CN101812132A - Humanized neutralizing antibody (RVFab3) against rabies virus glycoprotein - Google Patents
Humanized neutralizing antibody (RVFab3) against rabies virus glycoprotein Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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Abstract
The invention discloses a humanized neutralizing antibody (RVFab3) against rabies virus glycoprotein, which is obtained through screening by utilizing phage display technology. The antibody specifically identifies the granule antigen of the rabies virus, is against the rabies virus glycoprotein G, has obvious immunofluorescence reaction and enzyme linked immunosorbent assay with the rabies virus and has the neutralizing activity function against rabies virus infection. The antibody can be prepared into the specific antibody drugs for preventing and treating rabies, thereby being clinically used for preventing and treating rabies caused by the rabies virus.
Description
Technical field
The present invention relates to the genetic engineering antibody technology, particularly relate to a kind of human source anti-rabies virus glycoprotein neutrality antibody; The invention still further relates to this antibody in preparation prevention or treat application in the rabic medicine.
Background technology
Rabies are the worldwide Amphixenosises that caused by rabies virus, in case 100% death of falling ill.There is the rabies report in 87 countries in the world at present, have every year people more than 50,000 to die from rabies (Knobel DL, et al.2005) approximately.Prevention is antirabic major measure after the rabies exposure.For the people of serious exposure, (World HealthOrganization, WHO) suggestion adopts the rabies vaccine injection in conjunction with rabies poison immunoglobulin (Ig) (rabies immune globulin, method RIG) in the World Health Organization.The two class RIG that use at present as the human anti-rabies immunoglobulin (Ig) (human rabies immune globulin, HRIG) with the horse anti-rabies virus immunoglobulin (Ig) (Equine rabies immune globulin, ERIG).Because the ERIG side reaction is more serious, and has inhibition to the antibody response of some vaccine, and HRIG costs an arm and a leg, and supply is limited and have the potential cause of disease to threaten.Therefore preparing the passive immunization preparation efficient, inexpensive, that side reaction is little is our target.
The people source or the animal serum immunoglobulin (Ig) that contain specific antibody are with a long history in order to prevention and treatment transmissible disease.In the extracorporeal antivirus effect of monoclonal antibody and active and endogenous protective human body resist virus attack and obtained many experiment showed, and can be in vivo 100% watch for animals and avoid virus attack as neutralizing monoclonal antibodies such as mouse-anti hepatitis A virus (HAV), Hantaan virus, Measles virus, RSV virus, CMV viruses.The approach that obtains polyvalent antibody with antigen-immunized animal is the classical way that obtains antibody always, but lacks specificity and homogeneity.Then the B lymphocyte hybridoma technology of Jian Liing make numerous scientists by cell engineering can prepare in-vitro directedly various monoclonal antibodies (monoclonal antibody, McAb), its high specificity, the character homogeneous is easy to mass production.Yet McAb mostly is mouse source property, and the heterology reaction of mouse source McAb has greatly limited McAb as the application of treatment preparation at human body.Immunoglobulin (Ig) (Vaccinia immune globulin, VIG) as antibody component mainly from donor (convalescent) immune serum, all need spend great amount of manpower and financial resources from obtaining positive serum to detecting by security, this just makes its a large amount of preparations be restricted, so owing to derive from serum the pathophorous infection of haematogenous takes place easily simultaneously.Therefore end user source gene engineering product Blood substitute goods then can overcome these defectives, and deepening continuously of human genetically engineered antibody research brought new hope and bright prospects for the biological products development in this field.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 solution passive immunization formulation problems that is produced as of human source anti-rabies virus Study of Monoclonal Antibodies and phage antibody library technique provides new thinking.The monoclonal antibody cocktail that mad dog monoclonal antibody CR57 and CR4098 the make 26 kinds of typical street strains that neutralized.Protectiveness experiment to animal shows, utilizes the monoclonal antibody cocktail to treat and has feasibility and superiority (Goudsmit J, et al.2006).
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.People's source antivirus genetic engineering antibody, especially the research of people source whole antibody success, brought new hope for the specificity prevention and the treatment of various viral infectious, formed the new antiviral drug of a class gradually, promptly so-called antibody medicine (Antibody Drug) in the biological medicine of anti-virus infection field., also be badly in need of now substituting haematogenous VIG to the transformation of recombinant vaccine as haematogenous vaccine originally, as by chimeric antibody technology (Boulianne, G.L.et al., 1984 with genetic engineering antibody; Morrison, S.L.et al., 1984), humanized antibody technology (Jones, P.T.et al., 1986), the transgenic mice technology of carrier's monoclonal antibody (Green, L.L.et al., 1994), allosome hybridoma technology (James, K.et al., 1987), phage display technique (Barbas, C.F.et al., 1991) etc. produces humanized antibody, now become the great direction of domestic and international research, and progressively led to success.
Summary of the invention
First purpose of the present invention is to provide a kind of human source anti-rabies virus glycoprotein neutrality antibody and active fragments thereof.
Second purpose of the present invention is to provide the gene of the above-mentioned antibody of coding or its active fragments.
The 3rd purpose of the present invention is to provide above-mentioned antibody and the application of active fragments in preparation prevention or treatment rabies medicine or diagnostic reagent thereof.
The present invention uses phage surface to present technology, gather a plurality of vaccination person's peripheral blood lymphocytes with high titre rabies virus antibodies, made up human source anti-rabies virus genetic engineering antibody library by genetic engineering means, and screening obtains special rabies virus gene engineered antibody Fab section.The Fab section antibody called after RVFab3 that obtains.
This strain recombinant antibodies is to be determined by hypervariable region (CDRs) the specific gene sequence that is present in light chain of antibody and the heavy chain gene variable region, and obtains the functional antibodies of the specificity of effective expression in conjunction with rabies virus in prokaryotic cell prokaryocyte.Its specific recognition rabies virus particulate antigen, and all at rabies virus glycoprotein G has obvious immunofluorescence reaction (IFA) and enzyme linked immunological (ELISA) reaction with rabies virus, have that the rabies poison infects in and active function.
Specific light chain of RVFab3 and heavy chain variable region gene derive from the rich long-pending screening of the specificity in human source anti-rabies virus antibody gene storehouse, and the foundation of this antibody library derives from Chinese hydrophobia immune peripheral blood lymphocyte gene.The framework region sequence has been formed each antibody variable region sequence signature between corresponding three CDR region sequences combination of its light chain and variable region of heavy chain and the CDR district thereof, and RVFab3 is under the jurisdiction of the VH4 of heavy chain of antibody family.The antibody protein function is 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 and complementary decision the thereof, 6 corresponding C DR region amino acid sequences have constituted the specific antigens calmodulin binding domain CaM of antibody, and the antigen of antibody is in conjunction with feature and rabies poison functional character in the decision invention.The light chain of antibody of decision neutralizing antibody function and variable region of heavy chain amino acid detailed sequence and comparative result thereof are as shown in table 1:
Table 1
The aminoacid sequence of RVFab3 variable region of light chain is shown in SEQ ID No.1, and the aminoacid sequence of its variable region of heavy chain is shown in SEQ ID No.2.
The gene order of coding RVFab3 variable region of light chain is shown in SEQ ID No.3, and the gene order of variable region of heavy chain is shown in SEQ ID No.4.
Be to be understood that, under the prerequisite that does not influence the Fab antibody activity, those skilled in the art can carry out various replacements, interpolation and/or lack the aminoacid sequence that one or several amino acid obtains to have same function the aminoacid sequence shown in the SEQ ID No.1-2, for example the amino acid that will have similarity in non-hypervariable region is replaced, and replaces with Ala as the 8th Val with the heavy chain VH sequence of RVFab3.
In addition, consider the degeneracy of codon, for example can under the condition that does not change aminoacid sequence, the gene order of the above-mentioned Fab section antibody of encoding be made amendment, obtain the gene of coding same antibody in its coding region.Those skilled in the art can be according to expressing antibodies host's codon-bias, and the synthetic modifying gene is to improve the expression efficiency of antibody.
Further, the present invention recombinates the variable region of light chain and the variable region of heavy chain of above-mentioned Fab antibody, obtains the littler single-chain antibody (ScFv) of molecular weight, and this antibody equally can specific recognition rabies virus surface antigen, has the effect of intracellular immunity.The single-chain antibody penetration power is strong, is easy to enter local organization and plays a role.
Can be in expression vector with the gene of above-mentioned coding Fab antibody, ScFv gene clone, and then transform the host, obtain Fab antibody and single-chain antibody by abduction delivering.
In addition, the light chain encoding gene and the heavy Fd fragment gene of above-mentioned Fab antibody can be cloned in the complete anti-expression vector, and import in the host cell, obtain to express the full anti-immunoglobulin of rabies poison.
In an embodiment of the present invention, light chain and the heavy Fd fragment gene of above-mentioned Fab antibody RVFab3 are cloned into whole antibody expression vector pAC-L-Fc and transfection insect Sf9 cell respectively, utilize baculovirus/insect cell system to realize the secretion type expression of whole antibody, obtain whole antibody RVIgG3.
Utilize ELISA, IFA, SDS-PAGE that the whole antibody that obtains is carried out Function Identification, the result shows that humanized IgG whole antibody RVIgG3 all has the specificity combination at aG strain and CTN strain rabies virus particle, and expressing ERA, CVS, CTN and four kinds of rabies virus strain's glycoprotein of aG strain with baculovirus/insect cell system all has specificity to combine.Utilize rabies virus tachysynthesis fluorescence kitchen range to suppress experiment (RFFIT) whole antibody is carried out Function Identification, the result shows: it is active that RVIgG3 has neutralization preferably, can reach 813.3IU/mg, attack the ability of strain CVS-11 strain in having possessed fully with international standard.
The present invention uses phage antibody library technique, has successfully obtained the people source neutrality antibody of specificity at rabies virus glycoprotein; Utilize the full-antibody gene under people source neutrality rabies poison glycoprotein gene engineered antibody variable region gene, Fab antibody gene and above-mentioned each antibody gene feature of above-mentioned acquisition, can in prokaryotic cell prokaryocyte, yeast cell, eukaryotic cell and any recombination system, express and produce any other gene that contains this antibody gene after this antibody or the reconstruction based on this, during acquisition has and the antibody product of rabies virus infection, make and be used for prevention clinically and treat rabic specific antibody medicine.
Description of drawings
Fig. 1 is rabies poison people's source Fab antibody and ERA, CVS, CTN and aG strain rabies virus glycoprotein immunofluorescence analysis;
Fig. 2 is the SDS-PAGE electrophorogram of IgG behind the purifying;
Fig. 3 is that rabies poison humanized IgG antibody suppresses experiment at CVS-11 rabies virus tachysynthesis fluorescence kitchen range.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Material and method
1. virus, cell, carrier: rabies virus aG strain, the CTN strain, CVS strain and ERA strain are provided by China Sickness Prevention Control Center Virus Disease Prevention Control Institute.People's immunity is that the importer uses vaccine strain with strain PM strain, and the cell that is used for the external neutralization experiment of rabies virus is BHK-21 (ATCC).Bacterial strain is XLI-Blue (U.S. Stratagene), and carrier is pComb3 (40kb), is provided by U.S. Scripps institute.Rhabdovirus expression vector is pAC-L-Fc (German PROGEN PR3003) (Liang, M.F., Stefan, D., Li, D.X., Queitsch, I., Li, W., and Bautz, E.F.Baculovirusexpression cassettevectors for rapid production of complete human IgG from phagedisplayselected antibody fragments.Journal of ImmunologicalMethods.247:119-130.).Insect cell Sf9 is from the U.S. cell cultures center (ATCC).
2. antigen prepd
2.1 eukaryotic expression rabies virus glycoprotein (ERA, CVS, CTN, aG strain)
The rabies virus aG strain that utilizes the CDC virus disease to be provided respectively, the CTN strain, the cDNA of CVS strain and ERA strain is that template is carried out pcr amplification glycoprotein (G) gene, and 2 ends are all introduced the BamHI restriction enzyme site during design primer, and add the 6-His label at 3 ' section.The amplified production of purifying is cut with the BamHI enzyme, fragment after enzyme cut reclaims, directly be cloned into same in the rhabdovirus expression vector pAcUW51 that the BamHI enzyme is cut, make the HA gene under the control of polyhedron promotor, after PCR identified the goal gene direction of insertion, the acquisition recombinant plasmid was: pAc-HA.Carry out protein expression by the recombinant plasmid transfection insect cell is prepared recombinant baculovirus, express the BaculoGold cotransfection test kit that adopts U.S. Pharmogen company.It is as follows that working method outlines: after the BaculoGold linear DNA mixing with the recombinant plasmid dna of 5 μ g and 0.5 μ g, utilizing transfection reagent transfection stand density in the test kit is 50% Sf9 cell, cultivate after 4 days for 27 ℃, the collection supernatant carries out titration and amplification as the seed culture of viruses of recombinant virus.Baculovirusexpression vector system handbook is seen in concrete operations.Expression rabies virus glycoprotein (ERA, CVS, CTN, aG strain) the recombinate shape virus infection Sf9 cell that obtains, the results cells infected is made recombinant rabies poison glycoprotein (ERA, CVS, CTN, aG strain) antigen sheet after 4～5 days.
2.2 rabies virus purifying
Gather in the crops rabies virus aG strain and CTN strain respectively and infect culture supernatant behind the BHK-21 cell, after formalin-inactivated and safety inspection, with 20%～50% continuous sucrose density gradient 35000g, 4 ℃ of centrifugal 3h purified virus particles (Beckman SW28).
3. the structure of phage antibody library: with lymphocyte separation medium (U.S. Sigma) isolated lymphocytes from the anticoagulated blood of vaccination person with high titre rabies virus antibodies, extract total cell RNA with RNeasy Mini Kit (German QIAGEN), adopt the first chain synthetic agent box (the SuperScriptTMIII First-Strand Synthesis System for RT-PCR.CatNo.18080-051) reverse transcription of Invitrogen company to become cDNA the RNA that extracts with the Oligo-dT primer, with one group of amplification human antibody IgG1 heavy chain Fd and light chain Kappa and Lambda primer, people's endogenous light chain and heavy chain Fab gene are carried out pcr amplification.The PCR condition is: 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 2min, 35 circulations.Banking process carries out (Barbas by document substantially, C.FIII., Kang, A.S., and lamer, R.A.Assembly of combinatorial antibody libraries on phage surface:the geneIII site.Proc.Natl.Acad.Sci.USA.1991; 88 (18): 7978-7982).
4. the abduction delivering of the enrichment of phage antibody library screening and Fab section antibody: screening antigen is the inactivated virus particle aG and the CTN strain of ultracentrifugation purifying.Use 0.1m/LNaHCO during use
3(pH8.6) solution dilution, bag is by the immunity pipe, with 4% skimmed milk-PBS, behind 37 ℃ of sealing 2h, add above-mentioned phage antibody library, every pipe 1ml, hatch 2h for 37 ℃, wash repeatedly 20 times with 5%Tween-20-TBS, use glycine-hydrochloric acid elutriant wash-out of every pipe 1ml pH2.2 at last, the Tris liquid neutralization of pH9.6.Phage behind the wash-out continues to infect the fresh OD of 2ml
600XL1-Blu bacterium about=1.0 carries out the next round screening after helper phage VCSM13 (U.S. Stratagene) infects.So repeated screening is 4～5 times.The abduction delivering of concrete enrichment screening method and Fab section carries out (Barbas by document substantially, C.FIII., Kang, A.S., and larner, R.A.Assembly of combinatorial antibody libraries on phagesurface:the geneIII site.Proc.Natl.Acad.Sci.USA.1991; 88 (18): 7978-7982).
5. the ELISA of human source anti-rabies virus Fab antibody detects
5.1Fab the detection 0.1m/L NaHCO that expresses
3(pH9.6) in enzyme plate, 4 ℃ are spent the night solution bag by anti-human Fab's antibody (U.S. Sigma, dilution in 1: 2000 is used); The sealing of 4% skimmed milk, 37 ℃ of 1h add the Fab antibody of expressing, 37 ℃ of 1h; Add enzyme and mark anti-human Fab anti-(U.S. Sigma, dilution in 1: 2000 is used), 37 ℃ of 1h; The colour developing of colour developing liquid, 2M H
2SO
4Termination reaction, microplate reader detects the absorbance A value.
5.2 indirect enzyme-linked immunosorbent method detection Fab combines the inactivated rabies virus particle of activity usefulness purifying as envelope antigen with rabies virus, step subsequently is the same.
6. indirect immunofluorescence (IFA) detects: utilize the recombinate shape virus infection Sf9 cell of the expression rabies virus glycoprotein that has made up, the results cells infected is made recombinant rabies poison glycoprotein antigen sheet behind 4～5d.Add the Fab that expresses, 37 ℃ of incubation 30min, flushing, the anti-Fab antibody (U.S. Sigma) of adding FITC mark, 37 ℃ of incubation 30min, flushing is dried, and microscopically is observed.
7. the nucleic acid sequence analysis of people source Fab antibody variable gene: carry out nucleic acid sequence analysis with Qiagen MiniprepKit (German QIAGEN) preparation plasmid DNA.The sequencing primer of weight chain is respectively 5 '-AAACTAGCTAGTCGCCAAGGA-3 ' (shown in SEQ IDNo.5) and 5 '-CCGCGGTGGCGGCCGCAAAT-3 ' (shown in SEQ ID No.6).The IgG gene order relatively in sequencing result and the Internet V-Base gene pool.
8. the structure of whole antibody recombinant expression plasmid: the light chain of the Fab antibody that obtains is cut with the XbaI/SacI enzyme earlier, mend flat with end-filling enzyme (K fragment), be cloned into pAC-L-Fc carrier (German PROGEN PR3003) (Liang, M.F., Stefan, D., Li, D.X., Queitsch, I., Li, W., and Bautz, E.F.Baculovirusexpression cassette vectors for rapid productionof complete human IgG from phage displayselected antibody fragments.Journal ofImmunological Methods.247:119-130.), utilizes the XhoI/SpeI site to clone into heavy chain Fd section again, be built into the whole antibody expression vector.
9. transfection and recombinant virus infection and propagation: the BaculoGold cotransfection test kit that adopts U.S. Pharmogen company.It is as follows that working method outlines: with the recombinant plasmid dna of 5 μ g with after the BaculoGold linear DNA of 0.5 μ g mixes, utilizing transfection reagent transfection stand density in the test kit is 50% Sf9 cell, behind 27 ℃ of cultivation 4d, the collection supernatant carries out titration and amplification as the seed culture of viruses of recombinant virus.Concrete operations see Baculovirusexpression vector system handbook (BD Biosciences Pharmingen, USA).
10. whole antibody IgG secreting, expressing and purifying: the Sf9 cell of recombinant virus infection stand density about 70%, 27 ℃ of absorption 1h. use SF-900II SFM serum-free medium instead, collect supernatant after cultivating 3～5d for 27 ℃.Adopt the Protein-A affinity column direct purification of Amersham company to express supernatant (Harlow E, Lane D.Antibodies:A Laboratory Manual[M] .New York:Cold Spring Harbor Laboratory Press, 1988.).Utilize ELISA and IFA that the IgG antibody function characteristic of obtaining purifying is identified.Material method 5,6 parts are seen in concrete operations.
11. human source anti-rabies virus antibody tachysynthesis fluorescence kitchen range suppresses experiment (RFFIT): get each extent of dilution 50 μ l of antibody and antibody standard substance in 96 well culture plates, add the viral CVS-11 of neutralization, 50 μ l/ holes, establish the blank well contrast simultaneously, and the virus control hole is used in neutralization, in rearmounted 37 ℃ of the mixing and 1 hour, every hole adds 1 * 10
6/ ml BHK-21 cell suspension 50 μ l put 37 ℃ of 5%CO
2Cultivated 24 hours in the incubator.Wait to cultivate and finish to blot nutrient solution, after adding 100 μ l/PBS cleaned and blot in every hole, the adding of every hole was chilled to 4 ℃ 80% acetone, 50 μ l in advance, fix 10 minutes for-30 ℃, abandon acetone, treat to add behind the volatile dry fluorescent mark anti-rabies virus nucleoprotein antibody of working concentration, 50 μ l/ holes, hatched 30 minutes for 37 ℃, get rid of liquid, wash plate 2～3 times, dry liquid with PBS, every hole adds 80% glycerine, 50 μ l, fluorescence microscope.Can make the fluorescence kitchen range suppress the highly diluted multiple of 〉=50% antibody in the experimental group, be the NAT of tested antibody.According to Reed ﹠amp; The Muench formula, calculate each antibody sample and the mark product ED
50Thereby, draw tiring of each antibody to be checked.
12. the antibody after the sudden change of non-hypervariable region is to the research of rabies virus resistance
Based on RVIgG3 weight chain variable region amino acid sequence, the 8th Val of aminoacid sequence shown in the SEQ ID No.2 is replaced with Ala, the 10th Gly of RVIgG3 variable region of light chain (shown in the SEQ IDNo.1) is replaced with Gln.The heavy chain nucleic acid sequence encoding (codon gtg being replaced with gcc) and the light chain nucleic acid sequence encoding (codon ggg being replaced with cag) that synthesize RVIgG3 respectively in the corresponding position in the corresponding position.Method according to above-mentioned 8～11 is cloned into light chain gene and heavy chain Fd section among the pAC-L-Fc, and transfection insect Sf9 cell, utilizes baculovirus/insect cell system to realize the secretion type expression of whole antibody, and this mutant is carried out immunology detection.
The result
1. the screening of human source anti-rabies virus antibody library
With the rabies virus particle aG strain of purifying phage antibody library is carried out the enrichment screening, 2 take turns after the screening 6000 clones of picking at random.With anti-human Fab's antibody (sigma company, dilution in 1: 2000 is used), antigen coated 96 orifice plates of rabies virus particle aG strain, add the testing sample supernatant, mark anti-human Fab two anti-(Sigma company, dilution in 1: 2000 is used) with enzyme and detect.The result shows that common acquisition 2984 strain people source Fab express positive colony, and is as shown in table 2.2984 people source Fab express in the positive colony, 181 clones are arranged to the combination of rabies virus particle aG strain specificity, and wherein 36 strain Fab clone is confirmed as at rabies virus glycoprotein.
Table 2aG strain is screened the phage antibody library enrichment
| The Fab phage library | Storage capacity | The selected clone number | The Fab positive (ELISA) | AG virus-positive (ELISA) | The glycoprotein positive (IFA) |
| Fab phage library L word bank Fab phage library K word bank | ??2.4x10 8??2.1x10 8 | ??3000??3000 | ??1750??1234 | ??135??46 | ??30??6 |
| Sum | ??4.9x10 8 | ??6000 | ??2984 | ??181 | ??36 |
With the rabies virus particle CTN strain of purifying phage antibody library is carried out the enrichment screening, through 2 take turns screening after 2400 clones of picking at random.With anti-human Fab's antibody (sigma company, dilution in 1: 2000 is used), antigen coated 96 orifice plates of rabies virus particle aG strain, add the testing sample supernatant, mark anti-human Fab two anti-(Sigma company, dilution in 1: 2000 is used) with enzyme and detect.The result shows that common acquisition 1833 strain people source Fab express positive colony, and is as shown in table 3.1833 people source Fab express in the positive colony, 79 clones are arranged to the combination of rabies virus particle CTN strain specificity, and wherein 34 strain Fab clone is confirmed as at rabies virus glycoprotein.
Table 3 CTN strain is screened the phage antibody library enrichment
| The Fab phage library | Storage capacity | The selected clone number | The Fab positive (ELISA) | CTN virus-positive (ELISA) | The glycoprotein positive (IFA) |
| Fab phage library L word bank Fab phage library K word bank | ??2.4x10 8??2.1x10 8 | ??1500??1500 | ??838??995 | ??79??0 | ??34??0 |
| Sum | ??4.9x10 8 | ??3000 | ??1833 | ??79 | ??34 |
2. the sequential analysis of human source anti-rabies virus Fab antibody
Carry out analyzing and processing with the DNASTAR sequence analysis software, relatively the IgG sequence in the InternetV-Base gene pool in the people source Fab monoclonal antibody of above-mentioned 70 strain specificitys in conjunction with rabies virus glycoprotein, wherein has 11 strain sequence differences.Therefore this research is successfully screened and is cloned 11 strains and has the different antibody weight chain variable region sequences and the antibody of combination thereof, its variable region of heavy chain mainly is sorted in IgG VH4 and VH3 family, its variable region of light chain mainly is sorted in IgG VL1, VL2, VL3 and VK1 family, with the people source Fab monoclonal antibody called after RVFab1-11 of 11 strain specificitys in conjunction with rabies virus glycoprotein.It is as follows wherein to choose RVFab1, RVab3, RVFab5, RVFab8 and RVFab9 comparative sequences:
The aminoacid sequence of table 4 human source anti-rabies virus glycoprotein Fab antibody variable gene relatively
aVR, variable region (variable region); VH, heavy chain in VR (variable region of heavy chain); VL, light chain in VR (variable region of light chain).
bCDR, complementarity determining region (complementary determining region).
3. human source anti-rabies virus Fab antibody is to the specificity combination of rabies virus glycoprotein
The reorganization Fab antibody that obtains in order to confirm is to the binding specificity of different rabies virus strains glycoprotein, and we further identify the functionally active of prokaryotic expression Fab antibody by indirect immunofluorescence assay (IFA).As shown in Figure 1, RVFab1 and ERA, CVS, CTN and four kinds of rabies virus strains of aG strain glycoprotein immunofluorescence are all positive, negative with normal sf9 cell control reaction, all the other 10 strain people source Fab antibody are identical with RVFab1 with different mad dog strain glycoprotein immunofluorescence results.The 11 strain Fab positive antibodies that immunofluorescence result proof obtains from the antibody library screening are all at rabies virus glycoprotein, and react general wider.
4. whole antibody IgG expresses and purifying
5 strains have been finished the light chain and the heavy chain Fd fragment gene of the Fab antibody (RVFab1, RVFab3, RVFab5, RVFab8, RVFab9) of Function Identification, be cloned into transfection insect Sf9 cell behind the whole antibody expression vector pAC-L-Fc respectively, utilize baculovirus/insect cell system to realize the secretion type expression of whole antibody, difference called after RVIgG1, RVIgG3, RVIgG5, RVIgG8 and RVIgG9.Adopt the Protein-A affinity column direct purification of Amersham company to express supernatant, expression and purifying situation by SDS-PAGE check whole antibody IgG, the result confirms to obtain than pure protein, can clearly observe light, the heavy chain of antibody after unwinding, and lays respectively at about 28kD, 55kD place.As shown in Figure 2.
5. human source anti-rabies virus antibody tachysynthesis fluorescence kitchen range suppresses experiment (RFFIT)
In order further to study 5 strain whole antibody IgG (RVIgG1, RVIgG3, RVIgG5, RVIgG8, RVIgG9) neutralization activity, we have adopted tachysynthesis fluorescence kitchen range to suppress experiment and have detected antibody in neutralization reaction external and international standard attack strain CVS-11 strain, the result shows 5 strain human source anti-rabies virus whole antibody RVIgG1, RVIgG3, RVIgG5, the neutralization of RVIgG and RVIgG9 is tired and is respectively 866.6IU/mg, 813.3IU/mg, 689.8IU/mg, 876.6IU/mg, 428.5IU/mg, being equal to or higher than 0.5IU/ml according to WHO rabies Committee of Experts evaluation neutralizing antibody level, to represent that promptly this antibody has a neutralization active, thus the 5 strain people source monoclonal antibodies that obtain of this institute all to have a neutralization at rabies virus active.Wherein the neutralization activity of RVIgG9 a little less than, it is active that all the other 4 strains have preferably neutralization, in having possessed fully and international standard attack the ability of strain CVS-11 strain, as shown in Figure 3.
6. the antibody after the sudden change of non-hypervariable region is to influencing the rabies virus resistance
Method according to above-mentioned 8～11, to be cloned among the pAC-L-Fc based on amended light chain gene of RVIgG3 and heavy chain gene, and transfection insect Sf9 cell, utilize baculovirus/insect cell system to realize the secretion type expression of whole antibody, obtain mutant RVIgG3 '.This mutant is carried out immunology detection, indirect immunofluorescence experiment shows that RVFab3 ' energy specificity is at rabies virus glycoprotein G, adopted tachysynthesis fluorescence kitchen range to suppress experiment and detected antibody and attack the neutralization reaction of strain CVS-11 strain in external and international standard, the result shows that its character and RVIgG3 are basic identical.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Sequence table
＜110〉China Sickness Prevention Control Center Virus Disease Prevention Control Institute
＜120〉human source anti-rabies virus glycoprotein neutrality antibody (RVFab3)
<130>khp10112589.8
<160>6
<170>PatentIn?version?3.5
<210>1
<211>215
<212>PRT
<213>RVFab3-VL
<400>1
Glu?Leu?Thr?Gln?Pro?Pro?Ser?Ala?Ser?Gly?Thr?Pro?Gly?Gln?Arg?Val
1???????????????5???????????????????10??????????????????15
Thr?Ile?Ser?Cys?Ser?Gly?Ser?Ser?Ser?Asn?Ile?Gly?Ser?Asn?Thr?Val
20??????????????????25??????????????????30
Asn?Trp?Tyr?Gln?Gln?Leu?Pro?Gly?Thr?Ala?Pro?Lys?Leu?Leu?Ile?Tyr
35??????????????????40??????????????????45
Ser?Asn?Asn?Gln?Arg?Pro?Ser?Gly?Val?Pro?Asp?Arg?Phe?Ser?Gly?Ser
50??????????????????55??????????????????60
Lys?Ser?Gly?Thr?Ser?Ala?Ser?Leu?Ala?Ile?Ser?Gly?Leu?Gln?Ser?Glu
65??????????????????70??????????????????75??????????????????80
Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Ala?Ala?Trp?Asp?Asp?Ser?Leu?Asn?Ala
85??????????????????90??????????????????95
Trp?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro?Lys
100?????????????????105?????????????????110
Ala?Ala?Pro?Ser?Val?Thr?Leu?Phe?Pro?Pro?Ser?Ser?Glu?Glu?Leu?Gln
115?????????????????120?????????????????125
Ala?Asn?Lys?Ala?Thr?Leu?Val?Cys?Leu?Ile?Ser?Asp?Phe?Tyr?Pro?Gly
130?????????????????135?????????????????140
Ala?Val?Thr?Val?Ala?Trp?Arg?Ala?Asp?Ser?Ser?Pro?Val?Lys?Ala?Gly
145?????????????????150?????????????????155?????????????????160
Val?Glu?Thr?Thr?Thr?Pro?Ser?Lys?Gln?Ser?Asn?Asn?Lys?Tyr?Ala?Ala
165?????????????????170?????????????????175
Ser?Ser?Tyr?Leu?Ser?Leu?Thr?Pro?Glu?Gln?Trp?Lys?Ser?His?Arg?Ser
180?????????????????185?????????????????190
Tyr?Ser?Cys?Gln?Val?Thr?His?Glu?Gly?Ser?Thr?Val?Glu?Lys?Thr?Val
195?????????????????200?????????????????205
Ala?Pro?Thr?Glu?Cys?Ser?Phe
210?????????????????215
<210>2
<211>223
<212>PRT
<213>RVFab3-VH
<400>2
Leu?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu?Thr?Leu?Ser?Leu
1???????????????5???????????????????10??????????????????15
Thr?Cys?Thr?Val?Ser?Gly?Gly?Pro?Val?Ser?Ser?Val?Asn?Phe?Tyr?Trp
20??????????????????25??????????????????30
Ser?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Tyr
35??????????????????40??????????????????45
Ile?Tyr?Tyr?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg
50??????????????????55??????????????????60
Ile?Thr?Thr?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu
65??????????????????70??????????????????75??????????????????80
Ser?Ala?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Glu
85??????????????????90??????????????????95
Arg?Leu?Thr?Thr?Gly?Ala?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val
100?????????????????105?????????????????110
Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala
115?????????????????120?????????????????125
Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu
130?????????????????135?????????????????140
Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly
145?????????????????150?????????????????155?????????????????160
Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser
165?????????????????170?????????????????175
Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu
180?????????????????185?????????????????190
Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr
195?????????????????200?????????????????205
Lys?Val?Asp?Lys?Arg?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?Ser
210?????????????????215?????????????????220
<210>3
<211>652
<212>DNA
<213>RVab3-VL1
<400>3
gagctcacgc?agccgccctc?agcgtctggg?acccccgggc?agagggtcac?catctcttgt??????60
tctggaagca?gctccaacat?cggaagtaat?actgtaaact?ggtaccagca?gctcccagga??????120
acggccccca?aactcctcat?ctatagtaat?aatcagcggc?cctcaggggt?ccctgaccga??????180
ttctctggct?ccaagtctgg?cacctcagcc?tccctggcca?tcagtgggct?ccagtctgag??????240
gatgaggctg?attattactg?tgcagcatgg?gatgacagcc?tgaatgcctg?ggtgttcggc??????300
ggagggacca?agctgaccgt?cctaggtcag?cccaaggctg?ccccctcggt?cactctgttc??????360
ccgccctcct?ctgaggagct?tcaagccaac?aaggccacac?tggtgtgtct?cataagtgac??????420
ttctacccgg?gagccgtgac?agtggcctgg?agggcagata?gcagccccgt?caaggcggga??????480
gtggagacca?ccacaccctc?caaacaaagc?aacaacaagt?acgcggccag?cagctacctg??????540
agcctgacgc?ccgagcagtg?gaagtcccac?agaagctaca?gctgccaggt?cacgcatgaa??????600
gggagcaccg?tggagaagac?agtggcccct?acagaatgtt?cataattcta?ga??????????????652
<210>4
<211>669
<212>DNA
<213>RVab3-VH4
<400>4
ctcgagtcgg?gcccaggact?ggtgaagcct?tcggagactc?tgtctctcac?gtgcactgtc??????60
tctggtggcc?ccgtcagcag?tgttaatttc?tactggagct?ggatccggca?gcccccaggg??????120
aagggactgg?agtggattgg?gtatatctat?tacagtggga?gcaccaacta?taacccctcc??????180
ctcaagagtc?gaatcaccac?atcagttgac?acgtccaaga?accagttctc?cctgaagctg??????240
agcgctgtga?ctgctgcgga?cacggccgtg?tattactgtg?cgagagagag?gttgactact??????300
ggcgctatgg?acgtctgggg?ccaagggacc?acggtcaccg?tctcgtcagc?ctccaccaag??????360
ggcccatcgg?tcttccccct?ggcaccctcc?tccaagagca?cctctggggg?cacagcggcc??????420
ctgggctgcc?tggtcaagga?ctacttcccc?gaaccggtga?cggtgtcgtg?gaactcaggc??????480
gccctgacca?gcggcgtgca?caccttcccg?gctgtcctac?agtcctcagg?actctactcc??????540
ctcagcagcg?tggtgaccgt?gccctccagc?agcttgggca?cccagaccta?catctgcaac??????600
gtgaatcaca?agcccagcaa?caccaaggtg?gacaagagag?tcgagcccaa?atcttgtgac??????660
aaaactagt??????????????????????????????????????????????????????????????669
<210>5
<211>21
<212>DNA
＜213〉artificial sequence
<400>5
aaactagcta?gtcgccaagg?a??????????????????????????????????????????????????21
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<400>6
ccgcggtggc?ggccgcaaat????????????????????????????????????????????????????20
Claims (10)
1. a human source anti-rabies virus glycoprotein neutrality antibody is characterized in that, the aminoacid sequence of its light chain CDR1, CDR2 and CDR3 and heavy chain CDR1, CDR2 and CDR3 is as shown in the table:
2. antibody as claimed in claim 1 is characterized in that, its light chain variable region amino acid sequence and weight chain variable region amino acid sequence are shown in SEQ ID No.1 and SEQ ID No.2.
3. antibody as claimed in claim 2 is characterized in that, it is single-chain antibody ScFv or whole antibody immunoglobulin IgG.
4. each described antibody of claim 1-3 is through transforming the antibody of deriving that obtains, and described transformation comprises amino acid whose disappearance, replacement and/or insertion, and does not change the activity of antibody.
5. the gene of each described antibody of coding claim 1-4.
6. gene as claimed in claim 5 is characterized in that, the nucleotide sequence of the nucleotide sequence of encoded light chain variable region and encoding heavy chain variable region is shown in SEQ ID No.3 and SEQID No.4.
7. contain claim 5 or 6 described expression carrier.
8. the host of containing the described expression vector of claim 7.
9. each described antibody of claim 1-4 is in preparation prevention or treat application in the rabic medicine.
10. the medicine or the detection reagent that contain each described antibody of claim 1-4.
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| PCT/CN2010/001574 WO2011137569A1 (en) | 2010-05-06 | 2010-10-09 | Humanized neutralizing antibody rvfab3 against rabies virus glycoprotein |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011137569A1 (en) * | 2010-05-06 | 2011-11-10 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody rvfab3 against rabies virus glycoprotein |
| CN104603149A (en) * | 2012-05-24 | 2015-05-06 | 万机集团有限公司(英国) | Compositions and methods related to prevention and treatment of rabies infection |
| CN115260307A (en) * | 2021-05-08 | 2022-11-01 | 南昌大学 | High-affinity fully human monoclonal antibody for resisting rabies virus and application thereof |
| WO2023279803A1 (en) * | 2021-07-07 | 2023-01-12 | 高光 | Protein binding molecule of rbv and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101550189A (en) * | 2008-04-25 | 2009-10-07 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Anthropogenic antivirulin glycosidoprotein neutralizing genetic engineering antibody RD9 and preparation and application thereof |
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| CN1355253A (en) * | 2000-11-29 | 2002-06-26 | 中国预防医学科学院病毒学研究所 | Humanized genetically engineered neutralizing antibody of rabies virus |
| US20060134099A1 (en) * | 2003-06-02 | 2006-06-22 | Biotechnology Foundation, Inc. | Production of rabies antibodies in plants |
| CN101696242B (en) * | 2009-10-26 | 2011-12-28 | 中国人民解放军南京军区军事医学研究所 | Human anti rabies virus neutralizing antibody Fab as well as preparation method and application thereof |
| CN101812132B (en) * | 2010-05-06 | 2012-07-04 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody (RVFab3) against rabies virus glycoprotein |
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| 《European Journal of Immunology》 20050731 KRAMER RA等 The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries 第2131-2145页 1-10 第35卷, 第7期 2 * |
| 《Journal of Virological Methods》 20091130 HOUIMEI M等 Isolation and characterization of human neutralizing antibodies to rabies virus derived from a recombinant immune antibody library 第206-215页 1-10 第161卷, 第2期 2 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011137569A1 (en) * | 2010-05-06 | 2011-11-10 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody rvfab3 against rabies virus glycoprotein |
| CN104603149A (en) * | 2012-05-24 | 2015-05-06 | 万机集团有限公司(英国) | Compositions and methods related to prevention and treatment of rabies infection |
| CN115260307A (en) * | 2021-05-08 | 2022-11-01 | 南昌大学 | High-affinity fully human monoclonal antibody for resisting rabies virus and application thereof |
| CN115260308A (en) * | 2021-05-08 | 2022-11-01 | 南昌大学 | High-affinity rabies virus-resistant fully-humanized monoclonal antibody and application thereof |
| CN115260307B (en) * | 2021-05-08 | 2024-02-09 | 南昌大学 | High-affinity anti-rabies virus fully human monoclonal antibody and application thereof |
| CN115260308B (en) * | 2021-05-08 | 2024-02-09 | 南昌大学 | High-affinity anti-rabies virus fully human monoclonal antibody and application thereof |
| WO2023279803A1 (en) * | 2021-07-07 | 2023-01-12 | 高光 | Protein binding molecule of rbv and use thereof |
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| Publication number | Publication date |
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| CN101812132B (en) | 2012-07-04 |
| WO2011137569A1 (en) | 2011-11-10 |
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