CN1355253A - Humanized genetically engineered neutralizing antibody of rabies virus - Google Patents
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Abstract
A humanized neutralizing gene engineering antibody of rabies virus designated RVG10-, including the holoantibody gene of Fab fragments and IgG, its gene product and its application are disclosed. It features that the said recombinant antibody is determined by the specific gene sequence of hypervariable region (CORs) in the variable region of antibody light chain and heavy chain, and is effectively expressed in the eukaryotic and procaryotic cells. It can be used to prevent and cure rabies.
Description
The present invention relates to prevent and treat preparation and application, the especially specificity of personnel selection source gene engineering monoclonal antibody neutrality gene engineering monoclonal antibody at rabies virus glycoprotein.
Since B lymphocyte hybridoma cell-fusion techniques in 1975 came out, monoclonal antibody was used for fundamental research as a class, laboratory diagnosis, and the novel product of clinical treatment and prevention, its applying value and DEVELOPMENT PROSPECT have obtained general affirming.The researchdevelopment of molecular biology and molecular immunology has caused the generation and the development of genetic engineering antibody.Reorganization by the antibody molecule gene level can obtain diversified specific murine source and human antibody, and making has had breakthrough and more and more demonstrated its significance and practice prospect studies on Monoclonal Antibody.The phage antibody gene pool technology rise of rising the beginning of the nineties at the end of the eighties and the development in whole genetic engineering antibody technical study field make the development research of source, people from world today or genetic engineering antibody obtain remarkable progress and step into substantive applied research and development phase by the fundamental research stage.At present in the biological products that drugs approved by FDA is gone on the market or awaited the reply, various forms of monoclonal antibodies and genetic engineering antibody account for certain proportion, in 67 kinds of biological products with the approval listing, treatment and prevention have 9 kinds with monoclonal antibody and genetic engineering antibody at present.The medium antibody that awaiting the reply has 54 kinds.Wherein in four kinds of humanized genetic engineering antibodies of approved listing and generation tremendous economic and social benefit, a kind of antiviral gene engineered antibody that is is arranged wherein, its commodity are called " Synagis
TM"; be humanization preventing respiratory combination of syndromes poison (RSV) genetic engineering antibody; and that the anti-RSV virus gene engineering antibody in people source that derives from phage antibody library screening has gone through to enter the II phase is clinical, another kind of antiviral antibody anti-HBs antibody is also among examining.
Up to now, most of virus diseases do not have the specific treatment medicine.Rabic exposure back treatment is a global problem always, for serious resurrectionist (III), vaccine and rabies virus immunoglobulin (Ig) (Rabies Immune Globulin are used in World Health Organization's suggestion simultaneously, RIG) carry out active and passive immunotherapy, the antiserum(antisera) that can select for use has the rabies poison immunoglobulin (Ig) (HRIG) in horse anti-rabies virus immunoglobulin (Ig) (ERIG) and human blood source at present.But because ERIG has stronger side reaction after injecting human body, so AAI's suggestion preferentially selects for use HRIG to treat in HRIG and ERIG, but because the HRIG source is few, the price height, and not only non-specific foreign protein is many in the blood product, specific antibody content is very low, and maximum problem is the cause of disease pollution problem that blood source goods potential fails to detect, and considers and should abandon from long-term interest.Therefore become a general orientation of domestic and international research with human source gene engineering product Blood substitute goods, and progressively led to success.Still do not have the precedent of any approval in the world with prevention of pure mouse resource monoclonal antibody and treatment virus disease, mouse source antibody human is a heterologous protein with maximum disadvantage, causes easily that in human body inherited immunity repels and make the antibody inefficacy and cause immunological disease.Therefore, specificity antivirus people source neutrality antibody is one of the most promising biological products of virus disease prevention and treatment.The research of people's source antivirus genetic engineering antibody is except that successful anti-rsv antibodies, present system by phage surface, gene engineering antibody library technology and molecular biology method unite utilization, made substantial progress people's source antivirus whole antibody of having succeeded in developing at present of the research in this field has the antibody of anti-following virus: respiratory syncytial virus, HIV (human immunodeficiency virus) (HIV), hepatitis B virus, hsv (HSV), the anti-hepatitis A virus that Hantaan virus etc. and this institute that does not deliver have as yet succeeded in developing, rabies virus antibodies etc.Succeeding in developing of human source anti-rabies virus genetic engineering antibody preparation will be domestic and international initiative, might obtain a kind new medicine certificate.
The objective of the invention is by genetic engineering means and phage display technique in conjunction with utilization, directly from the human immunoglobulin gene storehouse, filter out the gene engineering monoclonal antibody of neutrality rabies poison, obtain its antibody gene, the specific antibody medicine that provides feasibility in the future possible clinical antiviral prevention and treatment.
The human source anti-rabies virus neutrality genetic engineering antibody of the present invention's statement comprises:
1, human source anti-rabies virus neutrality genetic engineering antibody RVG10 comprises Fab antibody and IgG full-antibody gene, gene product and application thereof.Be primarily characterized in that this recombinant antibodies be by be present in hypervariable region (CDRs) specific gene sequence decision in light chain of antibody and the heavy chain gene variable region and in protokaryon and eukaryotic cell, obtain the functional neutralizing antibody of the specificity of effective expression in conjunction with rabies virus.
2, human source anti-rabies virus neutrality genetic engineering antibody RVG10, be primarily characterized in that: the antibody protein function is determined by specificity nucleotide sequence among the complementary region CDR1 of decision family, the CDR2 that are present in antibody gene light chain and variable region of heavy chain and the CDR3, its amino acid sequence corresponding has constituted the specific antigens calmodulin binding domain CaM of antibody, as Fig. 9, be human source anti-rabies virus neutrality genetic engineering antibody variable region nucleotide and aminoacid sequence.
3, human source anti-rabies virus neutrality genetic engineering antibody RVG10, its oligogene is characterised in that: specific light chain and heavy chain variable region gene derive from the rich long-pending screening expression of the specificity in human source anti-rabies virus antibody gene storehouse, and corresponding separately three the CDR region sequences of its light chain (VL) and heavy chain are the distinctive brand-new sequence of this antibody.The aminoacid sequence in three CDR districts of heavy chain (VH) comprises: CDR1 is GGSITSHHYWS; CDR2 is YHSGTTNYNPSLKSRV; CDR3 is VRVTTGAFNL.The aminoacid sequence in three CDR districts of light chain (VL) comprises: CDR1 is RASQSVSSNLA; CDR2 is ASTRAA; CDR3 is QQYNTWPPYT.
4, human source anti-rabies virus neutrality genetic engineering antibody RVG10Fab antibody, it is characterized in that:, form by heavy chain Fd and Kappa chain for a kind of human source anti-rabies virus glycoprotein neutrality gene engineering monoclonal antibody Fab antibody that in prokaryotic cell prokaryocyte, obtains to stablize the gene recombination of effective expression.
5, human source anti-rabies virus neutrality genetic engineering antibody RVG10IgG whole antibody, it is characterized in that: form by total length IgG1 heavy chain gene and total length lambda chain light chain, its weight chain leader and heavy chain Fc gene source be in carrier pAc-L-Fc, states in number of patent application is 00105698.0 patent application.Between light chain gene leader and VL gene, contain SacI enzyme point of contact; Between heavy chain gene leader and VH gene, contain XhoI enzyme point of contact; Between heavy chain Fd gene and Fc gene, contain SpeI enzyme point of contact.
6, human source anti-rabies virus neutrality genetic engineering antibody RVG10, its major function is characterised in that: specific recognition rabies virus glycoprotein albumen, have that rabies poison infects in and active function.
7, the purposes of human source anti-hepatitis A virus neutrality engineered Fab antibody, it is characterized in that: the neutrality antibody variable region that utilizes above-mentioned acquisition, Fab or full-antibody gene, can in prokaryotic cell prokaryocyte, yeast cell, eukaryotic cell and any recombinant virus system, express and produce this antibody gene or reconstruction based on this after contain any other gene of this antibody gene, during acquisition has and the antibody product of rabies virus infection, its gene expression product is expected to be used to clinically prevent and treat the rabies that caused by rabies virus.
8, the purposes of human source anti-rabies virus neutrality genetic engineering antibody gene, it is characterized in that: according to CDR district specificity Nucleotide or the aminoacid sequence in the variable region of the rabies poison neutralizing antibody RVG10 that delivers, can be at the nucleotide sequence in identical therewith 6 the CDR districts of antibody weight chain of external synthetic or the nucleotide sequence of coding same amino acid, thereby obtain identical antibody gene or be used for the transformation of genes involved, and obtain neutralizing antibody or the associated protein or the polypeptide product of rabies poison glycoprotein.
Traditional hybridoma cell technology of utilizing obtains quite difficulty of human monoclonal antibody, and the clone of setting up is unstable usually, and gene is easily lost.The human source anti-rabies virus neutrality genetic engineering antibody of the present invention's statement, it is the expression that on the basis that obtains antibody gene, obtains gene product, this antibody gene can be along with plasmid DNA duplicating and stable duplicating in bacterium, and can reconstruct antibody gene arbitrarily, thereby obtain a kind of clinical treatment antibody preparation of feasibility according to different needs.
Following preferential embodiment elaborates to the present invention, load does not mean that restriction content of the present invention in these embodiments, be explanation the present invention, the employing phage expression vector is pComb3 (Barbas C.III et al, Proc.Natl.Acas.Sci USA 1991,89:10164-10168).Used main bacterial strain is commercial prod XLI-Blu (U.S. Strategene company).Used phage is VCSM13.Rabies virus is the AG strain of isolated in China.
Embodiment 1-6 is the screening preparation method of human source anti-rabies virus neutrality engineered Fab antibody and whole antibody RVG10; Embodiment 7 is the gene expression characteristics of human source anti-rabies virus neutrality genetic engineering antibody RVG10; Example 8-12 is albumen and the functional character of human source anti-rabies virus neutrality genetic engineering antibody RVG10.
Example 1, the pcr amplification of humanized IgG Fab fragment gene: with lymphocyte separation medium isolated lymphocytes from the donor periphery anticoagulation of crossing through the immunity of rabies virus PM strain Vero cell vaccine, with Tril-Zon (U.S. Gibco, BRL) extract total cell RNA, with the Olig-dT primer RNA that extracts is passed through reversed transcriptive enzyme (U.S. Gibco, BRL) reverse transcription becomes cDNA, with a group-specific IgGFabGamma chain, Kampa chain and Lamda strand primer (table 1), people's endogenous light chain and heavy chain Fab gene are carried out pcr amplification.The PCR condition is: 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 2 minutes, 35 circulations (PE 480), above-mentioned PCR product reclaims through sepharose respectively, through DNA purification column Spin-X (U.S. Gibco, BRL) behind the purifying, obtain Kamba, Lamda and Fd chain PCR product about 650-700bp.
Example 2, the foundation of phage antibody gene pool: different primer synthetic Kamba and Lamda chain PCR product are mixed, different primer synthetic Fd chains are mixed, be cloned into phage vector pComb3 with SacI/XbaI and XhoI/SpeI respectively, to be cloned into light, the pComb3 carrier DNA of heavy chain gene connects product behind ethanol sedimentation, hang with the 10ul aquae destillata, the 200ul electricity that it is good that prepared beforehand is gone in the electricity transduction changes bacterium XLI-Blu, (electric commentaries on classics condition is: the Bio-Red electroporation, 0.2cm electric revolving cup, 2.5K volt), add the 10mlSOC nutrient solution after electricity changes, 37 ℃ 1 hour, adding 10ml has the SB nutrient solution (3) of penbritin and tsiklomitsin, 37 ℃ 1 hour, add the aforementioned SB of 80-100ml, 37 ℃ added helper phage M13GCM 1 X10 12 after 2 hours, add kantlex (70ug/ml) after 1 hour, 37 ℃ of shaking table overnight incubation.With 4%PEG8000 and 3%NaCl precipitation phage supernatant, through 9000rpm, 20 minutes, 4 ℃ centrifugal after, with 2ml 0.02M PBS PH 7.4 resuspended precipitations, set up phage antibody library, packing is stored in-20 ℃ of refrigerator-freezers.
Example 3 is used for the preparation of the rabies virus antigen of antibody library enrichment screening: 1) rabies virus particulate purifying: used virus is the viral supernatant (stream grinds teacher Jia Keli of institute and is so kind as to give) that BHK-21 cell rolling bottle is cultivated.With supernatant centrifugal 10 minutes, discard cell debris at 4000rpm.It is centrifugal earlier to do bed course with 15%, 20%, 25%, 30% sucrose solution, to determine suitable sucrose concentration.Select the rotary head of SW40 for use.Every pipe adds 10ml virus culture supernatant, then the 1ml sucrose solution is added solution bottom, 4 ℃, centrifugal 2 hours of 25000rpm.The sample that will surpass then from purifying carries out the electrophoresis evaluation, determines the sucrose solution of suitable concn.With suitable concentration, adopt the SW28 rotary head, 4 ℃, centrifugal 2 hours purifying antigens of 25000rpm.With purifying antigen carry out that SDS-PAGE analyzes and Western Blot evaluation.2) preparation of cells infected lysate:,, make rabies virus antigen with 2%NP40 cell pyrolysis liquid lysing cell with the cell that rabies virus aG strain is infected.
Example 4, the screening of the specific enrichment of phage antibody library: with the rabies virus antigen (0.5-1 μ g/ml) that the solution bag of 0.1M NaHCO3 (pH8.6) is purified, every hole 55 μ l, 4 ℃ are spent the night; The antigen that inferior daily 1 * PBST flush away does not adsorb, confining liquid is abandoned in 37 ℃ of sealings of the skimmed milk with 4% 1 hour; Every hole adds 50 μ l phage antibody libraries, hatched 2 hours for 37 ℃, discard unconjugated phage in the hole, with 1 * TBST (50mM Tris-HCl, 150mMNaCl, 0.5%Tween 20, pH7.5) washing lotion is washed each hole, during flushing, blows and beats repeatedly with the pipettor that has suction nozzle, wash the phage of not adsorbing with abundant flush away altogether 10-20 time; Use ddH at last
2O washes twice, the liquid in the exhaustion hole; Every hole adds the elutriant of 50 μ l Gly-HCl (pH2.2), incubated at room 10 minutes.Add an amount of 2M Tris, the phage that elutes with neutralization; The phage of wash-out is added immediately (OD in the XLI-Blu bacterium liquid of 2ml prepared fresh
600=1), incubated at room 15-20 minute; Change over to then in the 250ml triangular flask, add SB10ml (penbritin: 20 μ g/ml, tsiklomitsin: 10 μ g/ml), get 10 μ l immediately and be coated with the penbritin plate, with the titration phage; Triangular flask and 37 ℃ of shaking culture 1 hour, (penbritin: 100 μ g/ml), 37 ℃ after 1 hour to add 100mlSB, add helper phage VCSM13 1ml, 37 ℃ of shaking culture 2 hours add kantlex (final concentration 70 μ g/ml), 37 ℃ of overnight incubation.So repeated screening is 4-5 time.
Example 5, the preparation of human IgG Fab antibody solubility expression product: extract plasmid DNA according to a conventional method after will having the positive colony amplification that positive antibody weight chain gene inserts, with the gIII in SpeI and the NheI excision carrier, become the FdgIII fusion rotein and be independent expressed proteins, connect the back and transform the XL1-Blu bacterium, the single bacterium colony of picking from the ammonia benzyl plate of overnight growth, inoculation SB or TB inoculum are when bacterium grows to OD
600=0.2-0.3 adds 1mM IPTG, at 30 ℃ of abduction delivering 10-12 hours.It is resuspended that results bacterium, centrifugal back add the PBS (0.02 M pH7.4) of original fluid 1/10 volume, multigelation three times, and centrifugal 10 minutes of 4 ℃ of 10000rpm, supernatant is the Fab antibody of expression.Be ready for use on further detection and evaluation.Negative control is that carrier pComb3 transformed bacteria is by the bacterial lysate with the quadrat method preparation.
Example 6, the clone of human source anti-rabies virus neutrality genetically engineered full-antibody gene reorganization: will present the rabies poison neutralizing antibody Fab gene clone that system obtains by phage surface and go in the baculovirus IgG expression vector of this laboratory invention.Wherein, light chain gene is cloned among the pAc-L-Fc with SacI and EcoRV, places under the control of p10 promotor, obtains intermediate carrier pAc-HA-L, and heavy chain Fd gene is cloned among the pAc-HA-L with XhoI and SpeI, places under the control of polyhedron promotor; It is light that acquisition has anti-hepatitis A antibody, the expression vector pAc-HA-HL of heavy chain full-antibody gene.This vector plasmid DNA is used for transfection insect cell behind column chromatography purification, obtain recombinant baculovirus.Behind the recombinate shape virus infection insect cell, obtain the expression of full humanized IgG antibody.
The nucleic acid sequence analysis of the variable region gene of example 7 human IgG Fab antibody RVG10: carry out nucleic acid sequence analysis with Qiagen MiniprepKit (U.S. Qiagen) preparation plasmid DNA.Order-checking is automatic sequencing.At least 3 clones are used to determine same identical sequence.The sequence that obtains all uses DNA Strider (MS) sequence analysis software to carry out analyzing and processing, and compares the IgG sequence in the gene pool on the Internet network.Confirmer's source anti-hepatitis A virus neutrality engineered Fab antibody HAFab16 gene is made up of human IgG γ chain Fd and λ chain, its gene expression characteristics is made of the specificity nucleotide sequence and the amino acid in 6 CDR districts in VH and the VL structural domain, sequence data such as Fig. 9 are human source anti-rabies virus neutrality genetic engineering antibody variable region nucleotide and aminoacid sequence.
Example 8: isolated lymphocytes from rabies virus immunity donor blood, with the round pcr humanized IgG Fab antibody gene that increased, set up the phage antibody gene pool, present with phage surface the Fab positive colony of technology screening and acquisition after the enrichment screening, select three strain positive colonies at last, wherein two strains are Fab antibody RVG10 and RVB2, and RVG10 derives from the screening of purifying antigen, and RVB2 then derives from the screening that monoclonal antibody is caught rabies virus antigen.The somatic cells cracking supernatant of expressing is detected simultaneously the active antigen-binding activity that reaches the rabies virus purifying antigen of combination of its antagonism human Fab antibody.ELISA result shows that obtaining 3 strain monoclonal antibodies is not only discerned by anti-human IgG Fab antibody, equally also discerns in conjunction with above-mentioned purifying rabies virus particulate antigen.As Fig. 1, be that the human source anti-rabies virus engineered Fab antibody combines with anti-human Fab's antibody and purifying rabies virus antigen ELISA.A monoclonal antibody RVG10 and an existing strain mouse source anti-rabies virus glycoprotein neutralization monoclonal antibody are competed, and the result shows, the mouse monoclonal antibody can blocked sugar albumen on the antigen binding site of G10 monoclonal antibody, as shown in Figure 2.Show that this strain antibody is to rabies virus glycoprotein.
Example 9 is cloned into whole antibody expression vector pAc-K-Fc with RVG10 strain antibody Fab base, is built into pAC-H (κ)-G10 insect cell expression plasmid.With RVG10 baculovirus expression plasmid transfection SF9 cell, results transfection supernatant after 5 days, cell detects respectively with the anti-human Fab's antibody and the anti-human IgG antibody of FITC mark, the result shows the expression that Fab and IgG are arranged in the cell after the transfection, and do not have in the blank cell, G10 expression plasmid transfection success, whole antibody obtains effective expression in cell.As shown in Figure 3, be the expression of RVG10 full-antibody gene in insect cell SF9 cell.Traget antibody is anti-human IgG fluorescence antibody.
Example 10, antibody purification: the supernatant of results is removed remaining impurity with the membrane filtration of 0.45 μ m earlier, with 1ml purification column (Hitrap
1ml Protein G) carry out purifying, and wash-out removes the salt component in the antibody purification, measure the content of purifying protein then.The antibody purification of getting 3-5 μ g carries out the SDS-PAGE electrophoretic analysis.As Fig. 4 is the protein electrophoresis figure of purifying RVG10 whole antibody molecule.Quantitative antibody is carried out avidity measure, the result shows that the antibody of 0.4 μ g/ml can be in conjunction with aG strain purifying antigen, so the avidity of the whole antibody that obtains is 2.67 * 10
-9M.Combine with the competition of mouse-anti rabies virus glycoprotein monoclonal antibody simultaneously, this specific specificity combination can be by mouse inhibition that monoclonal antibody is competed.As Fig. 5, be RVG10 whole antibody and mouse-anti rabies virus glycoprotein monoclonal antibody competition inhibition elisa assay to the purifying rabies virus antigen.
Example 11, external neutralization experiment: utilize in the G10 monoclonal antibody and the aG strain virus, thereby make virus no longer cells infected or metainfective sx.Sessile antibody amount (100 μ g/ml) is carried out serial log10 dilution with virus, and external neutralization virus is after 30～60 minutes, infect the new biography Vero cell in generation (24 orifice plates, Costar), with cell at 33 ℃, 5%CO
2Cultivate in the incubator, the metainfective cell conditioned medium of week back results changes into and keeps liquid, and continues to cultivate and observe.Virus infected cell is after three weeks, and the form of observation of cell is gathered in the crops the viral supernatant that infects simultaneously, and cell detects the rabies virus infection situation with fluorescent method.As shown in Figure 6, be the Vero cytopathy of rabies virus infection.With fail in compare (figure top) with control antibodies 100 IgG of virus infection, circle occurred by the cell of RGV10 neutralizing antibody neutral virus infection and contract, the refractive index variation, particle increases, cell detachment, and (figure below) appears obviously drawing in the net in iuntercellular.
Example 12, for further identify rabies virus in and situation, we are with the aseptic small white mouse of supernatant injection 11～13g (calibrating institute provides) of twice results, 30 μ l/ only detect cells infected with fluorescent method at last, detect the virus that goes up in the cleer and peaceful cell simultaneously.Shown in chart 7, with the cell conditioned medium injection mouse brain that infects after 7 days, by among the G10 and after virus (10
-1) mouse 100% survival of supernatant injection, and irrelevant none survival of monoclonal antibody neutral contrast.And in the supernatant after 21 days (7 days time cell change into keep liquid), by G10 neutral (10
-1) the mouse survival rate of viral supernatant injection be 1/3, this result shows that virus is not neutralized fully, but a small amount of virus existence is arranged, after after a while, virus has amplification again.
For in further determining and the needed antibody amount of aG strain virus, we pass through fixed virus again, the method of dilution antibody has repeated above-mentioned experiment once, and virus infected cell changes into and keeps liquid two days later, 33 ℃, be in further determining and the needed antibody amount of aG strain virus, we are again by fixed virus, and the method for dilution antibody has repeated above-mentioned experiment once, and virus infected cell two days later, change into and keep liquid, 33 ℃, 5%CO
2Incubator was cultivated after 7 days, the results culture supernatant, and the inoculation of mouse brain detects the virus in the supernatant, simultaneously with the virus in the fluorescent method detection cell, result such as chart 8, RVG10 antibody under 6ug/ml concentration still can in and rabies virus infection, confirm that RVG10 is the higher neutralizing antibody of a strain avidity.
Claims (9)
1, human source anti-rabies virus neutrality genetic engineering antibody RVG10 comprises Fab antibody and IgG full-antibody gene, gene product and application thereof.Be primarily characterized in that this recombinant antibodies be by be present in hypervariable region (CDRs) specific gene sequence decision in light chain of antibody and the heavy chain gene variable region and in protokaryon and eukaryotic cell, obtain the functional neutralizing antibody of the specificity of effective expression in conjunction with rabies virus.
2, human source anti-rabies virus neutrality genetic engineering antibody RVG10, be primarily characterized in that: the antibody protein function is by complementary region (the Complementarity-Determining Regions of decision family that is present in antibody gene light chain and variable region of heavy chain, what CDRs) the specificity nucleotide sequence determined among CDR1, CDR2 and the CDR3, its amino acid sequence corresponding has constituted the specific antigens calmodulin binding domain CaM of antibody, as Figure of abstract.
3, human source anti-rabies virus neutrality genetic engineering antibody RVG10, its oligogene is characterised in that: specific light chain and heavy chain variable region gene derive from the rich long-pending screening expression of the specificity in human source anti-rabies virus antibody gene storehouse, and corresponding separately three the CDR region sequences of its light chain (VL) and heavy chain are the distinctive brand-new sequence of this antibody.The aminoacid sequence in three CDR districts of heavy chain (VH) comprises: CDR1 is GGSITSHHYWS; CDR2 is YHSGTTNYNPSLKSRV; CDR3 is VRVTTGAFNL.The aminoacid sequence in three CDR districts of light chain (VL) comprises: CDR1 is RASQSVSSNLA; CDR2 is ASTRAA; CDR3 is QQYNTWPPYT.
4, human source anti-rabies virus neutrality genetic engineering antibody RVG10Fab antibody, it is characterized in that:, form by heavy chain Fd and Kappa chain for a kind of human source anti-rabies virus glycoprotein neutrality gene engineering monoclonal antibody Fab antibody that in prokaryotic cell prokaryocyte, obtains to stablize the gene recombination of effective expression.
5, human source anti-rabies virus neutrality genetic engineering antibody RVG10IgG whole antibody, it is characterized in that: form by total length IgG1 heavy chain gene and total length lambda chain light chain, its weight chain leader and heavy chain Fc gene source be in carrier pAc-L-Fc, states in number of patent application is 00105698.0 patent application.Between light chain gene leader and VL gene, contain SacI enzyme point of contact; Between heavy chain gene leader and VH gene, contain XhoI enzyme point of contact; Between heavy chain Fd gene and Fc gene, contain SpeI enzyme point of contact.
6, human source anti-rabies virus neutrality genetic engineering antibody RVG10, its major function is characterised in that: specific recognition rabies virus glycoprotein albumen, have that rabies poison infects in and active function.
7, according to above-mentioned 6 claims, the purposes of human source anti-hepatitis A virus neutrality engineered Fab antibody, it is characterized in that: the neutrality antibody variable region that utilizes above-mentioned acquisition, Fab or full-antibody gene, can be at prokaryotic cell prokaryocyte, yeast cell, express in eukaryotic cell and any recombinant virus system and produce and contain any other gene of this antibody gene after this antibody gene or the reconstruction based on this, during acquisition has and the antibody product of rabies virus infection, its gene expression product is expected to be used to clinically prevent and treat the rabies that caused by rabies virus.
8, according to claim 2 and 3, the purposes of human source anti-rabies virus neutrality genetic engineering antibody gene, it is characterized in that: according to CDR district specificity Nucleotide or the aminoacid sequence in the variable region of the rabies poison neutralizing antibody RVG10 that delivers, can be at the nucleotide sequence in identical therewith 6 the CDR districts of antibody weight chain of external synthetic or the nucleotide sequence of coding same amino acid, thereby obtain identical antibody gene or be used for the transformation of genes involved, and obtain neutralizing antibody or the associated protein or the polypeptide product of rabies poison glycoprotein.
9, according to claim 2,3 and 5, the purposes of human source anti-rabies virus neutrality genetic engineering antibody gene, it is characterized in that: according to claim 2, CDR region amino acid sequence described in 3 is the proprietary antibody sequence of this antibody, and any new engineered antibody variable region gene or associated products gene should not contain identical or 1-2 sequence that amino acid difference is easy only with above-mentioned any two kinds of CDR region sequences.Any new engineered antibody full-antibody gene should not contain the feature described in the claim 5.
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| CN101863978B (en) * | 2010-05-14 | 2013-04-10 | 中国人民解放军南京军区军事医学研究所 | Human source anti-rabies virus Fab antibody, cross linking method thereof with nanometer particles and application thereof |
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| CN103910796B (en) * | 2013-01-08 | 2016-04-20 | 广州泰诺迪生物科技有限公司 | A kind of neutralizing antibody of total man's source anti-rabies virus |
| WO2015062171A1 (en) * | 2013-10-28 | 2015-05-07 | 纪军 | Monoclonal antibody and antigen and gene thereof, and uses thereof |
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| CN105481979B (en) * | 2016-01-07 | 2019-03-12 | 中国疾病预防控制中心病毒病预防控制所 | Human source anti-rabies virus glycoprotein neutrality antibody RV3A5 and its application |
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