CN101538328A - Humanized neutralizing engineered antibody for targeting avian influenza virus H5N1 - Google Patents

Abstract

The invention discloses a humanized neutralizing engineered antibody for targeting avian influenza virus H5N1, which is derived from Chinese antibody recombination library of highly pathogenic avian influenza H5N1 convalescent and the main characteristics are as follows: the recombinant antibody is determined by specific gene sequences of complementarity determining regions (CDRs) being present in the antibody light-chain and heavy-chain gene variable region, and obtains effectively-expressed neutralizing functional antibody of specific binding AIVHA (avian influenza virus hemagglutinin) protein HA from prokaryotic and eukaryotic cells. The utilization of the antibody CDR or part of or whole gene can remodel and produce various forms of engineered antibodies in procaryon, yeast, insect and eucaryon cells, and any expression systems, and can prevent or treat related diseases of H5N1 avian influenza virus clinically.

Description

A kind of people source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibody

The application is to be on June 15th, 2007 applying date, and application number is dividing an application of 200710111098.8 application for a patent for invention " people source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibody ".

Technical field

The present invention relates to prevent and treat preparation and the application of using human genetically engineered antibody, especially specificity is at highly pathogenic bird flu virus H 5 N 1, in having and the neutrality anti-avian influenza virus genetic engineering antibody in China bird flu convalescent of avian influenza function source.

Background technology

(Highly pathogenic avian influenza HPAI) is the bird deadly infectious disease that is caused by the Influenza Virus A of orthomyxovirus section type influenza virus to high pathogenic avian influenza.Highly pathogenic bird flu virus H 5 N 1 is since generation 18 people in 1997 infect the dead epidemic situation of 6 people, be subjected to showing great attention to of international community always, particularly since two thousand three HPAI (High Pathogenic AI) virus H5N1 animal epidemics has become region popular in the Asia, and has spread to Europe And Africa.The countries and regions of people's cases of infection and generation people cases of infection constantly enlarge, and since the end of the year 2003, by on April 11st, 2007, global New Development human and bird fluenza (H5N1) case load rose to 291 examples, wherein dead 172 examples.China one has had 24 examples since November first in 2003, routine avian influenza virus H 5 N 1 cases of infection were made a definite diagnosis, wherein 15 examples are dead.Highly pathogenic bird flu virus H 5 N 1 has been broken through species barrier and not only bird has been brought grave danger, have a strong impact on the Economic development of country, and human health also brought huge threat, can or can not cause a flu outbreak to cause showing great attention to of the whole world by the H5N1 avian influenza virus.Influenza virus is a kind of sphere or shaft-like, tunicary sub-thread minus-stranded rna virus, and gene element is 8 sections.The transmembrane glycoprotein hemagglutinin (HA) on virus envelope surface and neuraminic acid zymoprotein (NA) are the main immunogenic antigen of influenza virus.HA discerns the target cell surface receptor and combines with acceptor, has and the host cell membrane fusion-activity, can induce body to produce the protectiveness neutralizing antibody.The HA antigenic variation that causes owing to the HA transgenation tends to cause immuning failure and variation strain popular, and the generation antigenic variation that influenza virus is frequent and lasting does not also have effective human-used avian influenza vaccine at present, does not have effective medicine yet.As the biotechnology preparation of a class novel specific anti-virus infection, neutrality antibody is being played the part of important role aspect anti-infective therapy and the urgent passive immunization prevention always.Therefore, the development specificity is very necessary and feasible at the antibody that different H5N1 strain isolateds have a wide spectrum neutralizing effect to be used for urgent prevention and treatment especially at the neutrality antibody of H5N1 avian influenza virus.

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.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.And the development of human genetically engineered antibody has brought new hope and long-range prospect for the biological products development in this field.Obtained many experimental results show that with active and endogenous protective human body opposing virus attack in the extracorporeal antivirus effect of monoclonal antibody; 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, we believe that the specificity neutralizing antibody should have good preventing and treatment effect to highly pathogenic bird flu virus H 5 N 1.

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.

Summary of the invention

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 anti-highly pathogenic bird flu virus H 5 N 1, obtain its antibody gene, the specific antibody medicine that provides feasibility in the future possible clinical antiviral prevention and treatment.

The present invention uses phage surface to present technology, therefrom obtain lymphocyte in the state Anhui bird flu patient decubation blood, made up anti-H5N1 avian influenza virus genetic engineering antibody library, people source by genetic engineering means, and screening obtains special anti-avian influenza virus genetic engineering antibody Fab section and whole antibody thereof.

People of the present invention source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibody comprises:

1, two strain Fab antibody, called after AVFluFab1 and AVFluFab3 respectively.

This two 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 obtains the functional antibodies of the specificity of effective expression in conjunction with highly pathogenic bird flu virus H 5 N 1 in prokaryotic cell prokaryocyte.Their specific recognition H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) particulate antigens, and all at avian flu virus hemagglutinin protein HA, have the reaction of obvious immunofluorescence reaction (IFA) and enzyme linked immunological (ELISA) with avian influenza virus H 5 N 1, have that anti virus H 5 N 1 of bird flu infects in and active function.

Specific light chain of AVFluFab1 and AVFluFab3 and heavy chain variable region gene derive from the rich long-pending screening of the specificity in anti-highly pathogenic bird flu virus H 5 N 1 antibody gene storehouse, people source, and the foundation of this antibody library derives from Chinese Anhui H5N1 bird flu convalescent 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, AVFluFab1 and AVFluFab3 are under the jurisdiction of VH4 of heavy chain of antibody family and VH3 respectively, VL2 of light chain of antibody family and VL1.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 each antibody is in conjunction with feature and anti-highly pathogenic bird flu virus H 5 N 1 functional character in the decision present patent application.Determine the light chain of antibody of every strain neutralizing antibody function and variable region of heavy chain amino acid detailed sequence and comparative result thereof as shown in Figure 1, "-" symbolic representation and the identical amino acid of the first row antibody sequence among the figure, dash area is the CDR district.

The gene order of AVFluFab1 variable region of light chain is shown in SEQ ID NO.5, and the gene order of variable region of heavy chain is shown in SEQ ID NO.6.The gene order of AVFluFab3 variable region of light chain is shown in SEQ ID NO.7, and the gene order of variable region of heavy chain is shown in SEQ ID NO.8.

According to specificity Nucleotide or aminoacid sequence in anti-highly pathogenic bird flu virus H 5 N 1 neutralizing antibody AVFluFab1 of the present invention and the AVFluFab3 variable region, can be at the nucleotide sequence of the identical therewith antibody weight chain gene 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 anti-Highly Pathogenic Avian Influenza Virus (HPAIV) neutralizing antibody or associated protein or polypeptide product.

2, two strain whole antibodies, called after AVFluIgG1 and AVFluIgG3 respectively.

Light chain and heavy Fd fragment gene with above-mentioned 2 strain Fab antibody (AVFluFab1, AVFluFab3), be 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 AVFluIgG1 and AVFluIgG3.

With ELISA, IFA, Western Blot and flow cytometry the functional performance of obtaining people source monoclonal antibody is identified.The result show this be 2 strain specificitys at the H5N1 avian influenza virus and with the people source monoclonal antibody of first 1 type and first 3 type human influenza virus no cross reactions.2 strain antibodies are all at H5N1 avian flu virus hemagglutinin protein HA, and wherein AVFluIgG1 can react with the inactivation of viruses of sex change and the HA that recombinates respectively, and reaction with it of AVFluIgG3.This prompting AVFluIgG1 at the antigenic determinant of HA be linear antigenic determinant, and the antigenic determinant that AVFluIgG3 discerned is a conformation dependent form.In addition, this 2 strain antibody all has higher and neutralization activity widely to the H5N1 avian influenza virus people source strain isolated in the Asia CHINESE REGION south and the north, the higher also wide spectrum more of the neutralization activity of AVFluIgG3 wherein, all has the neutralization activity for the isolating at present 8 strain H5N1 avian influenza virus people source strain isolateds of China, and at wherein some Local Isolates can be in nanogram level level neutralization virus, for provide effective candidate's epi-position to be significant for vaccine development.

The present invention obtains two strains in the world first and has the active anti virus H 5 N 1 of bird flu monoclonal antibody Fab section of neutralization, and in eukaryotic system, expressed its whole antibody, this result's acquisition has not only brought hope for the prevention of highly pathogenic bird flu virus H 5 N 1 and treatment, also provides new thinking for its vaccine development simultaneously.Utilize the people source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibody variable region gene of above-mentioned acquisition, full-antibody gene under Fab antibody gene and above-mentioned each antibody gene feature, can be at prokaryotic cell prokaryocyte, yeast cell, express and produce any other gene that contains this antibody gene after this antibody or the reconstruction based on this in eukaryotic cell and any recombination system, the antibody product that infects with highly pathogenic bird flu virus H 5 N 1 during acquisition has, make the specific antibody medicine that is used to prevent and treat the human and bird fluenza that causes by the H5N1 avian influenza virus clinically, thereby can be used to clinically prevent and treat the people who causes by avian influenza virus H 5 N 1 and infect bird flu.Based on above-mentioned engineered antibody gene and direct or indirect gene product thereof, can be made into spray-type antibody preparation and injection-type antibody preparation and be used for prevention and the treatment that the people infects the H5N1 high pathogenic avian influenza.

Description of drawings

Fig. 1 is an anti virus H 5 N 1 of bird flu people of the present invention source Fab antibody variable region amino acid sequence comparison diagram.

Fig. 23 takes turns the expression figure that ELISA after the enrichment detects the 45 strain people source Fab antibody that obtain.

Fig. 3 A is the 45 strain human Fab antibody of detect expressing with ELISA to the specificity of avian influenza virus (A/Anhui/1/2005) in conjunction with situation map;

Fig. 3 B is the 45 strain human Fab antibody of detect expressing with ELISA to the specificity of reorganization HA (A/Vietnam/1203/2004) in conjunction with situation map.

Fig. 4 is the Immunofluorescence Reactions result of anti virus H 5 N 1 of bird flu people source Fab monoclonal antibody and avian influenza virus and reorganization HA, and wherein: A, B, C, D are respectively positive serum, negative antibody, AVFluFab1, AVFluFab3 and reorganization HA (A/Anhui/1/2005) reaction result; E, F, G, H are respectively positive serum, negative antibody, AVFluFab1, AVFluFab3 and avian influenza virus (A/Anhui/1/2005) reaction result.

Fig. 5 is the SDS-PAGE electrophorogram of IgG behind the purifying.

Fig. 6 A be ELISA detect purifying humanized IgG antibody to the specificity of purifying avian influenza virus (A/Anhui/1/2005) in conjunction with situation map;

Fig. 6 B be ELISA detect purifying humanized IgG antibody to the specificity of reorganization HA (A/Vietnam/1203/2004) in conjunction with situation map.

Fig. 7 analyzes the specific result of anti virus H 5 N 1 of bird flu humanized IgG monoclonal antibody with Western blot, wherein: and A, react with purified virus (A/Anhui/1/2005); B reacts with recombinant baculovirus expression HA (A/Anhui/1/2005).

Fig. 8 be with fluidic cell indirect fluorescent classification (FACS) analyze humanized IgG antibody to the bird flu antigen-specific in conjunction with situation map, wherein: A, B, C, D are respectively the Sf9 cell response result of positive serum, negative antibody, AVFluIgG1, AVFluIgG3 and express recombinant HA (A/Anhui/1/2005).

Fig. 9 A is that microneutralization test detects the AVFluIgG1 antibody of purifying to the active result of the neutralization of Chinese H5N1 avian influenza virus people source strain isolated;

Fig. 9 B is that microneutralization test detects the AVFluIgG3 antibody of purifying to the active result of the neutralization of Chinese H5N1 avian influenza virus people source strain isolated.

Embodiment

Following preferential embodiment elaborates to the present invention, but does not mean that restriction content of the present invention.

The utilization phage surface presents technology, therefrom obtains lymphocyte in the state Anhui bird flu patient decubation blood, by genetic engineering means, has made up anti-H5N1 avian influenza virus genetic engineering antibody library, people source.H5N1 Anhui, people source strain isolated (A/Anhui/1/2005) with the purifying deactivation carries out the enrichment screening to the Fab phage antibody library, and carries out secreting, expressing in E.coli.Identify Fab antibody to H5N1 avian influenza virus specific bonded functionally active by ELISA, indirect immunofluorescence assay (IFA), and carry out sequencing.With the light chain and the heavy chain Fd fragment gene of positive colony, be cloned into whole antibody expression vector pAC-L-Fc transfection insect Sf9 cell respectively then, utilize baculovirus/insect cell system to realize the secretion type expression of whole antibody.With ELISA, IFA, Western Blot and flow cytometry the functional performance of obtaining people source monoclonal antibody is identified.

Materials and methods

1. virus, cell, carrier and antigen prepd

People source H5N1 isolate A/Anhui/1/2005, A/Guangxi/1/2005, A/Hunan/1/2006, A/Zhejiang/1/2006, A/Sichuan/1/2006, A/Fujian/1/2005, A/Guangdong/1/2006, A/xinjiang/1/2006 are provided by institute influenza chamber in the pre-prevention and control of CDC virus disease.Bacterial strain is XLI-Blu (U.S. Stratagene); Phage expression vector is pComb3 (40kb), is presented by U.S. Scripps institute; Used phage is VCSM13 (an American I nvitrogene company).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 cassette vectors for rapid production of complete human IgG from phagedisplayselected antibody fragments.Journal of Immunological Methods.247:119-130.), insect cell Sf9 and mdck cell are from the U.S. cell cultures center (ATCC).Wherein sieving storehouse antigen is that A/Anhui/1/2005 (H5N1) infects mdck cell, density gradient ultracentrifugation purified virus particle after formalin-inactivated and safety inspection.The rhabdovirus system expression reorganization hemagglutinin HA (A/Vietnam/1203/2004) of purifying is available from U.S.'s albumen scientific company (ProteinSciences Corp.).The recombinant fowl influenza hemagglutinin HA (A/Anhui/1/2005) of eukaryotic expression is made up and is expressed by this research department: pcr amplification avian influenza hemagglutinin albumen HA (A/Anhui/1/2005) gene order, it is cloned into rhabdovirus expression vector pAcUW51 (U.S. BD Pharmingen), carries out protein expression by the recombinant plasmid transfection insect cell is prepared recombinant baculovirus.

2. the structure of phage antibody library

With lymphocyte separation medium (U.S. Sigma) isolated lymphocytes from epidemic-stricken area, Anhui bird flu patient decubation anticoagulation, extract total cell RNA with RNeasy Mini Kit (German QIAGEN), adopt the first chain synthetic agent box (the SuperScriptTMIII First-Strand SynthesisSystem for RT-PCR.Cat No.18080-051) reverse transcription of Invitrogen company to become cDNA the RNA that extracts with the Oligo-dT primer, people's endogenous light chain and heavy chain Fab gene are carried out pcr amplification with the primer of one group of increase human antibody IgG1 heavy chain Fd and light chain Kappa and Lambda.The PCR condition is: 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 2 minutes, 35 circulations.Above-mentioned PCR product reclaims through sepharose respectively, through DNA purification kit QIAquick ^TMBehind kit (the German QIAGEN company) purifying, obtain Kappa, Lambda and Fd chain PCR product about 650-700bp.

Banking process is referring to document Barbas, C.FIII., Kang, A.S., and larner, R.A.Assembly ofcombinatorial antibody libraries on phage surface:the geneIII site.Proc.Natl.Acad.Sci.USA.1991; 88 (18): 7978-7982.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 10 μ l aquae destillatas, the 200 μ l electricity that it is good that prepared beforehand is gone in the electricity transduction changes bacterium XLI-Blu, and (electric commentaries on classics condition is: the Bio-Red electroporation, 0.2cm electric revolving cup, 2.5K volt), electricity adds 10ml SOC nutrient solution after changeing, 37 ℃ 1 hour, adding 10ml has the SB nutrient solution of penbritin and tsiklomitsin and cultivated 1 hour for 37 ℃, add the aforementioned SB of 80-100ml, 37 ℃ added helper phage VCSM131 * 10 after 2 hours ¹², add kantlex (70 μ g/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 PBSpH 7.4 resuspended precipitations, set up phage antibody library, packing is stored in-20 ℃ of refrigerator-freezers.

3. be used for the preparation of the H5N1 avian influenza virus antigen of antibody library enrichment screening

In three grades of laboratory Biohazard Safety Equipments of Biosafety, operate, use the protection of III level Biosafety.The amplification strain is A/Anhui/1/2005 (H5N1), is 10 with virus stock solution used as gradient dilution ^-1, 10 ^-2, 10 ^-3, 10 ^-4Stand-by.The mdck cell that growth conditions is good (cell attachment reaches 80%) is poured out cell growth medium, cleans cell 2-3 time with HankShi liquid.Respectively the dilution viral dilution liquid of difference 1ml is infected mdck cell, 35 ℃ of absorption 1h after absorption is finished, clean cell 2-3 time with HankShi liquid, add in culturing bottle and keep liquid, to 35 ℃ of cultivations.Treat that cells infected has 70%-80% pathology to occur and gathers in the crops, with cell freeze thawing 1-2 time, the results supernatant carries out hemagglutination test (HA test) (HA) and measures the blood clotting titre before the results.Behind formalin-inactivated, infect mdck cell, the blind passage three generations determines complete inactivation, carries out sucrose bed course ultracentrifugation purifying.

4. the enrichment of phage antibody library screening

Screening antigen is the deactivation H5N1 avian influenza virus particle (A/Anhui/1/2005) of ultracentrifugation purifying and the rhabdovirus system expression reorganization hemagglutinin HA (A/Vietnam/1203/2004) of purifying, uses 0.1M NaHCO during use ₃(pH8.6) solution dilution, bag is by 96 hole enzyme plates.The enrichment screening method carries out (Barbas by document substantially, C.FIII., Kang, A.S., and larner, R.A.Assembly of combinatorial antibody libraries on phage surface:thegeneIII site.Proc.Natl.Acad.Sci.USA.1991; 88 (18): 7978-7982), specific as follows:

Use 0.1M NaHCO ₃(pH8.6) the H5N1 avian influenza virus antigen that solution bag is purified (dilution in 1: 200), every hole 100 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 80 μ l phage antibody libraries, hatched 2 hours for 37 ℃, discard unconjugated phage in the hole, with 1 * TBST (50mM Tris-HCl, 150mM NaCl, 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, during piping and druming is for several times repeatedly.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 times.

5.Fab the abduction delivering of section antibody

The preparation of people source neutrality anti virus H 5 N 1 of bird flu engineered Fab antibody solubility expression product is carried out (Barbas by document substantially, C.FIII., Kang, A.S., and larner, R.A.Assembly of combinatorial antibody librarieson phage surface:the geneIII site.Proc.Natl.Acad.Sci.USA.1991; 88 (18): 7978-7982), be specially: 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 (0.02M pH7.4) of original fluid 1/10 volume, multigelation three times, and centrifugal 30 minutes of 4 ℃ of 12000rpm, 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.

6. the ELISA of the anti-H5N1 avian influenza virus in people source Fab antibody detects

4.1Fab the detection of expressing

Use 0.1M NaHCO ₃(pH9.6) solution bag is by anti-human Fab's antibody (U.S. Sigma, dilution in 1: 2000 is used), and 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, microplate reader detects the absorbance A value.

4.2 the indirect enzyme-linked immunosorbent method detects Fab and combines activity with H5N1 avian influenza virus and reorganization HA

As envelope antigen, step subsequently is the same with the deactivation H5N1 avian influenza virus particle (A/Anhui/1/2005) of purifying and reorganization hemagglutinin HA (A/Vietnam/1203/2004).

7. indirect immunofluorescence (IFA) detects

H5N1 avian influenza virus (A/Anhui/1/2005) infects mdck cell, cultivates about 48 hours for 35 ℃, and harvested cell drops on the glass negative, dries up, and acetone fixed 15min is also dry, makes the virus antigen sheet.Utilize expression avian influenza hemagglutinin albumen HA (A/Anhui/1/2005) the recombinate shape virus infection Sf9 cell that has made up simultaneously, the results cells infected is made reorganization HA antigen sheet after 4-5 days.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.

8.Western blot detects

Antigen is respectively the deactivation H5N1 avian influenza virus particle (A/Anhui/1/05) of purifying, the recombinant fowl influenza hemagglutinin HA (A/Anhui/1/2005) of eukaryotic expression.Positive control is a bird flu patient convalescent phase serum, and negative control is at hepatitis A virus (HAV) people source monoclonal antibody HAVIgG16.Antigen carries out the SDS-PAGE electrophoresis, and semidrying is carried out proteinic electrotransfer, and the protein transduction in the polyacrylamide gel is moved on to the NC film.After changeing film gel is carried out Coomassie brilliant blue dyeing to check whether protein shifts fully.The swimming lane of molecular weight standard is cut, mark molecular weight standard with reference to the albumen position.The NC film is sealed 5% skimmed milk room temperature sealing 2 hours.One anti-AVFluFab1, AVFluFab3 and H34 expression product stoste and antigen-reactive, positive serum dilutes and antigen-reactive room temperature reaction 2 hour with confining liquid at 1: 10.After the 1 * PBST washing 3 times with anti-human Fab (Sigma company uses at 1: the 500) room temperature reaction of HPR mark 1 hour.Put into the DAB substrate after 1 * TBST washs 3 times and show the liquid look, extremely in good time termination reaction appears in brown positive band, keeps in Dark Place.

Flow cytometry antigen-antibody binding specificity (flow cytometry, FCM)

Adopt method (the fluorescence activated cell sorting of indirect fluorescent classification, FACS) (Tara Heitner, AnneMoor.et al.Selection of cell binding and internalizing epidermal growth factor receptorantibodies from a phage library.Journal of Immunologicals Methods.2001; 248:17-30.), utilize and express avian influenza hemagglutinin albumen HA (A/Anhui/1/2005) recombinate shape virus infection sf9 cell, gather in the crops cells infected after 4-5 days, cell counting is set each reaction system and is about 2 * 10 ⁵Individual cell/50 μ l, add antibody 50 μ l to be measured, act on 1h on ice, the PBS that contains 0.25%BSA with 250 μ l washes 2 times, add the anti-Fab antibody dilution (U.S. Sigma) in 1: 100 of the anti-FITC mark of 100 μ l, black out acts on 30min on ice, and PBS washes 2 times, cell is resuspended among 1% Paraformaldehyde 96-PBS, to be measured.

10. the nucleic acid sequence analysis of people source Fab antibody variable gene

Carry out nucleic acid sequence analysis with Qiagen Miniprep Kit (German QIAGEN) preparation plasmid DNA.The sequencing primer of weight chain is respectively 5 '-AAACTAGCTAGTCGCCAAGGA-3 ' and 5 '-CCGCGGTGGCGGCCGCAAAT-3 '.The IgG gene order relatively in sequencing result and the Internet V-Base gene pool.

11. 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), utilize the XhoI/SpeI site to clone into heavy chain Fd section again, be built into the whole antibody expression vector.

12. transfection and expansion poison

Adopt the BaculoGold cotransfection test kit of 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.Baculovirus expression vector system handbook is seen in concrete operations.

13. 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, cultivate after 3～5 days for 27 ℃ and collect supernatant.Adopt the Protein-A affinity column direct purification of Amersham company to express supernatant (Harlow E, Lane D. " Antibodies:A Laboratory Manual " .Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, 1988.).With ELISA, IFA, Western Blot and flow cytometry obtain is identified the IgG antibody function characteristic of purifying.Concrete operations as above.

14. people source anti virus H 5 N 1 of bird flu antibody microneutralization functional examination

After people source anti-avian influenza virus IgG antibody purification and the filtration sterilization of yin and yang attribute control serum made serial dilution, mixed with 1: 1, put 37 ℃ of incubator effects 2 hours with the H5N1 avian influenza virus 50 μ l of 100TCID50.Select 8 holes to carry out viral residual titration, virus is made serial doubling dilution, make it to become 100TCID50,50TCID50 ... 0.7.Set up 4 positive cell contrasts of repeating hole, be about to mdck cell and 100TCID50 virus and carry out mixed culture; Set up 4 negative cell contrasts of repeating hole, be about to cell and dilution buffer liquid mixed culture.Add 100 μ l enchylema (1.5 * 10 ⁴) in the micro plate that contains virus-antibody (serum contrast) mixture and doubling dilution virus residual titration working fluid, 37 ℃ incubation 18-22 hour.Discard the enchylema in the microtest plate, PBS washes cell once, adds 100 μ l/ hole stationary liquids, in room temperature fixed cell 10 minutes, abandons stationary liquid, drying at room temperature.ELISA detects NAT: PBS flush away residual acetone, add anti-(resisiting influenza virus and albumen-NP monoclonal antibody) room temperature effect 1 hour, PBS washing culture plate 4 times, add dilution two anti-(sheep anti-mouse igg of HRP mark) room temperature effects 1 hour, wash plate 6 times, every hole adds OPD substrate 100 μ l (10mgOPD+20ml citrate buffer solution+10 μ l130% hydrogen peroxide, instant joining).Room temperature colour developing in 10 minutes, 1M sulfuric acid stops, the every hole OD of record 490nm.The result judges:

(cell positive contrasts average OD value-average OD value of cell negative control)/average OD value=X of 2+ cell negative control

X=cell half infects thresholding, when every hole OD value is lower than the X value, and judgement neutralization test reacting positive, the high dilution of neutralization reaction male antibody is NAT.

The result

1. the screening of people source anti virus H 5 N 1 of bird flu antibody library

Screen in rhabdovirus system expression reorganization hemagglutinin HA (A/Vietnam/1203/2004) antagonist storehouse with the deactivation H5N1 avian influenza virus particle (A/Anhui/1/2005) of ultracentrifugation purifying and purifying, through 3 take turns screening after 192 clones of picking at random.With anti-human Fab's antibody (sigma company, dilution use in 1: 2000), deactivation H5N1 avian influenza virus particle (A/Anhui/1/2005) antigen and reorganization hemagglutinin HA (A/Vietnam/1203/2004) bag are by 96 orifice plates, add the testing sample supernatant, marking anti-human Fab two anti-(sigma company, dilution in 1: 2000 is used) with enzyme detects.The result shows that common acquisition 45 strain people source Fab express positive colony, as shown in Figure 2.45 strain people source Fab express in the positive colony, 42 clones are arranged to the combination of H5N1 avian influenza virus specific, as shown in Figure 3A.The ELISA of anti-H5N1 avian influenza virus specific antibody in 45 Fab antibody positive clonal expression supernatants of results is detected by (Fig. 3 A) and reorganization hemagglutinin HA (A/Vietnam/1203/2004) antigen coated (Fig. 3 B) with avian influenza virus (A/Ahui/1/2005) purifying antigen bag, wherein 18 strain Fab clone and are confirmed as anti-H5N1 avian flu virus hemagglutinin protein HA (A/Vietnam/1203/2004) antibody.

2. people source anti virus H 5 N 1 of bird flu Fab antibody is to the antigenic specificity combination of bird flu

The reorganization Fab antibody that obtains in order to confirm is specific at avian influenza virus H 5 N 1, and the present invention further identifies the functionally active of prokaryotic expression Fab antibody by indirect immunofluorescence assay (IFA), Western Blot.H5N1 avian influenza virus (A/Anhui/1/2005) is made the virus antigen sheet after infecting mdck cell, utilize expression avian influenza hemagglutinin albumen HA (A/Anhui/1/2005) the recombinate shape virus infection Sf9 cell that has made up simultaneously, it is to be checked that the results cells infected is made reorganization HA antigen sheet after 4-5 days.The result conforms to fully with above-mentioned ELISA result, 42 clones and avian influenza virus (A/Anhui/1/2005) immunofluorescence are reacting positive, and wherein 18 strains are determined to be positive reaction with the antibody of reorganization HA (A/Vietnam/1203/2004) reaction with reorganization HA (A/Anhui/1/2005) immunofluorescence by ELISA; All the other 24 strains are not positive reaction at the Fab clone of reorganization HA (A/Vietnam/1203/2004) with reorganization HA (A/Anhui/1/2005) immunofluorescence.Immunofluorescence result's proof is screened the positive colony of acquisition all at avian flu virus hemagglutinin protein HA from antibody library, as shown in Figure 4, wherein: A, B, C, D are respectively positive serum, negative antibody, AVFluFab1, AVFluFab3 and reorganization HA (A/Anhui/1/05) reaction; E, F, G, H are respectively positive serum, negative antibody, AVFluFab1, AVFluFab3 and avian influenza virus (A/Anhui/1/05) reaction.

3. the sequential analysis of people source anti virus H 5 N 1 of bird flu Fab antibody

Carry out analyzing and processing with the DNASTAR sequence analysis software, compare the IgG sequence in the Internet V-Base gene pool, in the anti-highly pathogenic bird flu virus H 5 N 1 genetic engineering antibody in above-mentioned 42 strain people sources, the Fab clone that 18 strains and avian influenza virus reorganization HA (A/Vietnam/1203/2004) and reorganization HA (A/Anhui/1/2005) all react is same antibody sequence; All the other 24 strains are same antibody sequence with the Fab clone of reorganization HA (A/Anhui/1/2005) reaction, therefore find that altogether 2 strains have 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, and its variable region of light chain mainly is sorted in IgG VL2 and VL1 family.Fig. 1 is the aminoacid sequence and mutual comparison the thereof of the variable region gene of the anti-highly pathogenic bird flu virus H 5 N 1 genetic engineering antibody in 2 strain people sources, among the figure with first the row antibody sequence be the comparative standard sequence, "-" symbolic representation and the identical amino acid of the first row antibody sequence, dash area is the CDR district.

Concrete, the weight chain variable region amino acid sequence of people source anti virus H 5 N 1 of bird flu Fab antibody A VFluFab1 is seen SEQID NO.1, corresponding nucleotide sequence is seen SEQ ID NO.6; Its variable region of light chain amino sequence is seen SEQ ID NO.2, and corresponding nucleotide sequence is seen SEQ ID NO.5.The weight chain variable region amino acid sequence of AVFluFab3 is seen SEQ ID NO.3, and corresponding nucleotide sequence is seen SEQ ID NO.8; Its variable region of light chain amino sequence is seen SEQ ID NO.4, and corresponding nucleotide sequence is seen SEQ ID NO.7.

4. whole antibody IgG expresses and purifying

2 strains have been finished the light chain and the heavy Fd fragment gene of Function Identification Fab antibody (AVFluFab1, AVFluFab3), be cloned into whole antibody expression vector pAC-L-Fc transfection insect Sf9 cell respectively, utilize baculovirus/insect cell system to realize the secretion type expression of whole antibody.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, lay respectively at about 28KD, 55KD place, as shown in Figure 5.

5. people source anti virus H 5 N 1 of bird flu IgG antibody is at the antigenic specificity combination of bird flu

The reorganization IgG antibody (AVFluIgG1, AVFluIgG3) that obtains in order to confirm is specific at avian influenza virus H 5 N 1, and the present invention further identifies the functionally active of whole antibody IgG by ELISA, indirect immunofluorescence assay (IFA), Western Blot and flow cytometry.ELISA result is consistent with checking Fab antibody function activity, the whole antibody AVFluIgG1 of purifying and avian influenza virus (A/Anhui/1/2005) and reorganization HA (A/Vietnam/1203/2004) all react, and AVFluIgG3 can discern avian influenza virus (A/Anhui/1/2005), but do not react with reorganization HA (A/Vietnam/1203/2004), as shown in Figure 6.The 2 strains reorganization IgG antibody (AVFluIgG1, AVFluIgG3) that the indirect immunofluorescence assay demonstration obtains are all discerned avian influenza virus reorganization HA (A/Anhui/1/2005).Western Blotting result shows that AVFluIgG1 can react with the inactivation of viruses of sex change and the HA (A/Anhui/1/2005) that recombinates respectively, and reaction with it of AVFluIgG3.This prompting AVFluIgG1 at the antigenic determinant of HA be linear antigenic determinant, and the antigenic determinant that AVIgG3 discerned is a conformation dependent form.As shown in Figure 7.The present invention adopts fluidic cell indirect fluorescent classification (FACS) to analyze humanized IgG antibody to the combination of bird flu antigen-specific, use the Sf9 cell response of positive serum, negative antibody, AVFluIgG1, AVFluIgG3 and express recombinant HA (A/Anhui/1/2005) respectively, the result shows reorganization IgG antibody (AVFluIgG1, AVFluIgG3) but the Sf9 cell of equal specific recognition express recombinant HA (A/Anhui/1/2005).As shown in Figure 8.

6. people source anti virus H 5 N 1 of bird flu antibody microneutralization functional examination

Microneutralization test show this 2 strain reorganization IgG antibody (AVFluIgG1, AVFluIgG3) all have higher to the H5N1 avian influenza virus people source strain isolated in the Asia CHINESE REGION south and the north and neutralization widely active, the higher also wide spectrum more of the neutralization activity of AVFluIgG3 wherein, all has the neutralization activity for the isolating at present 8 strain H5N1 avian influenza virus people source strain isolateds of China, and at wherein some Local Isolates can be in nanogram level level neutralization virus, for provide effective candidate's epi-position to be significant for vaccine development.Test-results is shown in Fig. 9 and table 1.

The microneutralization test result statistics of table 1. IgG purification antibody

N: it is active that expression IgG antibody does not have neutralization

Sequence table (SEQUENCE LISTING)

＜110〉China Sickness Prevention Control Center Virus Disease Prevention Control Institute

＜120〉a kind of people source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibody

<130>JSP090056

<160>8

<170>PatentIn?version?3.1

<210>1

<211>112

<212>PRT

<213>Homo?sapiens

<400>1

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?Asn?Gly?Tyr?Tyr?Trp?Ser?Trp

20 25 30

Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Tyr?Leu?Phe

35 40 45

Asp?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro?Ser?Leu?Thr?Ser?Arg?Val?Thr

50 55 60

Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser?Ser

65 70 75 80

Val?Tbr?Ala?Ala?Asp?Tbr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?Phe?Trp

85 90 95

Gly?Leu?Asp?Gly?Phe?Asp?Ile?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val

100 105 110

<210>2

<211>107

<212>PRT

<213>Homo?sapiens

<400>2

Glu?Leu?Gln?Pro?Ala?Ser?Val?Ser?Gly?Ser?Pro?Gly?Gln?Ser?Ile?Thr

1 5 10 15

Ile?Ser?Cys?Thr?Gly?Thr?Ser?Ser?Asp?Val?Gly?Asp?Tyr?Asn?Tyr?Val

20 25 30

Ser?Trp?Tyr?Gln?Gln?His?Pro?Gly?Lys?Ala?Pro?Thr?Leu?Met?Ile?Tyr

35 40 45

Asp?Val?Asn?Lys?Arg?Pro?Ser?Gly?Asp?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?Asp?Tyr?Tyr?Cys?Ser?Ser?Tyr?Thr?Ser?Ser?Ser?Thr?Trp

85 90 95

Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu

100 105

<210>3

<211>122

<212>PRT

<313>Homo?sapiens

<400>3

Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly?Ser?Leu?Arg?Val

1 5 10 15

Ser?Cys?Thr?Asn?Ser?Gly?Phe?Thr?Phe?Ser?Asp?Tyr?Ala?Met?Ser?Trp

20 25 30

Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val?Ser?Ala?Ile?Ser

35 40 45

Gly?Asn?Gly?Gly?Ser?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg

50 55 60

Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Met?Tyr?Leu?Gln?Met

65 70 75 80

Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Val?Arg?Asp

85 90 95

Asp?Ser?Tyr?Asp?Gly?Gly?Gly?Gln?Tyr?Gly?Leu?His?Asn?Trp?Phe?Asp

100 105 110

Ser?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val

115 120

<210>4

<211>107

<212>PRT

<213>Homo?sapiens

<400>4

Glu?Leu?Thr?Gln?Pro?Pro?Ser?Val?Ser?Gly?Ala?Pro?Gly?Gln?Arg?Val

1 5 10 15

Thr?Ile?Ser?Cys?Thr?Gly?Ser?Ser?Ser?Asn?Ile?Gly?Ala?Gly?Tyr?Asp

20 25 30

Val?His?Trp?Tyr?Gln?Gln?Leu?Pro?Gly?Thr?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Tyr?Gly?Asn?Ser?Asn?Arg?Pro?Ser?Gly?Val?Pro?Asp?Arg?Phe?Ser?Gly

50 55 60

Ser?Lys?Ser?Gly?Thr?Ser?Ala?Ser?Leu?Ala?Ile?Thr?Gly?Leu?Gln?Ala

65 70 75 80

Glu?Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Gln?Ser?Tyr?Asp?Ser?Ser?Leu?Val

85 90 95

Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu

100 105

<210>5

<211>321

<212>DNA

<213>Homo?sapiens

<400>5

gagctccagc?ctgcctccgt?gtctgggtct?cctggacagt?cgatcaccat?ctcctgcact 60

gggaccagca?gtgacgttgg?tgattataac?tatgtctcct?ggtatcaaca?gcacccaggc 120

aaagccccca?cactcatgat?ttatgatgtc?aataagcggc?cctcagggga?ttctaatcgc 180

ttctctggct?ccaagtctgg?caacacggcc?tccctgacca?tctctgggct?ccaggctgag 240

gacgaggctg?attattactg?cagctcatat?acaagcagca?gcacttgggt?gttcggcgga 300

gggaccaagc?tgaccgtcct?g 321

<210>6

<211>336

<212>DNA

<213>Homo?sapiens

<400>6

ctcgagtcgg?gcccaggact?ggtgaagcct?tcggagaccc?tgtccctcac?ctgcactgtc 60

tctggtggct?ccatcaatgg?ttactactgg?agctggatcc?ggcagccccc?agggaaggga 120

ctggagtgga?ttggctatct?ctttgacagt?ggcagtacca?actacaaccc?ttcactcacg 180

agtcgagtca?ccatatcagt?ggacacgtcc?aagaaccagt?tctccctgaa?gctgagctct 240

gtgaccgctg?cggacacggc?cgtgtattac?tgtgcgagac?gattctgggg?tctcgatggt 300

tttgatatat?ggggccaagg?gacaacggtc?accgtc 336

<210>7

<211>321

<212>DNA

<213>Homo?sapiens

<400>7

gagctcacgc?agccgccctc?agtgtctggg?gccccagggc?agagggtcac?catctcctgc 60

actgggagca?gctccaacat?cggggcaggt?tatgatgtac?actggtacca?gcagcttcca 120

ggaacagccc?ccaaactcct?catctatggt?aacagcaatc?ggccctcagg?ggtccctgac 180

cgattctctg?gctccaagtc?tggcacctca?gcctccctgg?ccatcactgg?gctccaggct 240

gaggatgagg?ctgattatta?ctgccagtcc?tatgacagca?gccttgtggt?attcggcgga 300

gggaccaagc?tgaccgtcct?a 321

<210>8

<211>366

<212>DNA

<213>Homo?sapiens

<400>8

ctcgagtctg?ggggaggctt?ggtacaacct?ggggggtccc?tgagagtctc?ctgtacaaac 60

tctggattca?cctttagcga?ctatgccatg?agctgggtcc?gccaggctcc?agggaagggg 120

ctggagtggg?tctcagctat?tagtggtaat?ggtggtagta?gcacatacta?cgcagactcc 180

gtgaaggggc?ggttcaccat?ctccagagac?aattccaaga?acacgatgta?tctgcaaatg 240

aacagcctga?gagccgagga?cacggccgta?tattactgtg?tgagagatga?ctcctatgat 300

ggcggtggtc?agtacggtct?gcacaactgg?ttcgactcct?ggggccaggg?aaccctagtc 360

accgtc 366

Claims (7)

1. people source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibody, be under the jurisdiction of VH3 of heavy chain of antibody family and the VL1 of light chain of antibody family, three hypervariable region CDR1, CDR2 in its variable region of heavy chain and the aminoacid sequence of CDR3 are respectively DYAMS, AISGNGGSSTYYADSVKG and DDSYDGGGQYGLHNWFDS, and three hypervariable region CDR1, CDR2 in its variable region of light chain and the aminoacid sequence of CDR3 are respectively TGSSSNIGAGYDVH, GNSNRPS and QSYDSSLVVF.

2. people as claimed in claim 1 source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibody, it is characterized in that, this antibody is Fab antibody or whole antibody IgG, and its variable region of heavy chain has the aminoacid sequence of SEQ ID NO.3, and variable region of light chain has the aminoacid sequence of SEQ ID NO.4.

3. the gene of a people source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibody, coding claim 1 or 2 described people source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibodies.

4. gene as claimed in claim 3 is characterized in that the variable region of light chain of described gene has the nucleotide sequence of SEQ IDNO.7, and variable region of heavy chain has the nucleotide sequence of SEQ ID NO.8.

5. claim 1 or 2 described people source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibodies infect application in the medicine of bird flu the people that preparation prevention and treatment are caused by avian influenza virus H 5 N 1.

6. the gene of the described people of claim 3 source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibody infects application in the medicine of bird flu the people that preparation prevention and treatment are caused by avian influenza virus H 5 N 1.

7. application as claimed in claim 6, it is characterized in that, the variable region of light chain of the gene of described people source neutrality anti virus H 5 N 1 of bird flu genetic engineering antibody has the nucleotide sequence of SEQ ID NO.7, and variable region of heavy chain has the nucleotide sequence of SEQ ID NO.8.

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