CN104045709A - Humanized neutralizing antibody A6 against avian influenza virus (AIV) H7N9 as well as preparation method and application thereof - Google Patents
Humanized neutralizing antibody A6 against avian influenza virus (AIV) H7N9 as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a humanized neutralizing antibody A6 against the avian influenza virus (AIV) H7N9. The neutralizing antibody A6 is screened by utilizing the phage surface display technique. The amino acid sequences of light and heavy chain variable regions of the neutralizing antibody A6 are respectively shown in SEQ ID No.1 and SEQ ID No.2. The antibody can specifically recognize the antigens of H7N9 virions, can carry out obvious enzyme-linked immunosorbent assay with the virus H7N9, and has a neutralizing activity function of resisting infection with the virus H7N9. Besides, the antibody can be used for preparing specific antibody drugs for preventing and treating the AIV H7N9, and then the drugs are clinically used for preventing and treating acute respiratory infectious diseases caused by the virus H7N9.
Description
Technical field
The present invention relates to genetically engineered and phage display technique, specifically, relate to people source anti-H7N9 avian influenza virus neutrality antibody A6, its preparation method and application.
Background technology
H7N9 bird flu is the Acute respiratory infectious disease being caused by H7N9 avian influenza virus.Since in February, 2013, successively there is unknown cause severe pneumonia case in Shanghai City, Anhui Province, Jiangsu Province.Patient generally shows as influenza-like symptom, as heating, and cough, few phlegm, can be with headache, sore muscle and general malaise.Patient with severe symptoms's PD is rapid, shows as severe pneumonia, and body temperature continues mostly more than 39 ℃, occurs expiratory dyspnea, can be with spitting of blood phlegm; Can fast development occur adult respiratory distress syndrome, mediastinal emphysema, Sepsis, shock, the disturbance of consciousness and acute injury of kidney etc., mortality ratio is higher.
The Acute respiratory infectious disease being caused by H7N9 virus does not have corresponding vaccine and specific drugs at present, therefore prepares the focus that the passive immunization preparation efficient, inexpensive, side reaction is little becomes research.The people source that utilization contains specific antibody or animal serum immunoglobulin (Ig) prevent and to treat transmissible disease long-standing.In the extracorporeal antivirus effect of monoclonal antibody and active and endogenous protective body resist virus attack and obtained many experiment showed, and can be in vivo 100% watch for animals and avoid virus attack as neutralizing monoclonal antibodies such as Hantaan virus, Measles virus, RSV virus, rabies virus.
Immunoglobulin (Ig) (Immunoglobulin, Ig) as antibody component mainly from donor (convalescent) immune serum, consuming time longer to detecting by security from obtaining positive serum, and need to drop into a large amount of manpower and financial resources, this just makes its a large amount of preparations be restricted, because antibody is mainly derived from serum, therefore easily there is the pathophorous infection of haematogenous simultaneously.End user source gene engineering product Blood substitute goods can overcome these defects, along with deepening continuously of human genetically engineered antibody research, have brought new hope and bright prospects to the biological products development in this field.
The phage antibody gene pool technology (Barbas that rise the beginning of the nineties, C.F. etc., 1991) and the development in whole genetic engineering antibody technical study field, greatly promote the development research of people source or genetic engineering antibody, and by phase of basic research, stepped into substantive applied research and development phase.People's source antivirus genetic engineering antibody, the especially research of people source whole antibody success, has opened up new approaches to specificity prevention and the treatment of various viral infectious, and develop gradually the antiviral that a class is new in anti-virus infection biomedicine field.
Summary of the invention
The object of this invention is to provide a kind of people source anti-H7N9 avian influenza virus neutrality antibody A6, its preparation method and application.
In order to realize the object of the invention, a kind of people of the present invention source anti-H7N9 avian influenza virus neutrality antibody A6 or its active fragments, the aminoacid sequence of its light chain hypervariable region CDR1, CDR2 and CDR3 and heavy chain hypervariable region CDR1, CDR2 and CDR3 is as shown in table 1:
The aminoacid sequence of table 1 light chain and heavy chain hypervariable region
Aforesaid neutrality antibody A6, i) aminoacid sequence of its variable region of light chain is as shown in SEQ ID No.1, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function; Ii) aminoacid sequence of its variable region of heavy chain is as shown in SEQ ID No.2, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.
The active fragments of people source anti-H7N9 avian influenza virus neutrality antibody A6 described in the present invention refers to the Fab section of the people source anti-H7N9 avian influenza virus neutrality antibody A6 that can be combined with antigen.
The present invention also provides the gene of coding described people source anti-H7N9 avian influenza virus neutrality antibody A6.Wherein, the nucleotide sequence of encoded light chain variable region and encoding heavy chain variable region is respectively as shown in SEQ ID No.3 and SEQ ID No.4.
The present invention also provides carrier and the host cell of the gene that contains the described people source anti-H7N9 avian influenza virus neutrality antibody A6 that encodes.
The present invention also provides a kind of people of preparation source anti-H7N9 avian influenza virus neutrality antibody method, utilize phage display technique, gather a plurality of H7N9 bird flu patient decubation peripheral blood lymphocytes, by gene engineering method, built anti-H7N9 avian influenza virus genetic engineering antibody library, people source, and screening obtains the genetic engineering antibody Fab section of the anti-H7N9 avian influenza virus of specificity.
This antibody is to be determined by hypervariable region (CDRs) the specific gene sequence being present in light chain of antibody and heavy chain gene variable region, and can in prokaryotic cell prokaryocyte, obtain the functional antibodies of the specific binding H7N9 avian influenza virus of effective expression.Its specific recognition H7N9 avian influenza virus particulate antigen, have with H7N9 avian influenza virus that obvious enzyme linked immunological (ELISA) reacts and anti-H7N9 avian influenza in and active function.
The specific light chain of H7N9 avian influenza A 6 and heavy chain variable region gene derive from the rich long-pending screening of the specificity of people source anti-H7N9 avian influenza virus antibody gene pool, and the foundation of this antibody library derives from Chinese H7N9 avian influenza virus patient peripheral blood lymphocyte gene.Framework region sequence between corresponding three the CDR region sequences combination in its light chain and variable region of heavy chain and CDR district thereof has formed this antibody variable region sequence signature, and H7N9 avian influenza A 6 belongs to the VK1 of light chain of antibody family.Antibody protein function specificity nucleotide sequence and complementary sequence thereof in the decision family that is present in antibody gene light chain and variable region of heavy chain complementary district CDR1, CDR2 and CDR3 are determined, 6 corresponding CDR region amino acid sequences have formed the specific antigens calmodulin binding domain CaM of antibody, have determined that the antigen of antibody of the present invention is in conjunction with feature and anti-H7N9 avian influenza virus functional character.
In addition, consider the degeneracy of codon, for example, can not change under the condition of aminoacid sequence in its coding region, the gene order of the above-mentioned Fab section antibody of encoding is transformed, obtain the gene that coding has the antibody of identical function.Those skilled in the art can be according to the codon-bias of expressing antibody host, and synthetic modifying gene, to improve the expression efficiency of antibody.
Further, the present invention recombinates the variable region of light chain of above-mentioned Fab antibody and variable region of heavy chain, obtain the less single-chain antibody (ScFv) of molecular weight, this antibody equally can specific recognition H7N9 avian influenza virus surface antigen, has the effect of intracellular immunity.Single-chain antibody penetration power is strong, is easy to enter local organization and plays a role.With reference to Zhu, enter Wang Xin, Jiao Yongjun, Feng Zhenqing, Cao Bailiang, Guan Xiaohong. the screening of the anti-Met human genetically engineered antibody of high-affinity scFv and specificity analysis [J]. Chinese tumor biotherapy magazine, 2006,6:417-422.
Can be by the gene of above-mentioned coding Fab antibody, ScFv gene clone in expression vector, and then transform host, by abduction delivering, obtain Fab antibody and single-chain antibody.
In addition, the light chain encoding gene of above-mentioned Fab antibody and heavy Fd fragment gene can be cloned in complete anti-expression vector, and import in host cell, obtain the full anti-immunoglobulin of expressing anti-H7N9 avian influenza virus.With reference to Christoph Rader, Mikhail Popkov, John A.Neves, and Carlos F.Barbas III.Integrin α v β 3-targeted therapy for Kaposi.s sarcoma with an in vitro-evolved antibody.The FASEB Journal. (October 18,2002) 10.1096/fj.02-0281fje.
Utilize the methods such as ELISA, SDS-PAGE, neutralization experiment to carry out Function Identification to the Fab antibody (being neutrality antibody A6) obtaining, result shows that people source Fab antibody A 6 all has specific binding for H7N9 virus A/Anhui/1/2013 strain virus particle, utilize neutralization experiment to carry out Function Identification to Fab antibody, it is active that result shows that people source Fab antibody A 6 has good neutralization.
The present invention also provides the application in the medicine of disease, particularly respiratory tract disease that described neutrality antibody A6 or its active fragments cause by H7N9 avian influenza virus in preparation prevention or treatment.
The present invention further contains medicine or the detection reagent of described neutrality antibody A6 or its active fragments.
The present invention utilizes phage display technique, and successfully screening has obtained the people source neutrality antibody A6 of specificity for H7N9 avian influenza virus, utilize the anti-H7N9 avian influenza virus of the people source neutrality genetic engineering antibody variable region gene of above-mentioned acquisition, Fab antibody gene and the full-antibody gene based on this antibody gene, can be at prokaryotic cell prokaryocyte, in eukaryotic cell (comprising yeast cell) and any recombination system, express and produce this antibody or improved any other gene that contains this antibody gene based on this, during acquisition has and the antibody product of H7N9 avian influenza, make clinically for preventing and treat the disease being caused by H7N9 avian influenza virus, as the specific antibody medicine of Acute respiratory infectious disease.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE electrophorogram of H7N9 avian influenza A 6 antibody after purifying in the embodiment of the present invention 1; Wherein, 1 is antibody purification H7N9 avian influenza A 6, and M is albumen Marker.
Fig. 2 is that the ELISA of anti-H7N9 avian influenza virus people source H7N9 avian influenza A 6 antibody in the embodiment of the present invention 1 detects (A/Anhui/1/2013 strain) result; Wherein, positive control is business mouse-anti, and negative control is EV71 antibody.
Fig. 3 be in the embodiment of the present invention 1 anti-H7N9 avian influenza virus people source H7N9 avian influenza A 6 antibody in and experimental result.
Fig. 4 is the SDS-PAGE electrophorogram after H7N9 avian influenza A 6 antibody construction whole antibody IgG in the embodiment of the present invention 3; Wherein, 1 is H7N9 avian influenza A 6 whole antibody IgG, and M is albumen Marker.
Fig. 5 be in the embodiment of the present invention 3 anti-H7N9 avian influenza virus people source H7N9 avian influenza A 6 whole antibody IgG in and experimental result, negative control is EV71 antibody.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.
Embodiment 1 people source anti-H7N9 avian influenza virus neutrality antibody A6 and preparation thereof
1 materials and methods
1.1 viral, cell and carrier source: H7N9 virus A/Anhui/1/2013 strain is at document (Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z, Yang Y, Zhao X, Zhou L, Li X, Zou S, Zhang Y, Li X, Yang L, Guo J, Dong J, Li Q, Dong L, Zhu Y, Bai T, Wang S, Hao P, Yang W, Zhang Y, Han J, Yu H, Li D, Gao GF, Wu G, Wang Y, Yuan Z, Shu is infection with a novel avian-origin influenza A (H7N9) virus.N Engl J.Med.368:1888 – 1897 Y.2013.Human) in open, by Institute of Pathogen Biology, Chinese Academy of Medical Sciences, provided.Cell for the external neutralization experiment of H7N9 avian influenza virus is mdck cell, purchased from ATCC.The bacterial strain that structure phage antibody library is used is XL1-Blue (Stratagene, the U.S.), carrier pComb3H (being provided by U.S. Scripps institute).
1.2 antigen preparations
H7N9 viral purification: culture supernatant after results H7N9 virus A/Anhui/1/2013 strain infection mdck cell, after formalin-inactivated and safety inspection, with 30%-60% sucrose density gradient 35000g, 4 ℃ of centrifugal 3h purified virus particles (Beckman SW28).
The structure of 1.3 phage antibody libraries
With lymphocyte separation medium (Sigma, the U.S.) separated lymphocyte from H7N9 bird flu patient decubation anticoagulated blood, with RNeasy Mini Kit (QIAGEN, Germany) extract cell total rna, take Oligo-dT as primer, the RNA extracting is template, adopts the first chain synthetic agent box (SuperScriptTM III First-Strand Synthesis System for RT-PCR.Cat No.18080-051) reverse transcription of Invitrogen company to generate cDNA.With the primer of one group of amplification human antibody IgG1 heavy chain Fd and light chain Kappa (κ chain) and Lambda (λ chain), the primer sequence of use is as follows:
(1) heavy chain Fd district primer
3' end
CG1Z 5'-GCA TGT ACT AGT TTT GTC ACA AGA TTT GGG-3'
(2) light chain primer
κ chain variable region 5' end
VK1a 5'-GAC ATC GAG CTC ACC CAG TCT CCA-3'
VK2a 5'-GAT ATT GAG CTC ACT CAG TCT CCA-3'
VK3a 5'-GAA ATT GAG CTC ACG CAG TCT CCA-3'
κ chain variable region 3' end
CK1d 5'-GCG CCG TCT AGA ATT AAC ACT CTC CCC TGT TGA AGCTCT TTG TGA CGG GCG AAC TCA-3'
λ chain variable region 5' end
λ chain variable region 3' end
CL2 5'-CGC CGT CTA GAA TTA TGA ACA TTC TGT AGG-3'
People's endogenous light chain and heavy chain Fab gene are carried out to pcr amplification.PCR condition is: 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 2min, totally 35 circulations.Banking process is pressed document (Barbas substantially, C.F III., Kang, A.S., and larner, R.A.Assembly of combinatorial antibody libraries on phage surface:the gene III site.Proc.Natl.Acad.Sci.USA.1991; 88 (18): 7978-7982) carry out.Specific as follows:
First all different heavy chains and light chain PCR product are pressed respectively to cohort mixing separately.Learn from else's experience XbaI and SacI enzyme cut digestion, and the pComb3H carrier DNA 1.5-2 μ g after Purified in electrophoresis and light chain mixture 500-700ng, adds 2 μ l high density ligase enzymes (NEB 2000U/ μ l), adds connection damping fluid, the l6 ℃ of connection of spending the night.Add 100 μ l purified water next day, add 3M NaAc 16 μ l, add 2.5-3 times of dehydrated alcohol precipitation DNA, centrifugation, turns in competence bacteria XL1-Blue with joining electricity after the resuspended precipitation of 20 μ l pure water, and voltage 2.5kv, shocks by electricity 1 minute.After electricity turns, add immediately 2mL SOC nutrient solution, then proceed to immediately in bacteriological incubator, 37 ℃ of shaking culture 1 hour.Bacterium liquid is all applied on the LB plate containing penbritin to 37 ℃ of overnight incubation.Add 10-15mL nutrient solution next day in plate, and scraping bacterial plaque, is sub-packed in centrifuge tube, after 12000rpm is centrifugal, abandons supernatant, all with QIAGEN, carries greatly plasmid kit and extract plasmid pComb3H-L, frozen stand-by in-20 ℃ after mixing.By being cloned into pComb3H-L and the Fd chain purified pcr product of L chain, with XhoI and SpeI, carry out double digestion reaction respectively, 37 ℃ of 3-4h.Electrophoresis reclaims corresponding band, plasmid and Fd enzyme is cut to rear recovery product quantitative.Get enzyme and cut the rear carrier DNA 2 μ g that reclaim purifying of digestion, heavy chain PCR product 600ng left and right, adds 2 μ l high density ligase enzymes, adds corresponding connection damping fluid, and 16 ℃ of connections are spent the night.Add 100 μ l purified water next day, add 3M NaAc 16 μ l, add 2.5-3 times of dehydrated alcohol precipitation DNA, centrifugation, by the 20 resuspended precipitations of μ l pure water and join electricity and turn in competence bacteria XL1-Blue, voltage 2.5kv, shocks by electricity 1 minute.After electricity turns, add immediately 2mL SOC nutrient solution, then proceed to immediately in bacteriological incubator, 37 ℃ of 200rpm shaking culture 1h.Bacterium liquid is proceeded in a triangular flask, add the l0mL SB substratum containing 20 μ g/ml penbritins, 37 ℃ of 200rpm shaking culture 1 hour.Add 100ml SB nutrient solution (containing the Amp of 100 μ g/mL and the Tet of 20 μ g/mL), concussion is cultivated 1 hour.Add 10
12pfu helper phage VCSM13,37 ℃ of static infection 20min, then cultivate 2h to add final concentration are the Kan of 70 μ g/ml for 37 ℃, 37 ℃ of overnight incubation.Treat OD
600be about at 1 o'clock, by 4 ℃ of centrifugal 15min of 4000rpm of bacterium liquid, shift supernatant to aseptic triangular flask, add 4% (w/v) PEG8000 and 3% (w/v) NaCl, after dissolving completely, ice bath 30min is above with precipitation phage.4 ℃ of centrifugal 20-30min of 9000rpm, abandon supernatant and will precipitate with 2mL PBS resuspendedly, instantaneous centrifugal, and supernatant is Fab phage antibody library.
The enrichment screening of 1.4 phage antibody libraries and the abduction delivering of Fab section antibody
The inactivated virus particle A/Anhui/1/2013 strain of ultracentrifugation purifying of take is screening antigen.During use, use 0.1M NaHCO
3(pH8.6) solution dilution, coated immunity pipe, with the PBS liquid containing 4% skimmed milk, after 37 ℃ of sealing 2h, add above-mentioned phage antibody library, every pipe 1mL, hatch 2h for 37 ℃, with the TBS liquid containing 5%Tween-20, repeatedly wash 20 times, glycine-hydrochloric acid elutriant wash-out of last every effective 1mLpH2.2, and neutralize with the Tris liquid of pH9.6.Phage after wash-out continues to infect the fresh OD of 2mL
600be about 1 XL1-Blu bacterium, after helper phage VCSM13 (Stratagene, the U.S.) infects, carry out next round screening.So repeated screening is 3～4 times.The abduction delivering of concrete enrichment screening method and Fab section is pressed document (Barbas substantially, C.F III., Kang, A.S., and larner, R.A.Assembly of combinatorial antibody libraries on phage surface:the gene III site.Proc.Natl.Acad.Sci.USA.1991; 88 (18): 7978-7982) carry out.
The enrichment of phage antibody library: use 0.1M NaHCO
3(pH8.6) solution is pressed 1:100 dilution coated 96 orifice plates respectively by the A/Anhui/1/2013 strain of purifying, and 4 ℃ are spent the night.Inferior daily PBS-T (20mM PBS adds 0.05% tween 20) washes away the not antigen of absorption, the 37 ℃ of sealing 1h of skimmed milk with 3%.Abandon confining liquid, every hole adds 60 μ l phage antibody libraries, hatches 2h for 37 ℃.Discard unconjugated phage in hole, and use TBS-T (50mM Tris-HCl, 150mM NaCl, 0.5% tween 20, pH7.5) washing lotion is rinsed each hole, during flushing, with pipettor, repeatedly blows and beats, and washes altogether 20 times, fully to wash away the not phage of absorption.Finally use ddH
2o washes twice, remaining liq in exhaustion hole.Every hole adds the elutriant of 50 μ l glycine-HCl (pH2.2), and incubated at room 10min, in this process, blows and beats (noting not blowing out bubble) repeatedly with suction pipette head.Elutriant is concentrated on to a centrifuge tube, according to the ratio of every hole 3 μ l, adds 2M Tris, make pH be about 7, with in and wash-out under phage.The phage of wash-out is added to (OD in the XL1-Blue bacterium liquid that 2mL is fresh immediately
600=1), incubated at room 20min.Proceed in a 250mL triangular flask, add 10mL SB (containing penbritin 20 μ g/ml and tsiklomitsin 20 μ g/mL), get immediately 10 μ l and be applied to ammonia benzyl plate, for titration phage.37 ℃ of shaking culture 1h of all the other liquid.Add 100mL SB (containing penbritin 100 μ g/ml and tsiklomitsin 20 μ g/mL), 37 ℃ of shaking culture 2h.Afterwards, add helper phage VCSM13 (9 * 10
12pfu/mL) 1mL, 37 ℃ of static 20min, 37 ℃ of shaking culture 2h.Add kantlex (final concentration 70 μ g/mL), 37 ℃ of shaking culture are spent the night.Treat that bacterium grows to OD
600be about at 1 o'clock, by 4 ℃ of centrifugal 15min of 6500rpm of bacterium liquid, shift supernatant to aseptic triangular flask, add 4% (w/v) PEG8000 and 3% (w/v) NaCl, after dissolving completely, ice bath 30min is above fully to precipitate phage.4 ℃ of centrifugal 20-30min of 9000rpm, abandon supernatant.To precipitate and use 2mL PBS resuspended, instantaneous centrifugal, supernatant is first round enrichment screening Fab phage antibody library.
The expression of Fab positive colony: 1600 of the random chooses single bacterium colony after 3 enrichments screening in 96 deep-well plates, every hole 800 μ l substratum, 37 ℃ of overnight incubation.Next day, the ratio with 1:20 was forwarded in 96 orifice plates that contain 800 μ l SB substratum (containing Amp 100 μ g/mL), and 37 ℃ are cultured to OD
600during=0.2-0.3, adding final concentration is the IPTG of 1mM, 30 ℃ of abduction delivering 8-10h.4 ℃ of centrifugal 15min of 4000rpm, supernatant for detection of.
The ELISA of 1.5 people source anti-H7N9 virus Fab antibody detects
(1) detect the expression of Fab
Use 0.1M NaHCO
3(pH9.6) solution is coated in anti-human Fab antibody (using Sigma, the U.S. after 1:2000 dilution) on enzyme plate, and 4 ℃ are spent the night; 4% skimmed milk sealing, 37 ℃ of 1h, add the Fab antibody of expression, 37 ℃ of 1h; Add the anti-human Fab bis-of enzyme mark anti-(using Sigma, the U.S. after 1:2000 dilution), 37 ℃ of 1h; Nitrite ion colour developing, 2M H
2sO
4termination reaction, microplate reader detects absorbance A value.
(2) Dot-ELISA detects Fab and H7N9 virus combination activity
With the deactivation H7N9 virion of purifying, as envelope antigen, all the other steps are the same.
The nucleic acid sequence analysis of 1.6 people source Fab antibody variable genes
With Qiagen Miniprep Kit (QIAGEN, Germany), prepare plasmid DNA and carry out nucleic acid sequence analysis.The sequencing primer of weight chain is respectively 5 ＇-AAACTAGCTAGTCGCCAAGGA-3 ＇ and 5 ＇-CCGCGGTGGCGGCCGCAAAT-3 ＇.IgG gene order in sequencing result and Internet V-Base gene pool is compared.
The purifying of 1.7 people source anti-H7N9 virus Fab antibody
With the antibody (Sigma, the U.S.) of anti-human Fab, prepare 2mL affinity column, the bacterium liquid containing Fab antibody filtering is added in affinity column to iterative cycles 30min to 1h.The pre-wash buffer of PBS that adds pH value 6.8, washes away non-specific adsorption albumen.The Fab antibody protein that adds the glycine hydrochloride buffer solution elution combination of pH value 2.7, adds the Tris-HCl damping fluid of pH value 9.0 to neutralize the solution eluting, and then uses evaporating column centrifugal concentrating.The Fab Fragment Protein 10-20 μ g getting after purifying concentrates carries out SDS-PAGE electrophoresis.
The neutralization experiment of 1.8 people source anti-H7N9 virus Fab antibody detects
(1) experimental procedure
The antibody 50 μ l of 100 TCID50 H7N9 avian influenza A/Anhui/1/2013 strain virus 50 μ l and doubling dilution are hatched 2 hours jointly in 37 ℃, and then adding concentration is 2 * 10
5the cell culture fluid 100 μ l of individual mdck cell/mL.Observe 4 days, record cytopathy result.
(2) result is judged
Using and can protect pathology does not occur 50% cell the inverse of high dilution as the titre of neutralizing antibody.
2 results
The screening in 2.1 anti-H7N9 antiviral antibody storehouse, people sources
With the H7N9 virion A/Anhui/1/2013 strain of purifying, phage antibody library is carried out to enrichment screening, 3 take turns rear random 1600 clones of picking of screening.With anti-human Fab antibody (using Sigma, the U.S. after 1:2000 dilution) and antigen coated 96 orifice plates of H7N9 avian influenza virus particle A/Anhui/1/2013 strain, add testing sample supernatant, with the anti-human Fab bis-of enzyme mark anti-(using Sigma, the U.S. after 1:2000 dilution), detect.Result shows that obtaining altogether 1146 people source Fab expresses positive colony, and wherein, 935 clones can specific binding H7N9 avian influenza virus particle A/Anhui/1/2013 strain (table 2).
Table 2 A/Anhui/1/2013 strain is to phage antibody library enrichment the selection result
The sequential analysis of 2.2 people source anti-H7N9 avian influenza virus Fab antibody
With DNASTAR sequence analysis software, Fab fragment is carried out to analyzing and processing, compare the IgG sequence in Internet V-Base gene pool, in the Fab monoclonal antibody of the people source of above-mentioned 935 specific binding H7N9 avian influenza virus, there is the sequence of 23 Fab fragments different.Therefore the present invention successfully screens and clones 23 with different antibody weight chain variable region sequences and the antibody of combination thereof.Wherein, people source anti-H7N9 avian influenza virus neutrality antibody A6 belongs to the VK1 of light chain of antibody family, the aminoacid sequence of its variable region of light chain and variable region of heavy chain is respectively as shown in SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequence of encoded light chain variable region and encoding heavy chain variable region is respectively as shown in SEQ ID No.3 and SEQ ID No.4.
The purifying of 2.3 people source anti-H7N9 avian influenza virus Fab antibody
Antibody (Sigma with anti-human Fab, the U.S.) prepare 2mL affinity column, purifying Fab antibody expression supernatant, checks the Expression and purification situation of Fab antibody by SDS-PAGE, result confirms to obtain compared with pure protein, can clearly observe light chain of antibody and Fd chain (Fig. 1) after unwinding.
The ELISA of 2.4 people source anti-H7N9 avian influenza virus Fab antibody detects
Use respectively the deactivation H7N9 avian influenza virus particle A/Anhui/1/2013 strain of purifying as envelope antigen, the combination that detects antibody is active, and result as shown in Figure 2.
The ELISA of antigen-binding activity detects: use 0.1M NaHCO
3(pH9.6) the deactivation H7N9 avian influenza virus particle A/Anhui/1/2013 strain of the coated purifying of solution, 4 ℃ are spent the night, by PBS-T washing lotion, wash plate 3 times, fully remove liquid and add 5% skimmed milk 100 μ l, 37 ℃ of incubation 1h, PBS-T washing lotion is washed plate 3 times, fully remove liquid, the antibody supernatant 50 μ l that add prokaryotic expression, then add 5%PBS-skimmed milk 50 μ l slightly to rock and mix, 37 ℃ of incubation 1h in every hole.PBS-T washing lotion is washed plate 6 times, fully removes liquid, adds the anti-human Fab antibody of enzyme mark (Sigma company, 1:2000 dilutes use) 100 μ l, 37 ℃ of incubation 1h, PBS-T washing lotion is washed plate 6 times, fully remove liquid, add nitrite ion A, B color development at room temperature 10min, 2M H
2sO
4termination reaction, OD
450reading.
The neutralization experiment of 2.5 people source anti-H7N9 avian influenza virus Fab antibody
With H7N9 avian influenza virus A/Anhui/1/2013 strain, as attacking poison strain, the neutralization that detects antibody in mdck cell is active.Result is (ordinate zou is sample extension rate) as shown in Figure 3.
Experimental procedure: as attacking poison strain, the neutralization that detects antibody in mdck cell is active with H7N9 avian influenza virus A/Anhui/1/2013 strain.
The antibody A 6 of the 100TCID50H7N9 avian influenza virus A/Anhui/1/2013 strain of 50 μ l and 50 μ l doubling dilutions is added to 2 * 10 in 37 ℃ after jointly hatching 2 hours
5in the cell suspension 100 μ l of individual cell/mL.Observe 4 days, record cytopathy result.
Wherein, the H7N9 avian influenza virus using (A/Anhui/1/2013 strain) is through processing as follows: 1:100 dilution, then do serial half-log, and make it into 10
-2, 10
-2.5, 10
-3, 10
-3.510
-7.Every hole is containing 100 μ l virus liquids, and each extent of dilution repeats 4 holes, respectively adds in cell plate, every hole 100 μ l, and each extent of dilution adds 4 porocytes; Every hole adds cell suspension 100 μ l, cultivates 4d, observation of cell pathology for 35 ℃; When cytopathy stops developing, according to Reed-Muench Liang Shi method, virus titer is calculated, calculate viral TCID50/100 μ l.
The application of embodiment 2 people source anti-H7N9 avian influenza virus neutrality antibody A6
The Acute respiratory infectious disease that prevention at present and treatment are caused by H7N9 virus there is no specific vaccine and medicine, the immunoglobulin (Ig) that used clinically often derives from and infects viral patient's convalescent phase serum, this just makes its a large amount of preparations be restricted, so easily there is the pathophorous infection of haematogenous owing to deriving from serum simultaneously.Adopt the people source anti-H7N9 avian influenza virus genetic engineering antibody A6 that the present invention obtains to substitute haematogenous immunoglobulin (Ig), for the treatment of the Acute respiratory infectious disease that caused by H7N9 virus provide newly by way of.
Embodiment 3 utilizes neutrality antibody A6 to prepare the method for whole antibody immunoglobulin IgG
1, the expression of whole antibody IgG and purifying
1. the structure of whole antibody recombinant expression plasmid: first use primer (upstream primer 5'-CCCAAGCTTGTTGCTCTGGATCTCTGGTGCCTACGGGGACATCCAGATGACCC AGTCTCCATCCTCC-3', downstream primer 5'-CTAGTCTAGAATTAACACTCTCCCCTG-3') amplification obtains the light chain section of Fab antibody, with XbaI/HindIII enzyme, cut, be cloned into PIgG carrier (U.S. Scripps institute provides) (Christoph Rader, Mikhail Popkov, John A.Neves, and Carlos F.Barbas III.Integrin α v β 3-targeted therapy for Kaposi.s sarcoma with an in vitro-evolved antibody.The FASEB Journal. (October18, 2002) 10.1096/fj.02-0281fje.), obtain carrier PIgG-L.Use again primer (upstream primer 5'-GAGGAGCTCACTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGAC-3', downstream primer 5'-GAGGGGCCCTTGGTGGAGGCTGAGGAGACGGT-3') the heavy chain section of amplification Fab antibody, utilize SacI/ApaI enzyme cutting clone to enter carrier PIgG-L, be built into intact antibody carrier.
2. transfection: adopt the transfection reagent box purchased from American I nvitrogen company, working method outlines as follows: after 5 μ g recombinant plasmid dnas are mixed with transfection reagent, the 293T cell that transfection stand density is 70%, 37 ℃ of 5% CO
2cultivate.
3. whole antibody IgG purifying: collect supernatant after cultivating 3d, employing is expressed supernatant (Harlow E purchased from the Protein-A affinity column direct purification of Amersham company, Lane D.Antibodies:A Laboratory Manual[M] .New York:Cold Spring Harbor Laboratory Press, 1988).Adopt ELISA and IFA to identify the functional performance of the IgG purification antibody obtaining.
Fig. 4 is for building the SDS-PAGE electrophoresis result of the whole antibody IgG obtaining.
2, blood clotting suppresses experiment
Select avian influenza virus H7N9 to represent that strain A/Anhui/1/2013 carries out hemagglutination-inhibition test, analyze the blood clotting restraining effect of whole antibody IgG to strain.
(1) experimental procedure
Monoclonal antibody to be measured is carried out to doubling dilution, and every hole retains 25 μ l, and then every hole adds 25 μ l virus liquids (antigens of 4 aggegation units); Mix incubated at room 30min, then every hole adds 0.5% Ho RBC suspension 50 μ l, mixes incubated at room 30min, observes hemagglutination inhibition test (HI test) result.
(2) result is judged
After specific antibodies be combined with corresponding hemagglutinin antigen, can suppress the hemagglutination that virus causes, red cell agglutination suppress the to tire inverse of the high dilution that refers to serum when inhibition red cell agglutination occurs.Result shows, it is 0.8 μ g/ml that whole antibody IgG suppresses required minimum antibody amount to A/Anhui/1/2013 strain generation blood clotting.
Table 3 A6 whole antibody IgG suppresses experiment to A/Anhui/1/2013 strain blood clotting
3, in whole antibody IgG and experiment
Active in order further to verify the neutralization of whole antibody IgG, with H7N9 avian influenza virus A/Anhui/1/2013 strain, as attacking poison strain, the neutralization that detects antibody in mdck cell is active.
(1) experimental procedure
The whole antibody IgG 50 μ l of 100 TCID50 H7N9 avian influenza A/Anhui/1/2013 strain virus 50 μ l and doubling dilution are hatched 2 hours altogether in 37 ℃, and then adding concentration is 2 * 10
5the cell culture fluid 100 μ l of individual mdck cell/mL.Observe 4 days, record cytopathy result.
(2) result is judged
Using and can protect pathology does not occur 50% cell the inverse of high dilution as the titre of neutralizing antibody.Result shows, whole antibody IgG, to A/Anhui/1/2013 strain, neutralization occurs, and to suppress required minimum antibody amount be 0.04 μ g/ml.(Fig. 5)
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the anti-H7N9 avian influenza virus in people source neutrality antibody A6 or its active fragments, is characterized in that, the aminoacid sequence of its light chain and heavy chain hypervariable region CDR1, CDR2 and CDR3 is as shown in the table:
2. neutrality antibody A6 according to claim 1, is characterized in that,
I) aminoacid sequence of its variable region of light chain is as shown in SEQ ID No.1, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function; And
Ii) aminoacid sequence of its variable region of heavy chain is as shown in SEQ ID No.2, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.
3. the gene of neutrality antibody A6 described in the claim 2 of encoding.
4. gene according to claim 3, is characterized in that, the nucleotide sequence of encoded light chain variable region and variable region of heavy chain is respectively as shown in SEQ ID No.3 and SEQ ID No.4.
5. the carrier that contains gene described in claim 3 or 4.
6. the host cell that contains gene described in claim 3 or 4.
7. described in claim 1, neutrality antibody A6 or its active fragments obtain through transformation single-chain antibody ScFv or whole antibody immunoglobulin IgG.
8. prepare people source anti-H7N9 avian influenza virus neutrality antibody method for one kind, it is characterized in that, utilize phage display technique, gather H7N9 bird flu patient decubation peripheral blood lymphocyte, by gene engineering method, built anti-H7N9 avian influenza virus genetic engineering antibody library, people source, and screening obtains the genetic engineering antibody Fab section of the anti-H7N9 avian influenza virus of specificity.
9. the application in the medicine of the disease that described in claim 1 or 2, neutrality antibody A6 or its active fragments are caused by H7N9 avian influenza virus in preparation prevention or treatment.
10. the medicine or the detection reagent that contain neutrality antibody A6 described in claim 1 or 2 or its active fragments.
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| CN104312979A (en) * | 2014-09-30 | 2015-01-28 | 江苏省农业科学院 | Anti-H7 subtype avian influenza virus monoclonal antibody and application thereof |
| CN105985433A (en) * | 2015-02-09 | 2016-10-05 | 复旦大学 | Fully human monoclonal antibody by aiming at H7N9 subtype avian influenza virus and application thereof |
| CN107353340A (en) * | 2016-05-10 | 2017-11-17 | 深圳先进技术研究院 | The full human monoclonal antibody 2L11 of anti-H7N9 and its preparation method and application |
| CN107759688A (en) * | 2017-09-27 | 2018-03-06 | 中国医学科学院病原生物学研究所 | Anti- H7N9 avian influenza virus neutralizing antibody S28H and its application |
| WO2018086599A1 (en) * | 2016-11-11 | 2018-05-17 | 深圳先进技术研究院 | Anti-h7n9 fully-human monoclonal antibody 5j13, preparation method therefor, and application thereof |
| WO2020019222A3 (en) * | 2018-07-24 | 2020-03-19 | 深圳先进技术研究院 | Anti-h7n9 fully human monoclonal antibody hig311, preparation method therefor, and application |
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| CN107759688A (en) * | 2017-09-27 | 2018-03-06 | 中国医学科学院病原生物学研究所 | Anti- H7N9 avian influenza virus neutralizing antibody S28H and its application |
| WO2020019222A3 (en) * | 2018-07-24 | 2020-03-19 | 深圳先进技术研究院 | Anti-h7n9 fully human monoclonal antibody hig311, preparation method therefor, and application |
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