CN105001325A - Total-humanized anti-hepatitis B virus neutralizing antibody and preparing method and application thereof - Google Patents
Total-humanized anti-hepatitis B virus neutralizing antibody and preparing method and application thereof Download PDFInfo
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Abstract
The invention relates to a total-humanized anti-hepatitis B virus neutralizing antibody. The heavy chain variable region of the antibody has the amino acid sequences shown in the sequence tables of SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4. The light chain variable region of the antibody has the amino acid sequences shown in the sequence tables of SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8. The total-humanized anti-hepatitis B virus neutralizing antibody has the advantages that the peripheral blood of a volunteer with the high anti-hepatitis B virus immune globulin titer is collected, specific plasma cells are separated, antibody genes of single cells are fished and expressed in vitro, the functional experiment verification is carried out, and one strain of anti-hepatitis B virus antibody with the optimal neutralizing activity is obtained through screening. The antibody has the advantages of being total in human source, high in specificity, high in sensitivity and free of differential source seroreaction. Safety and high efficiency are achieved, side effects are avoided, mass preparation in vitro can be achieved, and the antibody is suitable for large-scale industrial production.
Description
Technical field
The present invention relates to field of pharmaceutical biology, be specifically related to a kind of total man source hepatitis B virus resisting neutralizing antibody and preparation method thereof and application.
Background technology
Monoclonal antibody, refers to only by the produced antibody of the cell of a type.Monoclonal antibody is produced by the cell after the immunocyte and cancer cells that can manufacture corresponding antibodies merge, and this fused cell had both had the ability that oncocyte constantly divides, and had again the ability that immunocyte can produce antibody.Hybrid cell after fusion, i.e. hybrid knurl, can produce antibody identical in a large number.When being applied in medical treatment, identifying that the display subtle change in antigen contributes to reducing side effect.The antibody medicine made by monoclonal antibody is the effective ways for the treatment of various diseases at present.The extracorporeal antivirus effect Neutralization effect of monoclonal antibody and endogenous protective body opposing virus attack have obtained many experiments to be proved, as mouse-anti hepatitis A virus (HAV), Hantaan virus, Measles virus, RSV is viral, CMV is viral etc., and neutralizing monoclonal antibody can 100% watch for animals from virus attack in vivo.Monoclonal antibody has three kinds of unique mechanism of action, is respectively target effect, blocking effect and intracellular signaling effect.Monoclonal antibody drug mainly in treatment tumour, autoimmune disorder and infectious diseases etc. curative effect give prominence to.
Monoclonal antibody drug development experience four-stage, is respectively: mouse monoclonal antibody, chimeric monoclonal antibodies, Humanized monoclonal antibodies and total man's resource monoclonal antibody.First stage, mouse resource monoclonal antibody is the common monoclonal antibody drug prepared with mouse, on the one hand antibody can not effect system that effectively complement is relevant with FC acceptor in human activin, human anti-murine antibodies (HAMA) is identified by human immune system, be eliminated very soon in human recycle system, on the other hand its toxic side effect is obvious, often causes the untoward reaction of human body, greatly constrains its development in clinical experiment and related application field; S-generation people mouse mosaic monoclonal antibody medicine carries out humanization modified to mouse antibodies surface amino groups acid residue, makes its humanization degree reach about 70%, still has very large toxic side effect; The humanization degree of the third generation humanization monoclonal antibody then occurred reaches about 95%, more close to the antibody of human body self, greatly reduces toxic side effect.Human antibody is directly separated from human immunity B cell, substantially do not have rejection, have irreplaceable advantage as therapeutic antibodies.Humanization and total man's resource monoclonal antibody due to side effect little, the residence time is long in vivo, is more conducive to treatment.
Hepatitis B virus causes the most commonly encountered diseases of viral hepatitis former, and it can pass through blood, mother and baby's close contact and Sex transmitted pathogen other people.At present, have an appointment 400,000,000 chronic HBVs (HBV) the infected in the whole world, and nearly 100,000,000 virus carriers of China, be in the 1st, the world.Carry HBV mother can vertical transmission to baby, cradle infect HBV person, more than 90% becomes chronic viral hepatitis B surface antigen (HBsAg) carrier.In Chinese chronic hepatitis B patients, about 25%-40% finally will die from liver cirrhosis or merge liver cancer, and the danger that male sex HBV infection person finally dies from related liver disease is 50%, and women is 15%.Unfortunately chronic HBV infection still lacks effective remedy measures so far, so prevent hepatitis B infected, blocks mother-to-baby transmission and just becomes abnormal important.
The medicine of blocking-up mother-to-baby transmission conventional clinically is at present anti-hepatis B immunoglobulin.Anti-hepatis B immunoglobulin (hepatitis B immune globulin, HBIG) be the immunoglobulin (Ig) of the high density specificity hepatitis B surface antibody be prepared into the serum of hepatitis B surface antibody positive human or blood plasma, can be combined with HBsAg, form immune complex, impel immunity system to remove hepatitis B virus.Research confirms, anti-hepatis B immunoglobulin adds Hepatitis B virus vaccine effectively can block maternal-fetal transmission, prevents infection of newborn hepatitis B virus.The pregnant woman of Hepatitis B carrier respectively injects anti-hepatis B immunoglobulin and Hepatitis B virus vaccine to its newborn infant in childbirth in 24 hours, can reach more than 90% to the blocking-up rate of spreading, be the most effective means of inhibition of hepatitis b virus mother-to-baby transmission of world health organisation recommendations.Current countries in the world are all widely using this way.In China, mother-to-baby transmission is that hepatitis B infects topmost approach, and the pregnant woman of national Hepatitis B carrier has exceeded 10%, so more should promote the use of this way.Although the preventative vaccine for HBV is proved to be effective, still has the individuality of 5-10% to traditional Hepatitis B virus vaccine nonreply or reply more weak.So, HBIG combined with lamivudine effect in the recurrence of prevention liver transplantation (liver transplantation, LT) HBV afterwards clinically significantly, also can be used for the passive immunization of high risk population, to HBV infection after blood transfusion, HBIG also can play certain preventive effect.
But anti-hepatis B immunoglobulin extracts from the blood of healthy blood donor, this just objectively limits the supply of raw material.Government department strengthens the management to blood sampling point in recent years, blood sampling program specification more, this also limits the output of blood products to a certain extent, and country controls very tight to blood products price at present, starting material are nervous, and retail price never adjustment, therefore most of producer all controls production quantity, thus cause the source of goods in short supply, and blood products, the risk infecting known or unknown virulence factor cannot be got rid of completely.
Therefore, need a kind of novel total man source of exploitation hepatitis B virus resisting neutralizing antibody badly, and improve to some extent in its Synthesis and applications and improve, thus solve the problems referred to above of prior art existence.
Summary of the invention
The object of this invention is to provide a kind of total man source hepatitis B virus resisting neutralizing antibody, to overcome currently available technology above shortcomings.
The object of the invention is to be achieved through the following technical solutions:
The invention provides a kind of total man source hepatitis B virus resisting neutralizing antibody, the variable region of the heavy chain of this antibody has the aminoacid sequence as shown in SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 in sequence table; The variable region of the light chain of this antibody has the aminoacid sequence as shown in SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 in sequence table.
The heavy chain of total man source of the present invention hepatitis B virus resisting neutralizing antibody has the aminoacid sequence as shown in SEQ ID NO 1 in sequence table, and the light chain of this antibody has the aminoacid sequence as shown in SEQ ID NO 5 in sequence table.
The present invention is also comprised the CDR region sequence including antibody of the present invention that formed by the interpolation to amino-acid residue, deletion, amendment the aminoacid sequence of above-mentioned antibody and has all antibody of identical function or transformation and optimization, comprises strand (scFv) antibody of people source and non-human source antibodies, transformation; And contain other antibody fragments in single heavy chain CDR district of the present invention or other antibody fragments containing single light chain CDR district of the present invention.
Present invention also offers a kind of method preparing described total man source hepatitis B virus resisting neutralizing antibody, comprise the following steps:
(1) choose volunteer, inject restructuring (yeast) hepatitis B vaccine respectively, when antibody titer and plasmocyte ratio reach maximum, blood drawing, utilize density gradient centrifugation method separating peripheral blood mononuclear cells (PBMC), the single B cell of sorting is in PCR plate, frozen for subsequent use;
(2) utilize PCR method separation antibody variable region gene from single B cell, method is as follows:
Reverse transcription synthesis cDNA Article 1 chain: the constant region primers and the ThermoScript II that 96 orifice plates containing single B cell are added each subtype heavy chain and light chain, vortex mixes, and reaction heating is placed on ice, and reverse transcription obtains cDNA;
PCR separation antibody gene: preparation PCR system, through denaturation, sex change, anneal, prolonged treatment, PCR circulates, PCR primer utilizes gel electrophoresis to identify, the gene fragment that qualification result is the positive is checked order, utilize IMGT software to analyze sequencing result, utilize TA cloning, screening from same sample hole is obtained all have a pair heavy chain of function, chain variable region gene is connected respectively on pcDNA3.3 carrier;
Product conversion will be connected in DH5 α competent cell, LB flat board containing penbritin is cultivated, the single bacterium colony Auele Specific Primer of random picking 12 carries out PCR qualification, identifies the transformant obtained containing heavy chain of antibody or light chain gene in positive transformant;
By ELISA Binding experiment, screening obtains the antibody HBV008 of the hepatitis B virus resisting of 1 strain binding activities the best;
(3) HBV008 monoclonal antibody expression and purification: the heavy chain matched and light chain gene expression vector are carried out transfection and cultivated, centrifugal, regulate pH, phosphoric acid buffer wash-out is neutralized to pH 7.2, dialysis, utilize ultraviolet spectrophotometer to survey OD260, OD280, calculate protein content, then cryopreservation;
Further, also comprise HBV008 monoclonal antibody and Hepatitis B virus vaccine specific binding and sensitivity technique step, its concrete steps are as follows:
By Hepatitis B virus vaccine (10ug/ml) bag by high-affinity elisa plate, PBS buffer solution, HBV008 albumen is diluted, is added and be coated with in the elisa plate of Hepatitis B virus vaccine, hatch rear PBS buffer solution, add hepatitis B virus resisting monoclonal antibody HBV008, hatch rear PBS buffer solution, then add the antibody that goat anti-human igg HRP marks, hatch rear PBS buffer solution, add TMB colour developing, detect after 2N sulfuric acid stops and read value under OD450 wavelength.
Further, in step (1), the choosing method of described antibody titer and plasmocyte ratio maximum is as follows: utilize flow cytometer to carry out staining analysis to the PBMC in HB vaccination volunteer 0-21 days peripheral bloods, comparison result, thus the PBMC choosing suitable time points carries out single plasmocyte sorting, therefrom separation antibody heavy chain, light chain gene and total man's resource monoclonal antibody gene.
Preferably, the PBMC in volunteer's difference HB vaccination peripheral blood of the 0th, 5,7,10,14 and 21 days, and carry out staining analysis.
Preferably, in step (1), Cytokines in Peripheral Blood Mononuclear (PBMC) utilizes BD selected by flow cytometry apoptosis CD3-after carrying out separation counting, CD14-, CD16-, CD19+, CD27+, the single B cell of CD38+ is in 96 hole PCR plate, and every Kong Hanyi B cell in PCR plate ,-80 DEG C frozen for subsequent use.
Present invention also offers total man source hepatitis B virus resisting neutralizing antibody or the application of their active fragments in the medicine preparing prevention or treatment hepatitis B virus related liver disease or diagnostic reagent.
Beneficial effect of the present invention is: the present invention is by collecting the peripheral blood of the higher volunteer of hepatitis B virus resisting immunoglobulin titer, be separated specific plasmocyte, by fishing the antibody gene getting individual cells, and express in vitro, do function experimental verification, screening obtains the antibody of the hepatitis B virus resisting of 1 strain Neutralization effect the best.This antibody have total man source, high specificity, highly sensitive, without the feature of allos sero-reaction, safe and efficient, have no side effect, external a large amount of preparation can be realized, be applicable to large-scale industrial production.
Accompanying drawing explanation
Below in order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment below, apparently, accompanying drawing in the following describes is only some embodiments of the application, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is different time points plasmocyte proportion grading schematic diagram after the immunity described in the embodiment of the present invention;
Fig. 2 is the gel electrophoresis schematic diagram of the antibody variable gene described in the embodiment of the present invention;
Fig. 3 is the protein electrophoresis figure of the monoclonal antibody HBV008 described in the embodiment of the present invention;
Fig. 4 be HBV008 antibody described in the embodiment of the present invention and Hepatitis B virus vaccine in conjunction with schematic diagram one;
Fig. 5 be HBV008 antibody described in the embodiment of the present invention and Hepatitis B virus vaccine in conjunction with schematic diagram two;
Fig. 6 be HBV008 antibody described in the embodiment of the present invention and Hepatitis B virus vaccine in conjunction with schematic diagram three;
Fig. 7 is the detectable level schematic diagram one of the antibody HBV008 described in the embodiment of the present invention to Hepatitis B virus vaccine;
Fig. 8 is the detectable level schematic diagram two of the antibody HBV008 described in the embodiment of the present invention to Hepatitis B virus vaccine;
Fig. 9 is the detectable level schematic diagram three of the antibody HBV008 described in the embodiment of the present invention to Hepatitis B virus vaccine;
Figure 10 is that HBV infection efficiency described in the embodiment of the present invention and many resistances are broken efficiency schematic diagram.
Embodiment
Embodiment one
The invention provides a kind of method preparing described total man source hepatitis B virus resisting neutralizing antibody, comprise the following steps:
(1) screening antibodies: choose 5-10 name normal human volunteers, signature " Informed Consent Form ", injects restructuring (yeast) hepatitis B vaccine respectively.Select antibody titer in above-mentioned sample the highest and the time point that plasmocyte ratio is the highest, extract volunteer blood 10-15ml, utilize the method separating peripheral blood mononuclear cells (PBMC) of density gradient centrifugation.Utilize BD selected by flow cytometry apoptosis CD3-after cell counting, the single B cell of CD14-, CD16-, CD19+, CD27+, CD38+ is in 96 hole PCR plate, and make each hole in PCR plate contain a B cell ,-80 DEG C frozen for subsequent use;
The choosing method of described antibody titer and plasmocyte ratio maximum is as follows: utilize flow cytometer to after volunteer inoculating's Hepatitis B virus vaccine the 0th day, 5th day, 7th day, 10th day, PBMC in 14th day and the 21st day peripheral blood carries out staining analysis, comparing result, result shows that plasmocyte ratio peaked the 14th day time, within 10th day, take second place, the ratio accounting for PBMC is respectively 1.61% and 1.51%, after immunity, as shown in Figure 1, wherein arrow represents the cell mass carrying out single B cell sorting to the result of different time points plasmocyte proportion grading.Selected 10th day and the 14th day PBMC carry out single plasmocyte sorting, and therefrom separation antibody heavy chain, light chain gene and total man's resource monoclonal antibody.
(2) PCR method separation antibody variable region gene from single B cell:
Reverse transcription synthesis cDNA Article 1 chain: the constant region primers and the ThermoScript II that 96 orifice plates containing single B cell are added each subtype heavy chain and light chain, vortex mixes, and 42 DEG C are reacted 1 hour, and 95 DEG C are heated 5 minutes, and be then placed on ice, reverse transcription obtains cDNA;
PCR separation antibody gene: preparation PCR system, comprises RT reaction product, Taq enzyme, dNTPs, and the Auele Specific Primer of each subtype heavy chain and light chain antibody, through 95 DEG C of denaturations 3 minutes, sex change 30 seconds, 62 DEG C of annealing 30 seconds, 72 DEG C extend and 30 seconds etc. carry out 30 PCR circulations.The PCR primer 96 hole agarose gel electrophoresis of 1.2% are identified, result as shown in Figure 2.Maker in Fig. 2 is the DL2000 DNA marker of TAKARA.
Gel electrophoresis is accredited as positive gene fragment to check order, by IMGT software, its sequencing result is analyzed, screening from same sample hole is obtained all have a pair heavy chain of function, method that chain variable region gene utilizes TA to clone respectively is connected to pcDNA3.3 and carries.
To connect in product conversion DH5 α competent cell, LB flat board containing penbritin is cultivated 16 hours for 37 DEG C, the single bacterium colony Auele Specific Primer of random picking 12 carries out PCR qualification, the transformant containing heavy chain of antibody or light chain gene is identified in positive transformant, by ELISA Binding experiment, screening obtains the antibody HBV008 of the good hepatitis B virus resisting of 1 strain binding activities.
(3) HBV008 monoclonal antibody expression and purification:
Its concrete steps are as follows:
Material: 1moL/L TRIS-HCL, 0.1moL/L phosphoric acid buffer, 293T cell, Protein A Sepharose CL 4B post albumen post, previous materials is from this yuan, Beijing Zhenyang Bioisystech Co., Ltd.
Method and result:
By the heavy chain of pairing and light chain gene expression vector PolyFect (Qiagen, Valencia, CA) transfection reagent cotransfection 293T cell, within after transfection 6-8 hour, change fresh culture, and at 37 DEG C of 5% CO
248-72 hour is cultivated in incubator.
Collect and express supernatant liquor, centrifugally discard cell debris, add 1mL pH 8.0,0.1moL/L phosphoric acid buffer and adjust pH to 9.0 with pH 9.0,1moL/L TRIS-HCL.0.1moL/L phosphoric acid buffer pH 8.0 is utilized to balance Protein A Sepharose CL 4B albumen post, cell conditioned medium is added in aforementioned Protein A Sepharose CL 4B albumen post, utilize above-mentioned damping fluid to wash in pillar to effluent liquid and can't detect foreign protein.Adopt the citrate buffer solution of pH 3.0 to carry out wash-out, collect effluent liquid, and immediately with the neutralization of 1moL/L pH 8.5 TRIS-HCL damping fluid, utilize pH 7.2,0.01M PBS damping fluid dialysis 72h subsequently.Then sampling is placed on ultraviolet spectrophotometer and surveys OD280, calculate protein content, then be placed in 4 DEG C of preservations, after purifying, the SDS electrophorogram of monoclonal antibody HBV008 as shown in Figure 3, and in figure, 1 is non-sex change HBV008 antibody, 2 is marker, 3 is sex change HBV008 antibody, and as can be seen from the figure, HBV008 antibody molecule amount is at 150KDa, heavy chain molecule amount 50KDa, light chain molecule amount 25KDa.
(4) HBV008 monoclonal antibody and Hepatitis B virus vaccine specific binding and sensitivity experiment
Material: bag is buffered liquid (PH9.6 0.05M carbonate buffer solution), lavation buffer solution (PH7.4 PBS), stop buffer (2M H
2sO
4), stop buffer (2M H
2sO
4).
Method and result:
Wrap the Hepatitis B virus vaccine (10ug/ml) produced by different process carrier in high-affinity elisa plate, 4 DEG C spend the night after with the PBS buffer solution 5 times containing 0.05% tween;
HBV008 albumen starting point concentration is 1ug/ml, 10 times of dilutions, and the HBV008 albumen got after 100ul dilution joins and is coated with in the elisa plate bar of Hepatitis B virus vaccine, hatches 1 hour for 37 DEG C, with the PBS buffer solution 5 times containing 0.05% tween;
Add the antibody that goat anti-human igg HRP marks, hatch 0.5 hour for 37 degree, with the PBS buffer solution 5 times containing 0.05% tween;
Finally add TMB colour developing, detect after 2N sulfuric acid stops and read value under OD450 wavelength, result as Figure 4-Figure 6, wherein, Hepatitis B virus vaccine in Fig. 4 is that Beijing institute of Biological Products is produced, Hepatitis B virus vaccine in Fig. 5 is the recombinant hepatitis B vaccine (Chinese hamster ovary celI) that North China Pharmaceutical Factory produces, and the Hepatitis B virus vaccine in Fig. 6 is that benefit can glad recombinant hepatitis B vaccine (yeast).As we can see from the figure, the combination of HBV008 antibody and Hepatitis B virus vaccine has dose-dependently, illustrates that the combination of antibody HBV008 and hepatitis B surface antigen is specific.
Utilize aforesaid method, Hepatitis B virus vaccine is carried out 1:100 doubly to dilute (saturated bag is by concentration), detect antibody HBV008 to the identification sensitivity of Hepatitis B virus vaccine, result as Figure 7-9, wherein, Hepatitis B virus vaccine in Fig. 7 is that Beijing institute of Biological Products is produced, and the Hepatitis B virus vaccine in Fig. 8 is the recombinant hepatitis B vaccine (Chinese hamster ovary celI) that North China Pharmaceutical Factory produces, and the Hepatitis B virus vaccine in Fig. 9 is that benefit can glad recombinant hepatitis B vaccine (yeast).Result display antibody HBV008 is 0.0093ug/ml to the minimal detectable concentration of Hepatitis B virus vaccine.
(5) neutralization test of HBV008 monoclonal antibody and hepatitis B virus
HBIG blocks HBV infection HepG2-NTCP
HepG2-NTCP cell is placed in 10%PBS DMEM culture medium culturing, after bed board, uses PEG to concentrate the HBV virus (2*10 of HepAD38 supernatant
9copy/ml) infect, add 4% PEG8000 simultaneously, HBV008 antibody and HBIG block with 1:200 dilution and infect, supernatant is abandoned after 16 hours, PBS washes three times, within every 3 days, collect culture supernatant, hepatitis B E antigen (HBeAg) test kit detects in supernatant the HBeAg content of secreting, to judge that HBV infection efficiency and many resistances are broken efficiency.As shown in Figure 10, after the 9th day, HBV008 success blocking virus infects result.
Derivative antibody fragment
Combination due to antibody and antigen molecule depends primarily on the complementary determining region (CDR district) of antibody, so also comprise other antibody fragments be derived by the CDR sequence of antibody HBV008 except antibody HBV008 via antibody described in the invention:
The people source that 1.CDR district transplants and non-human source antibodies's fragment
CDR implantation technique, refer to and the complementary determining region of a certain antibody (CDR district) is transplanted on the framework region of other antibody, to change the type of this antibody, and keep a kind of method of antibodies specific and avidity, the method is utilized to carry out humanization modified by antagonist, or the transformation of other animal sourceization, or change the hypotype of antibody.So the present invention also comprises the antibody obtained by the framework region that antibody CDR region sequence of the present invention is transplanted to other antibody, comprise people source and non-human source antibodies, and there is the antibody fragment with HBV008 antibody identical function.
2. the antibody fragment of the sudden change of antibody constant region a single point or multiple somes combinatorial mutagenesis transformations
The point mutation of antibody constant region especially on framework region is usually transformed for antagonist characteristic, to optimize the performance of antibody, comprises thermostability and water miscible further raising, optimizes the FC section of antibody to extend its transformation period etc.The transformation of antibody constant region comprises: a single point of constant region, framework region and FC section suddenlys change or multiple somes combinatorial mutagenesises, utilizes simple point mutation or multi-point combination sudden change antagonist constant region to carry out the antibody fragment after transforming and optimizing so the present invention is also included on antibody fragment that antibody HBV008 or its CDR transplant.
The antibody fragment of a single point sudden change of 3.CDR district or multiple somes combinatorial mutagenesis transformations
CDR district is the region of the specific binding of antibody and antigen, but not all cdr amino acid all participates in the specific binding of synantigen molecular epitope, so there is certain transformation space in CDR district, a single point sudden change or multiple somes combinatorial mutagenesises are utilized to transform to CDR district, likely when not affecting antibody function, further antagonist performance is optimized, and as improved humanization degree further, improves affinity of antibody etc. further.The present invention is also included in the antibody fragment after antibody HBV008 antibody or its CDR grafted antibody fragment utilize simple point mutation or multi-point combination sudden change to carry out transforming to CDR district partial amino-acid and optimize.
4. contain the antibody fragment in the single heavy chain of antibody described in the invention or single light chain CDR district
Usually and not all participate in the specific binding of synantigen, some antibody, as the neutralizing antibody M36 of HIV, are made up of single heavy chain for light chain and the heavy chain of composition antibody, and its combination with HIV gp120 molecule is relevant with three CDR districts of heavy chain.Strand displacement operation is carried out to some antibody, variable region of heavy chain or the variable region of light chain of the corresponding fragment of other antibody or single expression is replaced to by the heavy chain of a certain antibody or light chain, also likely have the function identical with original parental antibody, strand displacement is the important means of engineered antibody too.So the present invention also comprises containing antibody HBV008, or its CDR grafted antibody fragment, or the single heavy chain CDR district of antibody that transforms of its constant region and CDR district amino acid or the antibody fragment in single light chain CDR district.
5. the single-chain antibody scFv be made up of antibody heavy chain variable region of the present invention and variable region of light chain
Single-chain antibody (scFv) refers to and utilizes gene recombination technology, and utilized in the variable region of heavy chain of antibody and variable region of light chain a flexible Linker to couple together and the antibody fragment of expressing, the scFv antibody of expression has the function identical with original antibody.Because scFv antibody is less, so in expression and production, the aspects such as antibody penetration performance have certain advantage compared with conventional antibodies.So the present invention also comprises antibody HBV008, or its CDR grafted antibody, or the antibody that constant region and CDR district transform, or be connected with all antibody variable regions of single light chain containing the single heavy chain in antibody CDR district of the present invention, the scFv antibody obtained.
The present invention is not limited to above-mentioned preferred forms; anyone can draw other various forms of products under enlightenment of the present invention; no matter but any change is done in its shape or structure; every have identical with the application or akin technical scheme, all drops within protection scope of the present invention.
Sequence table
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Claims (10)
1. a total man source hepatitis B virus resisting neutralizing antibody, is characterized in that: the variable region of the heavy chain of this antibody has the aminoacid sequence as shown in SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 in sequence table; The variable region of the light chain of this antibody has the aminoacid sequence as shown in SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 in sequence table.
2. total man source according to claim 1 hepatitis B virus resisting neutralizing antibody, is characterized in that: the heavy chain of this antibody has the aminoacid sequence as shown in SEQ ID NO 1 in sequence table; The light chain of this antibody has the aminoacid sequence as shown in SEQ ID NO 5 in sequence table.
3. the people source containing total man source hepatitis B virus resisting neutralizing antibody sequence described in claim 1 or 2 and non-human source antibodies's fragment or single-chain antibody (scFv) fragment.
4. antibody fragment according to claim 3, is characterized in that: described antibody fragment has the CDR district of single heavy chain, and described antibody fragment has the CDR district of single light chain.
5. the nucleotide sequence of antibody amino acid according to any one of coding claim 1-4.
6. the DNA fragment containing the nucleotide sequence described in claim 5 or carrier.
7. prepare a method for the total man source hepatitis B virus resisting neutralizing antibody in claim 1-6 described in any one, it is characterized in that, comprise the following steps:
(1) choose volunteer, inject restructuring (yeast) hepatitis B vaccine respectively, when antibody titer and plasmocyte ratio reach maximum, blood drawing, utilize density gradient centrifugation method separating peripheral blood mononuclear cells (PBMC), the single B cell of sorting is in PCR plate, frozen for subsequent use;
(2) utilize PCR method separation antibody variable region gene from single B cell, method is as follows:
Reverse transcription synthesis cDNA Article 1 chain: the constant region primers and the ThermoScript II that 96 orifice plates containing single B cell are added each subtype heavy chain and light chain, vortex mixes, and reaction heating is placed on ice, and reverse transcription obtains cDNA;
PCR separation antibody gene: preparation PCR system, through denaturation, sex change, anneal, prolonged treatment, PCR circulates, PCR primer utilizes gel electrophoresis to identify, the gene fragment that qualification result is the positive is checked order, utilize IMGT software to analyze sequencing result, utilize TA cloning, screening from same sample hole is obtained all have a pair heavy chain of function, chain variable region gene is connected respectively on pcDNA3.3 carrier;
Product conversion will be connected in DH5 α competent cell, LB flat board containing penbritin is cultivated, the single bacterium colony Auele Specific Primer of random picking 12 carries out PCR qualification, identifies the transformant obtained containing heavy chain of antibody or light chain gene in positive transformant;
By ELISA Binding experiment, screening obtains the antibody HBV008 of the hepatitis B virus resisting of 1 strain binding activities the best;
(3) HBV008 monoclonal antibody expression and purification: the heavy chain matched and light chain gene expression vector are carried out transfection and cultivated, centrifugal, regulate pH, phosphoric acid buffer wash-out is neutralized to pH 7.2, dialysis, utilize ultraviolet spectrophotometer to survey OD260, OD280, calculate protein content, then cryopreservation.
8. the preparation method of total man source according to claim 7 hepatitis B virus resisting neutralizing antibody, is characterized in that, also comprise HBV008 monoclonal antibody and Hepatitis B virus vaccine specific binding and sensitivity technique step, its concrete steps are as follows:
By Hepatitis B virus vaccine (10ug/ml) bag by high-affinity elisa plate, PBS buffer solution, HBV008 albumen is diluted, is added and be coated with in the elisa plate of Hepatitis B virus vaccine, hatch rear PBS buffer solution, add hepatitis B virus resisting monoclonal antibody HBV008, hatch rear PBS buffer solution, then add the antibody that goat anti-human igg HRP marks, hatch rear PBS buffer solution, add TMB colour developing, detect after 2N sulfuric acid stops and read value under OD450 wavelength.
9. the preparation method of total man source according to claim 7 hepatitis B virus resisting neutralizing antibody, it is characterized in that, in step (1), the choosing method of described antibody titer and plasmocyte ratio maximum is as follows: utilize flow cytometer to carry out staining analysis to the PBMC in HBV004 HB vaccination 0-21 days peripheral bloods, comparison result, thus choose suitable PBMC and carry out single plasmocyte sorting, therefrom separation antibody heavy chain, light chain gene and total man's resource monoclonal antibody gene.
10. the total man source hepatitis B virus resisting neutralizing antibody described in claim 1 or 2 or the application of their active fragments in the medicine preparing prevention or treatment hepatitis B virus related liver disease or diagnostic reagent.
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