-Ala-Ser-Tyr-Tyr-Tyr-Met-Asp-Val; CDR
1the aminoacid sequence of ＇ is: Arg-Ser-Ser-Glu-Ser-Leu-Gln-His-Ser-Asp-Gly-Thr-Tyr-Tyr-Leu-Asp; CDR
2the aminoacid sequence of ＇ is: Ser-Ala-Ser-Asn-Thr-Ala-Pro; CDR
3the aminoacid sequence of ＇ is: Met-Gln-Ala-Leu-Glu-Thr-Pro-Phe-Thr.
The aminoacid sequence of heavy-chain variable domains is as shown in SEQIDNO.6, and the aminoacid sequence of light variable domains is as shown in SEQIDNO.8.
Meanwhile, antibody A
3d
5be selected from the monoclonal antibody of anti-hepatitis B virus surface antigen and monoclonal antibody fragment any one, this monoclonal antibody fragment is the Fab fragment of monoclonal antibody or F (ab ')
2fragment.
Further, present invention also offers a kind of antibody fragment for hepatitis B surface albumen, this antibody fragment is antibody A
3d
5fab fragment or F (ab ')
2fragment.
Further, the invention provides a kind of encoding antibody A
3d
5gene, the nucleotide sequence of encoding heavy chain variable domains as shown in SEQIDNO.5, coding light variable domains nucleotide sequence as shown in SEQIDNO.7.
Further, the invention provides a kind of expression vector of the said gene containing at least one copy.
Further, the invention provides a kind of host cell containing at least one above-mentioned expression vector.
Further, the invention provides antibody A
3d
5or its Fab fragment or F (ab ')
2the purposes of fragment in the medicine preparing prevention or treatment hepatitis B virus related liver disease or diagnostic reagent.Described medicine or diagnostic reagent except comprising antibody or antibody fragment, also comprise in pharmaceutically acceptable vehicle, thinner and carrier any one.
Invention effect and effect
The invention provides a kind of human antibody of anti-hepatitis B virus surface antigen, experiment shows that it can specific binding HBsAg albumen, has good HBV Neutralization effect, and then may stop the process of HBV infection and relevant hepatitis, liver cirrhosis and liver cancer.Meanwhile, because this antibody clones the human antibody obtained in the memory B cells that HBsAg is special from volunteer's peripheral blood of HB vaccination, it has compared with mouse source, the immunogenicity that chimeric and humanized antibody is lower.
Accompanying drawing explanation
Fig. 1 is human antibody A of the present invention
3d
5specific binding hepatitis B virus surface antigen detected result figure;
Fig. 2 is the position view of synthetic peptide of the present invention at hepatitis B surface antigen (HBsAg).
Fig. 3 is antibody A of the present invention
3d
5at HBsAg in conjunction with epi-position result figure;
Fig. 4 is antibody A of the present invention
3d
5affect the detection by quantitative result figure that HBsAg expresses;
Fig. 5 is antibody A of the present invention
3d
5affect HBVDNA copy number detection by quantitative result figure;
Fig. 6 is antibody A of the present invention
3d
5suppress the result figure of the release of liver cancer cell HBsAg.
Embodiment
Below in conjunction with embodiment, the present invention is further detailed, but should not be construed as limitation of the present invention.
Embodiment does not comprise the detailed description to traditional method, as: OverlappingPCR reacts, and restriction endonuclease reaction, is inserted into plasmid vector by gene, plasmid is introduced the method etc. of host cell.Such method is well-known for person having ordinary skill in the art, and described by having in many publications, such as Sambrook, J., Fritsch, E.F.andManiais, T. (1989) MolecularCloning:ALaboratoryManual, 2ndedition, ColdspringHarborLaboratoryPress.And material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Reagent and material
Lymphocyte separation medium, purchased from Beijing Ding Guo biotechnology limited liability company; Trizol reagent, purchased from Invitrogen company; Serum free medium EX-CELL302, purchased from SIGMA-ALDRICH company; Goatanti-humankappa-HRP is purchased from Invivogen company;
Embodiment one: the human antibody A of anti-hepatitis B virus surface antigen
3d
5preparation
One, the clone of antibody light chain constant region, weight chain constant area gene and the structure of carrier thereof
With lymphocyte separation medium separating health human lymphocyte, extract total serum IgE with Trizol reagent, design according to the sequence that document [4] and document [5] are reported heavy chain and the light chain constant region gene that primer adopts QIAGENOneStepRT-PCRKit amplification people antibody respectively.The heavy chain of people's antibody and light chain constant region gene are connected respectively in expression vector AbVec plasmid, construct heavy chain constant region nucleotide carrier IgG-AbVec plasmid and antibody light chain constant region nucleotide carrier Ig κ-AbVec plasmid respectively, confirm after sequence verification to obtain correct clone.Wherein, human antibody heavy chain's constant region nucleotide sequence and aminoacid sequence are respectively SEQIDNO:1 and SEQIDNO:2, and human antibody light chain constant region nucleotide sequence and aminoacid sequence are respectively SEQIDNO:3 and SEQIDNO:4.
Two, antibody heavy chain variable region, light-chain variable sequence obtain and the structure of recombinant expression vector
Particularly, adopt ultra-high speed streaming separation system to be separated the memory B cells (HBsAg+IgG+CD19+) that in volunteer's peripheral blood of HB vaccination, HBsAg is special, and be prepared into unicellular sample.And then, adopt SingleCellRT-PCR technology to clone light chain of antibody and weight chain variabl area sequence in each sample, because the cDNA of individual cells synthesis is rare especially, the method for Chao Shi PCR must be adopted to increase its antibody gene contained.
Visible significantly PCR band amplification (size is about 400bp) after two-wheeled PCR.Analyze specific PCR amplification antibody variable region band after its sequence, and introduce restriction enzyme site.Heavy chain of antibody and chain variable region gene are cloned into respectively IgG-AbVec plasmid and Ig κ-AbVec plasmid, form respectively containing A
3d
5the recombinant expression vector of complete heavy and light chain, is designated as IgG-AbVec-A respectively
3d
5with Ig κ-AbVec-A
3d
5.Wherein antibody A
3d
5weight chain variable region nucleotide sequence and aminoacid sequence are respectively SEQIDNO:5 and SEQIDNO:6; Light chain variable region nucleotide sequence and aminoacid sequence are respectively SEQIDNO:7 and SEQIDNO:8.
Three, the human antibody A of anti-hepatitis B virus surface antigen
3d
5expression and purification
5 × 10 are inoculated in 3.5cm Tissue Culture Dish
5cHO-K
1cell, cell cultures is carried out transfection when 90%-95% merges, and concrete steps are as follows: get 5 μ g plasmid IgG-AbVec-A
3d
5, 5 μ g plasmid Ig κ-AbVec-A
3d
5and 20 μ LLipofectamine2000Reagent, carry out transfection by Lipofectamine2000Reagent test kit specification sheets.After 24h is carried out in transfection, cell culture medium is changed to containing 600 μ g/mLG
418dMEM Screening of Media resistance clone.
The CHO-K of the stably express antibody obtained will be screened
1cell strain serum free medium EX-CELL302 enlarged culturing, use ProteinASepharose4FastFlow purification system, by specification affinity purification obtains antibody A
3d
5.The hepatitis B virus resisting surface protein total man resource monoclonal antibody A that purifying is obtained
3d
5dialyse with PBS, finally measure A with ultraviolet absorption method
3d
5the content of antibody.
Adopt polyacrylamide gel electrophoresis method, the structure of qualification antibody.Antibody after purifying detects its molecular size range by polyacrylamide gel electrophoresis respectively under non-reduced and reductive condition.Electrophoresis result shows: under the reducing conditions, A
3d
5antibody is rendered as heavy chain and the light chain bands that molecular size range is about 55KDa and 25KDa.Under non reducing conditions, A
3d
5antibody presents the single band that molecular weight is about 150KDa.
The human antibody A anti-hepatitis B virus surface antigen can be inferred thus
3d
5structure: this antibody is made up of the heavy chain that two identical light chains are identical with two, light chain is made up of variable region of light chain and constant region of light chain, heavy chain is made up of variable region of heavy chain and CH, wherein a light chain is connected by disulfide linkage with a heavy chain respectively, formation two near points, is connected to form described antibody by disulfide linkage between two heavy chains of two near points.So far, we build and give expression to complete total man's resource monoclonal antibody A
3d
5.
The human antibody A of embodiment two, anti-hepatitis B virus surface antigen
3d
5specificity
Total man's resource monoclonal antibody A is detected by Elisa method
3d
5specific binding HBsAg albumen.
Recombinant HBsAg albumen and reference protein cIg are wrapped by enzyme linked immunological plate, after confining liquid is closed, adds blank group, the A of the reference protein cIg of different concns and the different concns of expression and purification respectively
3d
5antibody incubation, adds Goatanti-humankappa-HRP after washing plate, and TMB nitrite ion develops the color, and microplate reader reads value under 450nm wavelength.
OD value according to antibody concentration and correspondence draws Fig. 1, can be obtained by Fig. 1 analysis, and along with increasing of antibody concentration, it wraps to be read by HBsAg protein groups correspondence that value is also corresponding to be increased.And bag is read value and blank group by reference protein group to read value close and do not change with antibody concentration and change, and reference protein cIg group is read value and is not relied on self change in concentration and change, therefore antibody A
3d
5can specific binding HBsAg albumen.
The human antibody A of embodiment three, anti-hepatitis B virus surface antigen
3d
5conjugated antigen Epitope Identification
The hepatitis B surface antigen (HBsAg) of comprehensive previous literature report has the antigenic epitopes region of Neutralization effect, synthesize biotin labeled hepatitis B surface antigen small peptide, measure the calmodulin binding domain CaM of total man source hepatitis B surface protein monoclonal antibody on HBsAg.As shown in the HBsAg mode chart in Fig. 2, four biotin labeling hepatitis B surface antigen small peptide P of synthesis
1(aa:104-120), P
2(aa:121-137), P
3(aa:139-148), P
4(aa:149-163), the aminoacid sequence of P1-P4 is as shown in SEQIDNO.9 ~ SEQIDNO.12.Wherein, antigen small peptide P
1and P
4for linear structure, P
2and P
3for ring texture
[6].
Adopt the methods analyst of ELISA A
3d
5the conjugated antigen epi-position of monoclonal antibody, as shown in Figure 3, A
3d
5antibodies is in P
3position.
The human antibody A of embodiment four, anti-hepatitis B virus surface antigen
3d
5with the qualification of HBV activity in external
Nearly decades, slow to HBV Infection in Vitro Models, with chimpanzee be mainly the HBV In vivo infection model of host because of expensive and more difficult acquisition, hinder HBV further investigation always
[7].In recent years, along with the foundation of HepaRG clone, there is greater advance to the research of HBV
[8].HepaRG clone is current unique putative HBV Infection in Vitro model.Therefore, we adopt HepaRG raji cell assay Raji A
3d
5in monoclonal antibody and the ability of HBV infection HepaRG cell, comprehensive HBsAg and HBVDNA copy number quantitative detecting analysis A
3d
5with HBV ability in monoclonal antibody.
One, antibody A
3d
5affect HBsAg detection by quantitative
Cultivate HepaRG cell with William ' sEmedium, add 2%DMSO and cultivate surrounding, within every three days, change liquid.After surrounding, differentiation-inducing good HepaRG cell presents typical liver cell sample tuftlet, and separates from the HepaRG cell of undifferentiated, and DMSO induction continued cultivation after two weeks, and cell about has half to present the change of liver cell sample.Differentiation-inducing good HepaRG cell continues with containing 10% foetal calf serum William ' sEmedium100units/mL penicillin, 100 μ g/mL Streptomycin sulphates, 5g/mL recombinant human insulin, 5 × 10
5mol/L hydrocortisone sodium salt continues to cultivate, for subsequent use.
Purifying degerming anti-hbs monoclonal antibodies (1 μ g/mL) and 2 × 10
6the virus quantity incubated at room of HBVDNA copy number 1 hour.Add in the differentiation-inducing good HepaRG cell of DMSO, and add PEG8000, final concentration is 4%, 37 DEG C of overnight incubation.Within second day, change liquid, wash three times with phosphate buffered saline buffer, add the fresh William ' sEmedium prepared, continue to cultivate.Collect the cell conditioned medium of after infecting the 6th day, the Electrogenerated chemiluminescent immunoassay instrument adopting Roche Holding Ag to produce and matched reagent weight per unit length thereof detect HBsAg.
As shown in Figure 4, infect the 6th day, same to control group (cIg group) and and antibody A
3d
5adopt antibody F prepared by identical method
2e
5, antibody B
2e
5, antibody D
3f
6compare: antibody F
2e
5hBsAg expression amount and control group almost indifference in cell conditioned medium in treatment group; Antibody B
2e
5, antibody D
3f
6and antibody A
3d
5group treatment group compared with control group (cIg group) in cell conditioned medium HBsAg expression amount occur declining, but A
3d
5obviously declining (* P < 0.05) appears in antibody comparatively other treatment group HBsAg expression amounts, therefore A
3d
5good HBV Neutralization effect is had more compared with other antibody.
Two, antibody A
3d
5affect HBVDNA copy number detection by quantitative
Detect HBVDNA copy number in the cell infecting latter 6th day according to HBVDNA PCR kit for fluorescence quantitative specification sheets, step is as follows:
(1) preparation work: sample treatment liquid A is placed in 70 DEG C of heating 5-10min, after melting, mixing is for subsequent use; In sample treatment liquid B, add dehydrated alcohol 6mL, mix for subsequent use; In sample treatment liquid C, add dehydrated alcohol 16mL, mix for subsequent use; Add 700 μ L sterilizing DEPC process water disinthibiting in agent, after dissolving, mixing is for subsequent use;
(2) nucleic acid extraction: draw 20 μ L and disinthibite agent in 0.5mL centrifuge tube; Add 100 μ L samples, repeatedly blow and beat 3 times with band filter core suction nozzle; Add 100 μ L sample treatment liquid A, vibration mixing, the brief centrifugation several seconds be placed on 70 DEG C of conditions under react 10min; The nucleic acid extraction column performing mark is put into 2ml centrifuge tube, all liquid in previous step is moved into nucleic acid extraction column.Centrifugal 10000rpm × 1min; Extraction column is put into a new 2mL centrifuge tube, add 500 μ L sample treatment liquid B, centrifugal 10000rpm × 1min; Extraction column is put into a new 2mL centrifuge tube, add 500 μ L sample treatment liquid C, centrifugal 10000rpm × 1min; Extraction column is put into a new 2mL centrifuge tube, centrifugal 14000rpmx1min; Extraction column is put into a new 2mL centrifuge tube, carefully 50uL sterilizing pure water is added in cylinder central authorities, leave standstill 1min; Centrifugal 10000rpm × 1min.The collection liquid 12 μ L got in 2mL centrifuge tube does PCR reaction template.Fluorescent value is detected according to HBVDNA PCR kit for fluorescence quantitative specification sheets.
As shown in Figure 5, infect the 6th day, antibody F
2e
5, antibody B
2e
5, antibody D
3f
6hBV-DNA copy number and control group (cIg group) almost indifference in treatment group cell, but A
3d
5antibody group treatment group occurs obviously declining (* P < 0.05) compared with HBV-DNA copy number in other treatment group cells, and A is described
3d
5antibody has the ability suppressing HBVDNA to copy preferably.
Embodiment five, antibody A
3d
5suppress the release of liver cancer cell HBsAg
Relation between HBV virus infection and primary hepatocellular carcinoma occur receives publicity day by day.Alexander in 1976 etc. are separated to a strain Bel7402 from Mozambique primary hepatocarcinoma male patient, called after PLC/PRF/5 (hereinafter referred to as PLC), this clone can produce HBsAg continually and steadily, since report, PLC has become the important experimental model studying HBV and Relations with Liver Cancer in the world.
HBsAg release in liver cancer cell whether can be suppressed with this hepatoma model research antibody.CIg, A
3d
5, B
2e
5, D
3f
6and F
2e
5antibody component other places reason PLC/RPF/5 cell, within 24 hours, recession is except antibody, and generation is with fresh substratum.3,6,12,24 hours points, detect the content of the HBsAg in cell conditioned medium.As shown in Figure 6, after antibody is removed, pass antibody D in time
3f
6and F
2e
5hBsAg and control group (cIg group) climbing speed almost indifference in cell conditioned medium in two treatment group.Antibody A
3d
5and B
2e
5the content climbing speed of the HBsAg in treatment group cell conditioned medium is starkly lower than cIg group, D
3f
6and F
2e
5treatment group, but A
3d
5antibody suppression HBsAg releasing effect is obviously better than B
2e
5antibody group (* p < 0.05), A
3d
5antibody can effectively suppress liver cancer cell HBsAg to discharge.
The effect of embodiment and effect
Embodiment provides a kind of human antibody of anti-hepatitis B virus surface antigen, and experiment shows that it can specific binding HBsAg albumen, has good HBV Neutralization effect, and then may stop the process of the infection of HBV and relevant hepatitis, liver cirrhosis and liver cancer.Meanwhile, because this antibody clones the human antibody obtained in the memory B cells that HBsAg is special from volunteer's peripheral blood of HB vaccination, it has compared with mouse source, the immunogenicity that chimeric and humanized antibody is lower.
Reference
[1]GanemD,PrinceAM,Hepatitis.Bvirusinfection-naturalhistoryandclinicalconsequences.NEnglJMed2004；350:1118–29.
[2]YangPL,AlthageA,ChungJ,ChisariFV.HydrodynamicinjectionofviralDNA:amousemodelofacutehepatitisBvirusinfection.ProcNatlAcadSciUSA2002；99:13825–30.
[3]SandgrenEP,PalmiterRD,HeckelJL,etal.Completehepaticregenerationaftersomaticdeletionofanalbumin-plasminogenactivatortransgene.Cell1991；66:245–56.
[4]HieterPA，MaxEE，SeidmanJG，MaizelJVJr，LederP.ClonedhumanandmousekappaimmunoglobulinconstantandJregiongenesconservehomologyinfunctionalsegments.Cell.1980；22(1Pt1):197-207.
[5]EllisonJW，BersonBJ，HoodLE.ThenucleotidesequenceofahumanimmunoglobulinCgamma1gene.NucleicAcidsRes.1982Jul10；10(13)：4071-9.
[6]GriponP,CannieI,UrbanS.EfficientinhibitionofhepatitisBvirusinfectionbyacylatedpeptidesderi-vedfromthelargeviralsurfaceprotein.JVirol2005；79:1613–22.
[7]EngelkeM,MillsK,SeitzS,etal.CharacterizationofahepatitisBandhepatitisdeltavirusreceptorbindingsite.Hepatology2006；43:750–60.
[8]LeistnerCM,Gruen-BernhardS,GlebeD.RoleofglycosaminoglycansforbindingandinfectionofhepatitisBvirus.CellMicrobiol2008；10:122–33.
The invention is not restricted to the scope of embodiment; to those skilled in the art; as long as various change to limit and in the spirit and scope of the present invention determined in described claim; these changes are apparent, and all innovation and creation utilizing the present invention to conceive are all at the row of protection.
Claims (11)
1. the human antibody A of an anti-hepatitis B virus surface antigen
3d
5, it is characterized in that having:
Heavy-chain variable domains, comprises hypervariable region CDR
1, CDR
2, CDR
3; And
Light variable domains, comprises hypervariable region CDR
1＇, CDR
2＇, CDR
3＇,
Wherein, described CDR
1aminoacid sequence be: Met-Tyr-Ala-Phe-Ser;
Described CDR
2aminoacid sequence be: Gly-Ile-Ile-Pro-Ser-Phe-Asp-Thr-Thr-Asn-Tyr-Ala-Gln-Lys-Phe-Gln-Cys;
Described CDR
3aminoacid sequence be: Ser-Val-Ile-Thr-Asp-Leu-Asp-Thr-Val-Gly-Asn-Asp-Glu-Ser-Gly-Asp-Ala-Ser-Tyr-Tyr-Tyr-Met-Asp-Val;
Described CDR
1the aminoacid sequence of ＇ is: Arg-Ser-Ser-Glu-Ser-Leu-Gln-His-Ser-Asp-Gly-Thr-Tyr-Tyr-Leu-Asp;
Described CDR
2the aminoacid sequence of ＇ is: Ser-Ala-Ser-Asn-Thr-Ala-Pro;
Described CDR
3the aminoacid sequence of ＇ is: Met-Gln-Ala-Leu-Glu-Thr-Pro-Phe-Thr.
2. the human antibody A of anti-hepatitis B virus surface antigen according to claim 1
3d
5, it is characterized in that:
Wherein, the aminoacid sequence of described heavy-chain variable domains is as shown in SEQIDNO.6, and the aminoacid sequence of described light variable domains is as shown in SEQIDNO.8.
3. the human antibody A of anti-hepatitis B virus surface antigen according to claim 2
3d
5, it is characterized in that:
Wherein, described antibody A
3d
5be selected from the monoclonal antibody of anti-hepatitis B virus surface antigen and described monoclonal antibody fragment any one,
Described monoclonal antibody fragment is the Fab fragment of described monoclonal antibody or F (ab ')
2fragment.
4. a human antibody fragment for anti-hepatitis B virus surface antigen, is characterized in that:
The human antibody A that described antibody fragment is the anti-hepatitis B virus surface antigen described in claim 1 or 2
3d
5fab fragment or F (ab ')
2fragment.
5. the human antibody A of an anti-hepatitis B virus surface antigen of encoding described in claim 1 or 2
3d
5gene, it is characterized in that:
Encode the nucleotide sequence of described heavy-chain variable domains as shown in SEQIDNO.5, and the nucleotide sequence of described light variable domains of encoding is as shown in SEQIDNO.7.
6. an expression vector, is characterized in that:
Described expression vector contains the gene according to claim 5 of at least one copy.
7. a host cell, is characterized in that:
Described host cell contains at least one expression vector according to claim 6.
8. the human antibody A of the anti-hepatitis B virus surface antigen described in any one of claims 1 to 3
3d
5purposes in the medicine preparing prevention or treatment hepatitis B virus related liver disease or diagnostic reagent.
9. the purposes of human antibody fragment in the medicine preparing prevention or treatment hepatitis B virus related liver disease or diagnostic reagent of anti-hepatitis B virus surface antigen according to claim 4.
10. the human antibody A containing the anti-hepatitis B virus surface antigen described in any one of claims 1 to 3
3d
5medicine or diagnostic reagent, it is characterized in that:
Wherein, described medicine or diagnostic reagent also comprise any one in pharmaceutically acceptable vehicle, thinner and carrier.
The medicine of 11. 1 kinds of human antibody fragments containing anti-hepatitis B virus surface antigen according to claim 4 or diagnostic reagent, is characterized in that:
Wherein, described medicine or diagnostic reagent also comprise any one in pharmaceutically acceptable vehicle, thinner and carrier.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107556381A (en) * | 2017-10-12 | 2018-01-09 | 南京京达生物技术有限公司 | A kind of hepatitis B virus surface antigen detection antibody IgM and application |
| CN112165974A (en) * | 2018-05-31 | 2021-01-01 | 诺华股份有限公司 | Hepatitis B antibodies |
| CN115160433A (en) * | 2022-06-28 | 2022-10-11 | 吉林大学 | Humanized HBV B and C genotype pre-S1 protein antibody and application thereof |
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| CN112165974A (en) * | 2018-05-31 | 2021-01-01 | 诺华股份有限公司 | Hepatitis B antibodies |
| CN115160433A (en) * | 2022-06-28 | 2022-10-11 | 吉林大学 | Humanized HBV B and C genotype pre-S1 protein antibody and application thereof |
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