CN102718864B - Human anti-EV71 (enterovirus 71) neutralizing antibody EV71FabK7 and preparation method and application thereof - Google Patents

Abstract

The invention provides human anti-EV71 (enterovirus 71) neutralizing antibody EV71FabK7 which is obtained by screening by means of phage display. Amino acid sequences of light chain and heavy chain variable areas are shown as SEQ ID No.1 and SEQ ID No.2 respectively. The antibody can specifically recognize EV71 virus particle antigens, can have evident enzyme-linked immune reaction with EV71 viruses, and has a function of neutralizing activity resistant to EV71 virus infection. Further, the antibody can be made into specific antibody medicine for preventing and treating the hand-foot-mouth disease and clinically used for preventing and treating the hand-foot-mouth disease caused by EV71 viruses.

Description

People source anti-EV71 virus neutrality antibody EV71FabK7, its preparation method and application

Technical field

The present invention relates to the genetic engineering antibody technology, specifically, relate to a kind of people source anti-EV71 virus neutrality antibody EV71FabK7, its preparation method and application.

Background technology

Hand foot mouth disease (Hand foot mouth disease, HFMD) be the transmissible disease caused by enterovirus, the multiple infant who is born in below 5 years old, can cause fash, the ulcer at the positions such as heating and hand, foot, oral cavity, indivedual patients can cause the complication such as myocarditis, pulmonary edema, AME.The enterovirus that causes hand foot mouth disease has kind more than 20, and wherein Coxsackie virus (Cox Asckievirus) A16 type (Cox A16) and enterovirns type 71 (Enterovirus71.EV71) are the most common.Be separated to EV71 in the patient body that shows as neurological symptom disease (1969 ~ 1973 years) that people's reported first such as Schmidt in 1974 are broken out from California, USA, subsequently, many countries have reported the popularity of EV71 virus in different areas in succession in the world, and people recognize that EV71 virus is the main pathogen of hand foot mouth disease gradually.

Because hand foot mouth disease does not have corresponding vaccine and specific drugs at present, therefore prepare the focus that the passive immunization preparation efficient, inexpensive, that 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 human body resist virus attack and obtained many experiment showed, and can be in vivo 100% watch for animals and avoid virus attack as neutralizing monoclonal antibodies such as 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 the pathophorous infection of haematogenous easily occurs 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, opened up new approaches to specificity prevention and the treatment of various viral infectious, and develop gradually the antiviral drug that a class is new in the anti-virus infection biomedicine field.

Summary of the invention

The purpose of this invention is to provide the anti-EV71 virus neutrality antibody EV71FabK7 in a kind of people source or its active fragments.

Another object of the present invention is to provide the preparation method of described antibody EV71FabK7 or its active fragments.

A further object of the present invention is to provide the application of described antibody EV71FabK7 or its active fragments.

In order to realize the object of the invention, the anti-EV71 virus neutrality antibody EV71FabK7 in a kind of people of the present invention source 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 heavy chain of table 1 antibody EV71FabK7 and the aminoacid sequence of light chain hypervariable region

	CDR1	CDR2	CDR3
				Heavy chain VH	SGYYWG	SIYHSGSTYYNPSLKS	DSSYNGFDP
Light chain VL	RASQSISSSFLA	SASSRAT	QQYATSLRT

Aforesaid antibody EV71FabK7, 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.For example, the Arg of the 16th is replaced with to Lys and can not affect the function of albumen.

Aforesaid antibody EV71FabK7, ii) the aminoacid sequence of its variable region of heavy 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.For example, the Val of the 8th is replaced with to Ala and can not affect the function of albumen.

The present invention also provides the gene of encoding said antibody EV71FabK7.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 host cell that the present invention also provides the carrier of the gene that contains encoding said antibody EV71FabK7 and contains this carrier.

Single-chain antibody ScFv or whole antibody immunoglobulin IgG that the present invention also provides described antibody EV71FabK7 or its active fragments to obtain through transformation.

The present invention also provides the preparation method of described antibody EV71FabK7 or its active fragments, utilize phage display technique, gather a plurality of patient's EV71 decubation peripheral blood lymphocytes, built anti-EV71 virus gene engineering antibody library, people source by gene engineering method, and screening obtains the genetic engineering antibody Fab section of the anti-EV71 virus of specificity.

This antibody determines by being present in light chain of antibody and Zhong hypervariable region, heavy chain gene variable region (CDRs) specific gene sequence, and can in prokaryotic cell prokaryocyte, obtain the functional antibodies of the specific binding EV71 virus of effective expression.Its specific recognition EV71 virion antigen, have with EV71 virus that obvious enzyme linked immunological (ELISA) reacts and anti-EV71 virus infection in and active function.

The specific light chain of EV71FabK7 and heavy chain variable region gene derive from the rich long-pending screening of the specificity of people source anti-EV71 antiviral antibody gene pool, and the foundation of this antibody library derives from Chinese EV71 virus disease human peripheral lymphocyte gene.Framework region sequence between corresponding three the CDR region sequences combination of its light chain and variable region of heavy chain and CDR district thereof has formed this antibody variable region sequence signature, and EV71FabK7 belongs to the VL1 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-EV71 viral function feature.

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 transformed in its coding region, 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 the antibody host, and the synthetic modifying gene, to improve the expression efficiency of antibody.

Further, the present invention is recombinated the variable region of light chain of above-mentioned Fab antibody and variable region of heavy chain, obtains the less single-chain antibody (ScFv) of molecular weight, and this antibody equally can specific recognition EV71 viral surface antigen, has the effect of intracellular immunity.The single-chain antibody penetration power is strong, is easy to enter local organization and plays a role.

Can be by the gene of above-mentioned coding Fab antibody, ScFv gene clone in expression vector, and then transform the host, obtain Fab antibody and single-chain antibody by abduction delivering.

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-EV71 virus.

Utilize the methods such as ELISA, SDS-PAGE, neutralization experiment to carry out Function Identification to the Fab antibody obtained, result shows that people source Fab antibody EV71FabK7 all has specific binding for SHZH98 strain and FUYANG-0805 strain EV71 virion, utilize the neutralization experiment to carry out Function Identification to Fab antibody, result shows that EV71FabK7 has neutralization activity preferably.

The present invention also provides described antibody EV71FabK7 or the application of its active fragments in the medicine of preparation prevention or treatment hand foot mouth disease.

The present invention further contains medicine or the detection reagent of described antibody EV71FabK7 or its active fragments.

The present invention utilizes phage antibody library technique, has successfully obtained the people source neutrality antibody EV71FabK7 of specificity for EV71 virus; Utilize the full-antibody gene under the anti-EV71 virus gene engineering antibody of people source neutrality variable region gene, Fab antibody gene and this antibody gene feature of above-mentioned acquisition, can express and produce any other gene that contains this antibody gene after this antibody or reconstruction based on this in prokaryotic cell prokaryocyte, yeast cell, eukaryotic cell and any recombination system, during acquisition has and the antibody product of EV71 virus infection, make clinically the specific antibody medicine for prevention and treatment hand foot mouth disease.

The accompanying drawing explanation

The SDS-PAGE electrophorogram that Fig. 1 is EV71FabK7 antibody after purifying; Wherein, 1 is antibody purification EV71FabK7.M is albumen Marker.

The ELISA that Fig. 2 is anti-EV71 virus people source EV71FabK7 antibody detects (SHZH98 strain) result, and wherein positive control is the business mouse-anti, and negative control is COxsackie A4 antiviral antibody.

The ELISA that Fig. 3 is anti-EV71 virus people source EV71FabK7 antibody detects (FUYANG-0805 strain) result, and wherein positive control is the business mouse-anti, and negative control is COxsackie A4 antiviral antibody.

Embodiment

Following examples are used for the present invention is described, 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.

The structure in embodiment 1 anti-EV71 antiviral antibody storehouse, people source and the screening of Fab antibody

1.1 materials and methods

1.1.1 viral, cell and carrier source: EV71 virus SHZH98 strain and FUYANG-0805 strain are at document (Yang F, Jin Q, He Y, Li L, Hou is complete genome of Enterovirus 71 China strain.Sci China C Life Sci and Wu Z Y.2001.The, Yang F, Zhao R, Zhao L, Guo D, Jin is of small interfering RNAs which inhibit the replication of several Enterovirus 71 strains in China.J Virol Methods Q.2009.Identification) in open, by Institute of Pathogen Biology, Chinese Academy of Medical Sciences, provided.The cell that neutralizes experiment for the EV71 virosome outward is the RD cell, purchased from ATCC.Bacterial strain is XL1-Blue(Stratagene, the U.S.), carrier pComb3H (being provided by U.S. Scripps institute).

1.1.2 antigen preparation

EV71 viral purification: gather in the crops respectively the SHZH98 strain of EV71 virus and FUYANG-0805 strain and infect culture supernatant after the RD cell, after formalin-inactivated and safety inspection, with 20% sucrose density gradient 35000g, 4 ℃ of centrifugal 3h purified virus particles (Beckman SW28).

1.1.3 the structure of phage antibody library

With lymphocyte separation medium (Sigma, the U.S.) separate lymphocyte from patient's EV71 decubation anticoagulated blood, with RNeasy Mini Kit (QIAGEN, Germany) extract total cell RNA, take Oligo-dT as primer, and the RNA of extraction is that masterplate 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 and Lambda, the primer sequence of use is as follows:

(1) heavy chain Fd district primer

The 5' end

VH1a 5'-CAG GTG CAG CTC GAG CAG TCT GGG-3'

VH1f 5'-CAG GTG CAG CTG CTC GAG TCT GGG-3'

VH2f 5'-CAG GTG CAG CTA CTC GAG TCG GG-3'

VH3a 5'-GAG GTG CAG CTC GAG GAG TCT GGG-3'

VH3f 5'-GAG GTG CAG CTG CTC GAG TCT GGG-3'

VH4f 5'-CAG GTG CAG CTG CTC GAG TCG GG-3'

VH6f 5'-CAG GTG CAG CTA CTA GAG TGG GG-3'

VH6a 5'-CAG GTA CAG CTC GAG CAG TCA GG-3'

The 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 AGC TCT TTG TGA CGG GCG AAC TCA-3'

λ chain variable region 5-end

VL1 5'-AAT TTT GAG CTC ACT CAG CCC CAC-3'

VL2 5'-TCT GCC GAG CTC CAG CCT GCC TCC GTG-3'

VL3 5'-TCT GTG GAG CTC CAG CCG CCC TCA GTG-3'

VL4 5'-TCT GAA GAG CTC CAG GAC CCT GTT GTG TCT GTG-3'

VL5 5'-CAG TCT GAG CTC ACG CAG CCG CCC-3'

VL6 5'-CAG ACT GAG CTC ACT CAG GAG CCC-3'

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.The PCR condition is: 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 2min, totally 35 circulations.Banking process is pressed document (Barbas substantially; the 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:

At 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 (NEB2000U/ μ l), adds the 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, turn in competence bacteria XL1-Blue with after the resuspended precipitation of 20 μ l pure water, joining electricity, and voltage 2.5kv, shock by electricity 1 minute.Add immediately 2ml SOC nutrient solution after electricity turns, then proceed to immediately in bacteriological incubator, 37 ℃ of shaking culture 1 hour.Bacterium liquid all is applied on the LB plate containing penbritin to 37 ℃ of overnight incubation.Add the 10-15ml nutrient solution next day in plate, and the scraping bacterial plaque, be sub-packed in centrifuge tube, after 12000rpm is centrifugal, abandons supernatant, all with the large upgrading grain of QIAGEN test kit, extracts 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, carry out the double digestion reaction with XhoI and SpeI 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, add 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, shock by electricity 1 minute.Add immediately 2ml SOC nutrient solution after electricity turns, 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-A+ 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 1012pfu helper phage VCSM13,37 ℃ of static infection 20min, then 37 ℃ of cultivation 2h add the Kan that final concentration is 70 μ g/ml, 37 ℃ of overnight incubation.Be about at 1 o'clock until OD600, 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 fully, ice bath 30min is above with the precipitation phage.4 ℃ of centrifugal 20-30min of 9000rpm, abandon supernatant and will precipitate with 2ml PBS resuspendedly, instantaneous centrifugal, and supernatant is the Fab phage antibody library.

1.1.4 the abduction delivering of the enrichment of phage antibody library screening and Fab section antibody

Take the inactivated virus particle SHZH98 of ultracentrifugation purifying and FUYANG-0805 strain is screening antigen.Use 0.1M NaHCO during use 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, the glycine of last every effective 1ml pH2.2-hydrochloric acid elutriant wash-out, and neutralize with the Tris liquid of pH9.6.Phage after wash-out continues to infect the fresh OD of 2ml ₆₀₀be the XL1-Blu bacterium of 1.0 left and right, after helper phage VCSM13 (Stratagene, the U.S.) infects, carry out the 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; the 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 SHZH98 strain of purifying, and 4 ℃ are spent the night.Inferior daily PBS-T(20mM PBS adds 0.05%Tween-20) wash away not the antigen of absorption, 37 ℃ of skimmed milks with 3% seal 1h.Abandon confining liquid, every hole adds 60 μ l phage antibody libraries, hatches 2h for 37 ℃.Discarding unconjugated phage in hole, use TBS-T(50mM Tris-HCl, 150mM NaCl, 0.5%Tween-20, pH7.5) washing lotion rinses each hole, during flushing, with pipettor, repeatedly blows and beats, wash altogether 20 times, fully to wash away the not phage of absorption.Finally use ddH ₂o washes twice, remaining liq in the exhaustion hole.Every hole adds 50 μ l Glycine-HCl(pH2.2) elutriant, incubated at room 10min, in this process, blow and beat (note do not blow 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 phage under wash-out.The phage of wash-out is added in the XL1-Blue bacterium liquid that 2ml is fresh (OD600=1) immediately to 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 the 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.Grow to OD600 until bacterium and be 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 fully, 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: 2000 of the random chooses single bacterium colonies after 3 enrichments screening in 96 deep-well plates, every hole 800 μ l substratum, 37 ℃ of overnight incubation.Be forwarded to the ratio of 1:20 next day in 96 orifice plates that contain 800 μ l SB substratum (containing Amp 100 μ g/ml), 37 ℃ while being cultured to OD600=0.2-0.3, add the IPTG that final concentration is 1mM, 30 ℃ of abduction delivering 8-10hr.4 ℃ of centrifugal 15min of 4000rpm, supernatant for detection of.

1.1.5 the ELISA of people source anti-EV71 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 dilutions) 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 dilutions), 37 ℃ of 1h; The nitrite ion colour developing, 2M H ₂sO ₄termination reaction, microplate reader detects the absorbance A value.

(2) Dot-ELISA detects Fab and EV71 virus combination activity

With the deactivation EV71 virion of purifying, as envelope antigen, all the other steps are the same.

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

Prepare plasmid DNA with Qiagen Miniprep Kit (QIAGEN, Germany) 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.

1.1.7 the purifying of people source anti-EV71 virus Fab antibody

Prepare the 2ml affinity column with the antibody (Sigma, the U.S.) of anti-human Fab, the bacterium liquid containing Fab antibody filtered is added in affinity column to iterative cycles 30min to 1h.Add the pre-wash buffer of PBS of pH value 6.8, wash away non-specific adsorption albumen.The Fab antibody protein that adds the glycine hydrochloride buffer solution elution combination of pH value 2.7, add the Tris-HCl damping fluid of pH value 9.0 to neutralize the solution eluted, and then uses the evaporating column centrifugal concentrating.Fab Fragment Protein 10 ~ 20 μ g that get after purifying concentrates carry out the SDS-PAGE electrophoresis.

1.1.8 the neutralization experiment of people source anti-EV71 virus Fab antibody detects

(1) experimental procedure

The antibody 25 μ l of 100TCID50EV71 virus 25 μ l and doubling dilution are hatched 2 hours jointly in 37 ℃, and then adding concentration is 2 * 10 ⁵the cell culture fluid 50 μ l of individual RD cell/ml.Observe 7 days, record the cytopathy result.

(2) result is judged

In 2 holes of high dilution antibody, have 1 hole cytopathy to occur, cytopathy does not appear in another hole, and this dilution inverse is the NAT of this antibody; When the high dilution 2 complete pathologies in hole, adjacent low extent of dilution 2 holes are pathology not fully, and the inverse of both Average dilutions is the NAT of this antibody; When 1 porocyte pathology all appears in two adjacent extent of dilution antibody, cytopathy does not appear in another 1 hole, and the inverse of both Average dilutions is the NAT of this antibody.

1.2 result

1.2.1 the screening in anti-EV71 antiviral antibody storehouse, people source

With the EV71 virion SHZH98 strain of purifying, phage antibody library is carried out to the enrichment screening, 3 take turns rear random 2000 clones of picking of screening.With anti-human Fab antibody (using Sigma, the U.S. after 1 ﹕ 2000 dilutions) and antigen coated 96 orifice plates of EV71 virion SHZH98 strain, add the testing sample supernatant, with the anti-human Fab bis-of enzyme mark anti-(using Sigma, the U.S. after 1 ﹕ 2000 dilutions), detect.Result shows that obtaining altogether 816 people source Fab expresses positive colony, and wherein, 413 clones can specific binding EV71 virion SHZH98 strain (table 2).

Table 2SHZH98 strain is to phage antibody library enrichment the selection result

1.2.2 the sequential analysis of people source anti-EV71 virus Fab antibody

With the DNASTAR sequence analysis software, the Fab fragment is carried out to analyzing and processing, relatively the IgG sequence in Internet V-Base gene pool, in the Fab monoclonal antibody of the people source of above-mentioned 413 specific binding EV71 viruses, have the sequence difference of 30 Fab fragments.Therefore the present invention successfully screens and clones 30 with different antibody weight chain variable region sequences and the antibody of combination thereof, and its variable region of heavy chain mainly belongs to IgG VH3 and VH4 family, and its variable region of light chain mainly belongs to IgG VL1, VK1 and VK3 family.Wherein, EV71FabK7 belongs to the VK3 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.

1.2.3 the purifying of people source anti-EV71 virus Fab antibody

Antibody (Sigma with anti-human Fab, the U.S.) prepare the 2ml affinity column, purifying Fab antibody expression supernatant, check the Expression and purification situation of Fab antibody by SDS-PAGE, result confirms to obtain than pure protein, can clearly observe light chain of antibody and Fd chain (Fig. 1) after unwinding.

1.2.4 the ELISA of people source anti-EV71 virus Fab antibody detects

Use respectively the deactivation EV71 virion SHZH98 strain of purifying and FUYANG-0805 strain as envelope antigen, detect antibody in conjunction with active, result is as shown in Figure 2.

The ELISA of antigen-binding activity detects: use 0.1M NaHCO ₃the solution of (pH 9.6) is the SHZH98 strain of deactivation EV71 virion and the FUYANG-0805 strain of coated purifying respectively, 4 ℃ are spent the night, wash plate 3 times by the PBS-T washing lotion, fully remove liquid and add 5% skimmed milk 100 μ l, 37 ℃ of incubation 1h, the 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.The 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, and the PBS-T washing lotion is washed plate 6 times, fully removes liquid, adds nitrite ion A, B color development at room temperature 10min, 2MH ₂sO ₄termination reaction, the OD450 reading.

1.2.5 the neutralization experiment of people source anti-EV71 virus Fab antibody

Respectively with the SHZH98 strain of EV71 virus and FUYANG-0805 strain as attacking poison strain, detect the neutralization activity of antibody in the RD cell.Result as shown in Figure 3.

Experimental procedure: respectively with the SHZH98 strain of EV71 virus and Fuyang-0805 strain as attacking poison strain, detect the neutralization activity of antibody in the RD cell.

37 ℃ of the antibody of the 100TCID50EV71 of 50 μ l virus (SHZH98 strain and Fuyang-0805 strain) and 50 μ l doubling dilutions (prepared by embodiment 6) are jointly hatched after 2 hours and added 2 * 10 ⁵in the cell suspension 100 μ l of individual cell/ml.Observe 7 days, record the cytopathy result.

Wherein, the EV71 virus (SHZH98 strain and Fuyang-0805 strain) of using is through processing as follows: by the virion of embodiment 1 preparation, by 10 times of gradient dilutions, be 10 ^-1to 10 ^-10virus liquid, respectively add in cell plate, every hole 50 μ l, and each extent of dilution adds 4 porocytes; Every hole adds cell suspension 50 μ l, establishes cell contrast (50 μ l diluents+50 μ l cell suspensions) simultaneously, cultivates 7d, observation of cell pathology for 37 ℃; Calculate the TCID50 of isolated viral strain by the Karber formula; Log TCID50=L-d (S-0.5).Wherein: the minimum dilution log value of using in the L=experiment; The log value of d=dilution gradient; The summation (the shared ratio sum of cell hole that CPE occurs) of positive part when S=sentences eventually.

1.2.6 the impact of the antibody after the sudden change of non-hypervariable region on the EV71 virus resistance

Adopt the method for site-directed point mutation, the Arg of the 16th of the light chain VL sequence of EV71FabK7 is replaced with to Lys, the Val of the 8th of heavy chain VH sequence replaces with Ala.After light chain after synthetic sudden change and heavy chain nucleic acid sequence encoding, the weight chain gene is cloned in pComb3H respectively, obtains mutant EV71FabK7 '.This mutant is carried out to immunology detection, and the ELISA experiment shows that EV71FabK7 ' can specific binding EV71 virus.Adopt the neutralization experiment detect antibody in vitro with the neutralization reaction of SHZH98 strain and FUYANG-0805 strain, result shows its character and EV71FabK7 basic identical (extension rate that in table 3, data are antibody).

Table 3 antibody EV71FabK7 and mutant EV71FabK7 ' thereof the neutralization activity to SHZH98 strain and FUYANG-0805 strain

The application of embodiment 2 people source anti-EV71 virus neutrality antibody EV71FabK7

Prevention at present and treatment hand foot mouth disease there is no specific vaccine and medicine, the immunoglobulin (Ig) that used clinically all derives from the positive serum of the anti-EV71 of people, this just makes its a large amount of preparations be restricted, so owing to deriving from serum, the pathophorous infection of haematogenous easily occurs simultaneously.Adopt the anti-EV71 virus gene engineering antibody in people source that the present invention obtains to substitute the haematogenous immunoglobulin (Ig), for the treatment of hand foot mouth disease provide newly by way of.

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-EV71 virus in people source neutrality antibody EV71FabK7, is characterized in that,

The aminoacid sequence of its variable region of light chain is as shown in SEQ ID No.1, and the aminoacid sequence of variable region of heavy chain is as shown in SEQ ID No.2.

2. the gene of coding claim 1 described antibody EV71FabK7.

3. gene according to claim 2, is characterized in that, the nucleotide sequence of encoding heavy chain variable region is as shown in SEQ ID No.4.

4. the anti-EV71 virus in people source neutrality antibody EV71FabK7 ', is characterized in that, the aminoacid sequence of its variable region of light chain is as shown in SEQ ID No.33, and the aminoacid sequence of variable region of heavy chain is as shown in SEQ ID No.34.

5. the gene of coding claim 4 described antibody EV71FabK7 '.

6. the carrier that contains claim 2, the described gene of 3 or 5 any one.

7. the host cell that contains the described carrier of claim 6.

8. the application of the anti-EV71 virus in the described people of claim 1 or 4 source neutrality antibody in the medicine of preparation prevention or treatment hand foot mouth disease.

9. the medicine that contains the described people of claim 1 or 4 source anti-EV71 virus neutrality antibody.

10. the detection reagent that contains the described people of claim 1 or 4 source anti-EV71 virus neutrality antibody.

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