CN101812131B - Humanized neutralizing antibody (RVFab8) against rabies virus glycoprotein - Google Patents
Humanized neutralizing antibody (RVFab8) against rabies virus glycoprotein Download PDFInfo
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- CN101812131B CN101812131B CN2010101715431A CN201010171543A CN101812131B CN 101812131 B CN101812131 B CN 101812131B CN 2010101715431 A CN2010101715431 A CN 2010101715431A CN 201010171543 A CN201010171543 A CN 201010171543A CN 101812131 B CN101812131 B CN 101812131B
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
Abstract
The invention discloses a humanized neutralizing antibody (RVFab8) 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 gp neutrality antibody; The invention still further relates to this antibody and prevent or treat the application in the rabic medicine in preparation.
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) rabies vaccine injection bonded rabies poison Tegeline (rabies immune globulin, method RIG) are adopted in suggestion in the World Health Organization.Two types of RIG that use at present as the human anti-rabies Tegeline (human rabies immune globulin, HRIG) and the horse anti-rabies virus Tegeline (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 Tegeline that contain specific antibody are with a long history in order to prevention and treatment transmissible disease.Resisting virus attack with active and endogenous protective human body in the extracorporeal antivirus effect of monoclonal antibody has obtained many experiment showed, like neutralizing monoclonal antibodies such as mouse-anti HAV, Hantaan virus, Measles virus, RSV virus, CMV viruses and can be in vivo 100% watches for animals and avoid virus attack.The approach that obtains polyvalent antibody with antigen-immunized animal is the classical way that obtains antibody always, but lack of specific and homogeneity.Then the B lymphocyte hybridoma technology of setting up make numerous scientists through 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 mostly McAb 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.Tegeline (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 through 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 through 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 meliority (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 one type of new antiviral drug 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 through 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 of carrier's monoclonal antibody technology (Green, L.L.et al., 1994), allosome hybridoma technology (James; K.et al., 1987), phage display technique (Barbas, C.F.et al.; 1991) etc. produce humanized antibody, become the great direction of domestic and international research at present, 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 gp 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 through genetic engineering means, and screening obtains special rabies virus gene engineered antibody Fab section.The Fab section antibody called after RVFab8 that obtains.
This three strains 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 in prokaryotic cell prokaryocyte, obtains the functional antibodies of the specificity combination rabies virus of effective expression.Their specific recognition rabies virus particulate antigens, and all to rabies virus glycoprotein G have 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 RVFab8 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 RVFab8 is under the jurisdiction of the VL2 of light chain of antibody family.The antibody protein function is by specificity nucleotide sequence among the complementary region CDR1 of decision family, CDR2 and the CDR3 that are present in antibody gene light chain and variable region of heavy chain 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 each antibody combines characteristic and rabies poison functional character in the decision invention.Determine that light chain of antibody and variable region of heavy chain amino acid detailed sequence and the comparative result thereof of every strain neutralizing antibody function are as shown in table 1:
Table 1
The aminoacid sequence of RVFab8 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 RVFab8 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; The amino acid that for example will have similarity in non-hypervariable region is replaced, and replaces with Ala like the 8th Val with the heavy chain VH sequence of RVFab8.
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 get into 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 through abduction delivering.
In addition, can the light chain encoding sox of above-mentioned Fab antibody be cloned in the complete anti-expression vector with heavy Fd fragment gene, and import in the host cell, obtain to express the full AIG of rabies poison.
In an embodiment of the present invention; The light chain of above-mentioned Fab antibody RVFab8 is cloned into whole antibody expression vector pAC-L-Fc and transfection insect Sf9 cell respectively with heavy Fd fragment gene; Utilize baculovirus/insect cell system to realize the secretion type expression of whole antibody, obtain whole antibody RVIgG8.
Utilize ELISA, IFA, SDS-PAGE that the whole antibody that obtains is carried out Function Identification; The result shows that humanized IgG whole antibody RVIgG8 all has specificity to combine to aG strain and CTN strain rabies virus particle, and expressing ERA, CVS, CTN and four kinds of rabies virus strain's gp 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 RVIgG8 has neutralization preferably; Can reach 876.6IU/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 to 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 characteristic 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; With the antibody product of rabies virus infection, process and be used for prevention clinically and treat rabic specific antibody medicine during acquisition has.
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 to CVS-11 rabies virus tachysynthesis fluorescence kitchen range.
Embodiment
Below in conjunction with accompanying drawing and embodiment, specific embodiments of the invention describes in further detail.Following examples are used to explain the present invention, but are not used for limiting scope of the present 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 uses strain PM strain to use vaccine strain as the importer, 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 gp (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 through the recombinant plasmid transfection insect cell is prepared recombinant baculovirus, express the BaculoGold cotransfection test kit that adopts U.S. Pharmogen company.Working method runs over as follows: after the BaculoGold linear DNA mixed 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 processed recombinant rabies poison gp (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 vaccination person's with high titre rabies virus antibodies anticoagulated blood; 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) rt 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 basically; C.FIII., Kang, A.S.; And larner, 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 encapsulates 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 glycocoll-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 basically; 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 0.1m/L NaHCO is used in the detection of expressing
3(pH9.6) solution encapsulates anti-human Fab's antibody (U.S. Sigma, dilution in 1: 2000 is used), and in enzyme plate, 4 ℃ are spent the night; 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 two 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, ELIASA detects the absorbance A value.
5.2 the indirect enzyme-linked immunosorbent method detect Fab combine with rabies virus activity with the inactivated rabies virus particle of purifying as envelope antigen, 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 processed 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 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, put down, be cloned into pAC-L-Fc carrier (German PROGEN PR3003) (Liang with end-filling enzyme (K fragment) benefit; 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 BaculoGo1d cotransfection test kit that adopts U.S. Pharmogen company.Working method runs over as follows: 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 neutralization and use viral CVS-11; 50 μ l/ holes establish the blank well contrast simultaneously, and the virus control hole are used in neutralization; With 1 hour, every hole added 1 * 10 in rearmounted 37 ℃ of the mixing
6/ ml BHK-21 cell suspension 50 μ l put 37 ℃ of 5%CO
2Cultivated 24 hours in the incubator.Wait cultivate to finish to blot nutrient solution, add in every hole after 100 μ l/PBS clean and blot, every hole adds 80% acetone, the 50 μ l of precooling to 4 ℃ ,-30 ℃ fixing 10 minutes; 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 with PBS; Dry liquid, 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 seized antibody.According to Reed & Muench formula, calculate the ED of each antibody sample and mark article
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 RVIgG8 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 5th Ala of RVIgG8 variable region of light chain (shown in the SEQ IDNo.1) is replaced with Gly.The heavy chain nucleic acid sequence encoding (codon gtg being replaced with gcc) and the light chain nucleic acid sequence encoding (codon gcc being replaced with ggg) that synthesize RVIgG8 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 two mutants 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, have 181 clones that rabies virus particle aG strain specificity is combined, and wherein 36 strain Fab clone is confirmed as to rabies virus glycoprotein.
Table 2aG strain is screened the phage antibody library enrichment
| The Fab phage library | Storage capacity | The selected clone number | Fab positive (ELISA) | AG virus-positive (ELISA) | Gp 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, have 79 clones that rabies virus particle CTN strain specificity is combined, and wherein 34 strain Fab clone is confirmed as to rabies virus glycoprotein.
Table 3CTN strain is screened the phage antibody library enrichment
| The Fab phage library | Storage capacity | The selected clone number | Fab positive (ELISA) | CTN virus-positive (ELISA) | Gp 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 combination rabies virus glycoprotein, wherein has 11 strain sequences different.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, 11 strain specificitys is combined the people source Fab monoclonal antibody called after RVFab1-11 of rabies virus glycoprotein.It is following wherein to choose RVFab1, RVab3, RVFab5, RVFab8 and RVFab9 comparative sequences:
The aminoacid sequence of table 4 human source anti-rabies virus gp 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 combines the specificity of rabies virus glycoprotein
The reorganization Fab antibody that obtains in order to confirm is to the binding specificity of different rabies virus strains gp, and we further identify the functionally active of prokaryotic expression Fab antibody through indirect immunofluorescence assay (IFA).As shown in Figure 1; RVFab1 and ERA, CVS, CTN and four kinds of rabies virus strains of aG strain gp 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 gp immunofluorescence results.The 11 strain Fab positive antibodies that immunofluorescence result proof obtains from the antibody library screening are all to rabies virus glycoprotein, and react general wider.
4. whole antibody IgG expresses and purifying
5 strains have been accomplished 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 through 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 the neutralization activity of 5 strain whole antibody IgG (RVIgG1, RVIgG3, RVIgG5, RVIgG8, RVIgG9); 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 that the neutralization of 5 strain personnel rabies poison whole antibody RVIgG1, RVIgG3, RVIgG5, RVIgG and RVIgG9 is tired and is respectively 866.6IU/mg; 813.3IU/mg; 689.8IU/mg, 876.6IU/mg, 428.5IU/mg; Be equal to or higher than 0.5IU/ml according to WHO rabies Committee of Experts evaluation neutralizing antibody level and represent that promptly this antibody has the activity of neutralization, so the 5 strain people source monoclonal antibodies that this institute obtains all have the neutralization activity to rabies virus.Wherein the neutralization activity of RVIgG9 a little less than, it is active that all the other 4 strains have preferably neutralization, attacks the ability of strain CVS-11 strain in having possessed fully with international standard, 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 RVIgG8 and heavy chain gene; And transfection insect Sf9 cell, utilize baculovirus/insect cell system to realize the secretion type expression of whole antibody, obtain two mutants RVIgG8 '.This two mutants is carried out immunology detection; Indirect immunofluorescence experiment shows that RVFab8 ' can specificity be directed against 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 RVIgG8 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 know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded 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 gp neutrality antibody (RVFab8)
<130>khp10112291.7
<160>6
<170>PatentIn version 3.5
<210>1
<211>215
<212>PRT
<213>RVFab8-VL
<400>1
Glu Leu Gln Pro Ala Ser Val Ser Gly Ser Leu Gly Gln Ser Ile Thr
1 5 10 15
Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Asn Tyr Asn Leu Val
20 25 30
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Ile Ile Tyr
35 40 45
Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser
50 55 60
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu
65 70 75 80
Asp Glu Ala Asn Tyr Tyr Cys Ser Ser Tyr Thr Ala Thr Lys Asn Tyr
85 90 95
Trp Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Arg 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 Lys 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>RVFab8-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 Ser Ile Ser Ser Val Asn Ser Tyr Trp
20 25 30
Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Asn
35 40 45
Phe Tyr Tyr Ser Gly Asn Thr His Tyr Asn Pro Ser Leu Lys Ser Arg
50 55 60
Val Thr Ile Ser Val Gly Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu
65 70 75 80
Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln
85 90 95
Ser Thr Ile Gly Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 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 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr Ser
210 215 220
<210>3
<211>652
<212>DNA
<213>RVab8-VL2
<400>3
gagctccagc ctgcctccgt gtctgggtct cttggacagt cgatcaccat ctcctgcact 60
ggaaccagca gtgatattgg gaattataac cttgtctcct ggtaccaaca acacccaggc 120
aaagccccca aactcataat ttatgaggtc actaagcggc cctcaggggt ttctaatcgc 180
ttctctggct ccaagtctgg caacacggcc tccctgacca tctctgggct ccaggctgag 240
gacgaggcta attactactg cagctcatat acagccacca agaattactg gattttcggc 300
ggagggacca agctgaccgt ccgaggtcag cccaaggctg ccccctcggt cactctgttc 360
ccgccctcct ctgaggagct tcaagccaac aaggccacac tggtgtgtct cataagtgac 420
ttctacccgg gagccgtgac agtggcctgg aaggcagata gcagccccgt caaggcggga 480
gtggagacca ccacaccctc caaacaaagc aacaacaagt acgcggccag cagctatctg 540
agcctgacgc ctgagcagtg gaagtcccac agaagctaca gctgccaggt cacgcatgaa 600
gggagcaccg tggagaagac agtggcccct acagaatgtt cataattcta ga 652
<210>4
<211>669
<212>DNA
<213>RVab8-VH4
<400>4
ctcgagtcgg gcccaggact ggtgaagcct tcggagaccc tgtccctcac ctgcactgtc 60
tctggtggct ccatcagcag tgttaattcc tactggggct ggatccgcca gcccccaggg 120
aaggggctgg agtggattgg gaatttctat tatagtggga acacccacta caacccgtcc 180
ctcaagagtc gagtcaccat atccgtaggc acgtccaaga accagttctc cctgaagctg 240
aactctgtga ccgccgcaga cacggctgta tattactgtg cgagacagtc gaccataggg 300
ggcttctttg actactgggg ccagggaacc ctggtcaccg tctcctcagc ctccaccaag 360
ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 420
ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cagtgtcgtg gaactcaggc 480
gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 540
ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 600
gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa 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 (9)
1. a human source anti-rabies virus gp 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 respectively 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. the gene of each described antibody of coding claim 1-3.
5. gene as claimed in claim 4 is characterized in that, the nucleotide sequence of the nucleotide sequence of encoded light chain variable region and encoding heavy chain variable region is respectively shown in SEQ ID No.3 and SEQ ID No.4.
6. contain the said expression carrier of claim 4.
7. the host of containing the said expression vector of claim 6.
8. each described antibody of claim 1-3 prevents or treats the application in the rabic medicine in preparation.
9. the medicine or the detection reagent that contain each described antibody of claim 1-3.
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| PCT/CN2010/001575 WO2011137570A1 (en) | 2010-05-06 | 2010-10-09 | Human-derived neutralizing antibody rvfab8 against rabies virus glycoprotein |
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| CN101812131B (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 (RVFab8) against rabies virus glycoprotein |
| CN103323588A (en) * | 2012-03-19 | 2013-09-25 | 郑州中道生物技术有限公司 | Method for detecting canine rabies virus antibody and detection kit |
| BR112014029274B1 (en) * | 2012-05-24 | 2022-02-15 | Mountgate Innotech (Hk) Limited | ISOLATED ANTIBODY, PHARMACEUTICAL COMPOSITION, ANTIBODY USE, AND, KIT TO TREAT RABIC INFECTION |
| CN103505729B (en) * | 2013-05-24 | 2015-10-28 | 华北制药集团新药研究开发有限责任公司 | A kind of stable rabies virus human antibody combination preparation |
| CN104193823B (en) * | 2014-08-22 | 2019-06-11 | 兰州生物制品研究所有限责任公司 | A kind of anti-rabies virus specificity human antibody and application |
| CN104829724B (en) * | 2015-04-27 | 2019-01-11 | 华北制药集团新药研究开发有限责任公司 | A kind of source of mouse monoclonal antibody and its preparation method and application |
| CN104804083B (en) * | 2015-04-27 | 2019-03-05 | 华北制药集团新药研究开发有限责任公司 | A kind of source of mouse monoclonal antibody and its preparation method and application |
| CN115260307B (en) * | 2021-05-08 | 2024-02-09 | 南昌大学 | High-affinity anti-rabies virus fully human monoclonal antibody and application thereof |
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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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| Title |
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| HOUIMEI M等.Isolation and characterization of human neutralizing antibodies to rabies virus derived from 1-10a recombinant immune antibody library.《Journal of Virological Methods》.2009,第161卷(第2期),第206-215页. * |
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