CN103864924A - Middle east and respiratory syndrome coronavirus antibody and preparation method thereof - Google Patents
Middle east and respiratory syndrome coronavirus antibody and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a middle east and respiratory syndrome coronavirus antibody and a preparation method thereof, and belongs to the technical field of cell-mediated immunity. RDB protein of an MERS-CoV virus S protein receptor binding domain is obtained by using in-vitro expression and purification of a baculovirus expression vector system, and Bab/c mice are immunized by using high-purity MERS RBD protein, an efficient antiviral neutralizing anti-body is screened by the methods such as an enzyme-linked immuno sorbent assay (ELISA), a false virus neutralization experiment and the like. The antibody and the crystal structure of the MERS RBD protein are analyzed, and a crucial amino acid of the antibody combined with viral proteins is determined from the molecular level. Thus, the antibody is further humanized, and the possibility is provided for clinical detection, prevention and treatment of MERS-CoV virus infection.
Description
Technical field
The present invention relates to a kind of Middle East respiration syndrome coronavirus antibody and preparation method thereof, belong to cellular immunization technical field.
Background technology
Middle East respiration syndrome coronavirus (Middle East Respiratory Syndrome Cononaviruse, MERS-CoV) belongs to coronaviridae together, beta coronavirus genus with SARS-CoV.For normal chain, single strand RNA virus.Its surperficial spike protein-S albumen plays keying action in the time of virus infection humans and animals.Once there is scientist to infer that this virus may derive from bat.Sequential analysis finds, MERS-CoV virus is very high with bat and pig coronavirus sequence homology, with bat coronavirus HKU4 and HKU5 can be up to 60%.Still source and the natural host of not determining at present MERS-CoV virus, cannot take effective measures to prevent and stop this virus disseminating.
Due to the similarity between MERS-CoV in clinical disease and SARS-CoV, propose them and may use identical cell receptor-Zinc metallopeptidase Zace1 2(ACE2).But further research is found: dipeptidyl peptidase-4 (DPP4 is also referred to as CD26) is the acceptor of MERS-CoV, express at human bronchial and renal epithelial cell.When virus infection people, virus S protein receptor binding domain (Receptor Binding Domain, RBD) meeting and this protein binding, virus, take them as " debarkation point ", is attached on respiratory tract cell, further invades in human body thereupon.The aminoacid sequence of CD26 albumen is high conservative, is also present in bat and other many animal bodies, and therefore, MERS-CoV virus may utilize this protein between multiple species, to continue to propagate, and has larger potential threat.At present, infecting MERS-CoV and after virus, there is no effective medicine, is mainly symptomatic treatment, also there is no preventative vaccine.Monoclonal antibody is the specific antibody producing for specific antigen, and it has height specificity, can take accurate aim, target acquisition target spot, reacts specifically, thereby directly remove viral object with target.
MERS-CoV virus surface has a membranin to be referred to as S albumen, virus copy with life cycle in play an important role.On S albumen, have subregion, be referred to as receptor binding domain (Receptor Binding Domain, RBD) can with the CD26 protein binding of cell surface, mediation virus infected cell.Thereby, find and prepare effective antibody prevention MERSRBD protein binding CD26 albumen, and then suppressing virus infection cell, become one of method of detection, prevention and treatment MERS-CoV virus infection.By preparing anti-MERS RBD protein monoclonal antibody, screening is the neutralizing antibody of specific binding with it, analyzes the antibody critical sites of combination with it on molecular level, further to its humanization, has become the effective means of preparation prevention or therapeutic antibodies.At present also not about the report of anti-MERS-CoV virucidin.
Owing to lacking at present anti-MERS-CoV virucidin, and prepare lot of antibodies and be applied to and clinically need the long period, can not detect effectively clinically fast.Therefore this antibody not only can be applied to clinical detection, and to providing architecture basics and possibility for preparing prevention or therapeutic antibodies after its humanization.
The invention describes the technology that screening has the active MERS-CoV mouse resource monoclonal antibody of neutralization, and the humanization modified technology of neutralizing antibody based on sequence and structural information.For detection, prevention and the treatment of MERS-CoV.
Summary of the invention
First technical problem that the present invention will solve is to provide a kind of Middle East respiration syndrome coronavirus (MERS-CoV) antibody, described antibody is MERS-CoV mouse resource monoclonal neutralizing antibody, and its heavy chain, light-chain amino acid sequence are respectively as shown in SEQ ID NO.1, SEQ ID NO.2.
Second technical problem that the present invention will solve is to provide the preparation method of described MERS-CoV mouse resource monoclonal neutralizing antibody, first be to prepare high purity, activated MERS RBD(MERS-CoV virus receptor land albumen), then obtain the mouse resource monoclonal neutralizing antibody of anti-MERS-CoV virus take this albumen as antigen selection.
Described MERS-CoV mouse resource monoclonal neutralizing antibody preparation method's concrete steps are:
(1) gene order in coding MERS-CoV virus surface S protein receptor binding district is connected in carrier pFastBac1, construction recombination plasmid pFastBac-MERS RBD, and transform DH10Bac competent cell, Screening and Identification positive colony, extract recombinant baculovirus plasmid, transfection sf9 cell, cultivates and obtains a large amount of infectious titers; Then infect Hi5 cell, the concentrated target protein MERS-RBD that obtains of purifying from cell culture supernatant.
(2) using MERS-RBD albumen as antigen immune Balb/c mouse, the splenocyte of getting the mouse that serum antibody titer is the highest merges by 5:1 ratio with myeloma cell SP2/0, obtains hybridoma by HAT screening culture medium and limiting dilution assay; And further detect the efficiently monoclonal cell strain of secretion MERS-RBD protein antibodies of screening acquisition with ELISA, and injecting mouse peritoneal and prepare ascites monoclonal antibody, purifying obtains mouse source antibody.
(3) gained mouse source antibody is detected and is suppressed RBD in conjunction with the effect of CD26 by flow cytometer, and in confirming to be had by pseudovirus neutralization test with the antibody of pseudovirus activity; Described pseudovirus is that plasmid pCAGGS-MERS S and HIV pNL4-3.luc.RE cotransfection 293T cell cultures are obtained.
The 3rd technical problem that the present invention will solve is to provide a kind of humanization MERS-CoV mouse resource monoclonal neutralizing antibody.
The heavy chain of described humanization MERS-CoV mouse resource monoclonal neutralizing antibody, the aminoacid sequence of light chain are respectively as shown in SEQ ID NO.3, SEQ ID NO.4.
The 4th technical problem that the present invention will solve is to provide the preparation method of described humanization MERS-CoV mouse resource monoclonal neutralizing antibody, first to prepare high-purity activity MERS-CoV virus receptor land albumen, take this albumen as antigen selection, obtain the mouse resource monoclonal neutralizing antibody of anti-MERS-CoV virus; Then resist the crystalline structure of mouse resource monoclonal neutralizing antibody and MERS-RBD albumen and analyze and sequence alignment, obtain this mouse source antibody and the protein bound key amino acid of MERS-RBD, finally mouse source antibody is carried out to humanization modified design.
Described humanization MERS-CoV mouse resource monoclonal neutralizing antibody preparation method's concrete steps are:
(1) gene order in coding MERS-CoV virus surface S protein receptor binding district is connected in carrier pFastBac1, construction recombination plasmid pFastBac-MERS RBD, and transform DH10Bac competent cell, Screening and Identification positive colony, extract recombinant baculovirus plasmid, transfection sf9 cell, cultivates and obtains a large amount of infectious titers; Then infect Hi5 cell, the concentrated target protein MERS-RBD that obtains of purifying from cell culture supernatant.
(2) using MERS-RBD albumen as antigen immune Balb/c mouse, the splenocyte of getting the mouse that serum antibody titer is the highest merges by 5:1 ratio with myeloma cell SP2/0, obtains hybridoma by HAT screening culture medium and limiting dilution assay; And further detect the efficiently monoclonal cell strain of secretion MERS-RBD protein antibodies of screening acquisition with ELISA, and injecting mouse peritoneal and prepare ascites monoclonal antibody, purifying obtains mouse source antibody.
(3) gained mouse source antibody is detected and is suppressed RBD in conjunction with the effect of CD26 by flow cytometer, and in confirming to be had by pseudovirus neutralization test with the antibody of pseudovirus activity; Described pseudovirus is that plasmid pCAGGS-MERS S and HIV pNL4-3.luc.RE cotransfection 293T cell cultures are obtained.
(4) compare by order-checking the Fab complete sequence that obtains described mouse source antibody, preparation and antibody purification Fab fragment; MERS RBD albumen and Fab fragments are hatched altogether, by the analysis to compound crystal structure, determined in Fab fragments for the amino acid playing a crucial role in conjunction with MERS RBD albumen; The Fab fragment of described mouse source antibody and the human antibody of reporting are compared, look for the highest sequence of homology with it, retain in CDR district for the amino acid playing a crucial role in conjunction with MERS RBD albumen, other amino acid substitution is the corresponding aminoacid sequence of human antibody, finally obtains humanization MERS-CoV mouse resource monoclonal neutralizing antibody.
The present invention successfully screens Liao Kang MERS-CoV virucidin, and by crystallographic structural analysis the accurate binding site of antibody on virus S protein.This antibody is the neutralizing antibody of finding first, can effectively suppress MERS-CoV Virus entry, MERS-CoV surface protein is had to high-affinity, therefore this antibody not only can be applied to clinical detection, and to providing architecture basics and possibility for preparing prevention or therapeutic antibodies after its humanization.
Accompanying drawing explanation
Fig. 1: MERS RBD protein purification molecular sieve and SDS-PAGE figure; Molecular sieve figure center line is 280nm absorption value; In SDS-PAGE figure, No. 1 swimming lane is low molecular weight protein (LMWP) Marker, MERS RBD dimer and monomer in the corresponding molecular sieve figure of all the other swimming lanes difference.
Fig. 2: monoclonal cell strain supernatant is to MERS RBD protein binding CD26 restraining effect streaming figure.
Fig. 3: in antibody 4C2 and pseudovirus curve; X-coordinate is antibody dilution multiple, and ordinate zou is neutralization ratio.
Fig. 4: antibody 4C2 is in conjunction with MERS RBD protein surface plasma resonance kinetic curve; X-coordinate is in conjunction with Dissociation time, and unit is second; Ordinate zou is the combination response intensity of chip surface, and unit is that RU(is Response unit).
Fig. 5: MERS RBD albumen and antibody 4C2Fab are hatched rear molecular sieve and SDS-PAGE figure altogether; In SDS-PAGE figure, 1 is MERS RBD albumen, and 2 is 4C2Fab, and M is low molecular weight protein (LMWP) Marker, and 3 is MERS RBD albumen and 4C2Fab.
The compound crystal structure of Fig. 6: MERS-RBD and antibody 4C2Fab.
Fig. 7: in humanized antibody 4C2 and pseudovirus curve; X-coordinate is antibody dilution multiple, and ordinate zou is neutralization ratio.
Embodiment
We are by DNA fragmentation (the amino acid E367-Y606 of coding MERS RBD albumen, NCBI accession number JX869059) be connected in pFastBac1 carrier with EcoR I and Xho I restriction enzyme site, and before the encode fragment of albumen, add signal peptide gp67 to contribute to protein excretion to express, after the encode fragment of albumen, insert encoding sequence and translation stop codon of 6 histidine-tagged (hexa-His-tag), can obtain like this C-holds with histidine-tagged recombinant protein, be beneficial to follow-up protein purification, called after pFastBac-MERS RBD.By MERS RBD gene (amino acid E367-Y606, NCBI accession number JX869059) insert between pCAGGS carrier EcoR I and Xho I restriction enzyme site, between Xho I and Bgl II restriction enzyme site, add mouse Fc gene fragment (amino acid 239G-469K, NCBI accession number AY311599), be beneficial to follow-up protein purification and cell dyeing, called after pCAGGS-MERS RBD mFc.CD26 gene order (amino acid S39-P766, NCBI accession number NP_001926) is inserted between pEGFPC1 carrier Xho I and Sma I restriction enzyme site to called after pEGFPC1-CD26.ACE2 gene order (amino acid S19-D615, NCBI accession number BAJ21180) is inserted between pEGFPN1 carrier Xho I and Sma I restriction enzyme site to called after pEGFPN1-ACE2.MERS S gene (amino acid M1-H1353, NCBI accession number JX869059) is inserted between pCAGGS carrier EcoR I and Sma I restriction enzyme site, after gene, adds Flag label SEQ ID NO.5, called after pCAGGS-MERS S.Recombinant plasmid cuts evaluation by PCR, enzyme and order-checking confirms that the external source fragment of inserting is entirely true.
Embodiment 2MERS RBD protein expression and purifying
First, recombinant plasmid pFastBac-MERS RBD is transformed to DH10Bac competent cell, and 37 ℃ of incubated overnight, identify positive colony by blue hickie screening and PCR, and extract restructuring Bacmid(baculovirus plasmid), contain the plasmid of MERSRBD gene order.The Bacmid transfection sf9 cell of recombinating, collects culture supernatant after transfection 3d, obtains P1 for baculovirus.Amplification 3 generation virus can obtain a large amount of infectious titers continuously.Then infect Hi5 cell, the centrifugal cell conditioned medium of collecting of 48h, spends the night cell culture supernatant and Ni-NTA Resin (GE) in 4 ℃ of combinations.With buffer A (20mM Tris, 150mM NaCl, pH8.0) washing resin, to remove the albumen of non-specific binding.Finally target protein is eluted from resin with buffer B (20mM Tris, 150mM NaCl, pH8.0,300mM imidazole), and with the evaporating pipe that 10K holds back (10K cutoff), elutriant is concentrated into 5ml.By the protein solution after concentrated further with molecular exclusion chromatography purifying, use AKTA-purifier(GE) and Superdex200Hiload16/60 pillar (GE), use buffer A (20mM Tris, 150mM NaCl, pH8.0), monitor the ultraviolet absorption value of 280nm simultaneously, collect target protein, and identify purity of protein by SDS-PAGE.The molecular sieve collection of illustrative plates of typical target protein and SDS-PAGE analysis chart are as shown in Figure 1.
MERS-RBD albumen, as 3 of antigen immune Balb/c mouse, is got 100g protein dissolution in the aseptic PBS solution of 50 μ l, with 50 μ l not formula Freund's complete adjuvant evenly mix, subcutaneous multi-point injection.After 2 weeks, booster immunization once, after 3 days, get tail vein, detect serum antibody titer, the splenocyte of getting the highest mouse of tiring merges by 5:1 ratio with myeloma cell SP2/0, select to cultivate by HAT screening culture medium, obtain hybridoma with limiting dilution assay.The MERS-RBD albumen of getting purifying is envelope antigen, detects through ELISA, obtains the monoclonal cell strain of efficient secretion MERS-RBD protein antibodies.According to hybridoma (1-5) × 10
6the amount of/mouse is injected mouse peritoneal and is prepared ascites monoclonal antibody, after 10 days, collects ascites, centrifugal removal precipitation.0.22M membrane filtration ascites, by Protein G affinity column (GE) antibody purification.
Embodiment 4 screens neutralizing antibody
(1) the monoclonal cell strain of the anti-MERS-RBD antibody of ELISA screening secretion
4 μ g MERS-RBD albumen are dissolved in to coated elisa plate in the carbonate buffer solution of 1ml pH9.6,400ng/ hole, 4 ℃ are spent the night.With containing the PBS solution of 3% bovine serum albumin BSA in 37 ℃ of sealings 1 hour.With the PBS of PBST solution (containing 0.05%(volumn concentration) Tween20) wash 3 times, add respectively the Hybridoma Cell Culture supernatant of gradient dilution, 100 μ l/ holes, hatch 1 hour for 37 ℃, if the positive contrast of mouse heart serum, the negative contrast of bovine serum albumin.With after PBST washing 3 times, add two anti-(1:2000 dilution, Santa Cruz, the Inc.) of the anti-mouse of HRP mark, 100 μ l/ holes, hatch 40 minutes for 37 ℃.With after PBST washing 3 times, add TMB (tetramethyl benzidine, Sigma.) to develop the color, hatch 15-30 minute for 37 ℃, then add 0.1M H
2sO
4termination reaction.Finally, detect the absorbance value at OD450nm place by microplate reader.Result shows: in 1000 clones of screening, have 67 Elisa reacting positives, titre reaches 10
4above.
(2) antibody suppression MERS RBD is combined with acceptor CD26
First, utilize Lipofectamine2000 transfection plasmid pEGFPC1-CD26 and pEGFPN1-ACE2 to BHK21 cell, after 24 hours, under fluorescent microscope, observe CD26-GFP and ACE2-GFP fusion rotein is mainly distributed on cytolemma.Collect cell, and counting, resuspended to 1 × 10 after PBS washed twice
7cell/ml, packing cell is to EP pipe, and 100ul/ manages.PCAGGS-MERS RBD mFc is utilized to Lipofectamine2000 transfecting eukaryotic cells 293T, after 72 hours, collect supernatant and obtain MERD RBD-Fc fusion rotein by ProteinA affinitive layer purification, MERS RBD-Fc fusion rotein is mixed with monoclonal cell strain culture supernatant, room temperature is hatched altogether after 30 minutes and is joined in the BHK21 cell of expressing CD26-GFP or ACE2-GFP fusion rotein, and room temperature is hatched 30 minutes again.PBS washed cell 2 times, adds two anti-(the 1:200 dilutions) of the anti-mouse of TRITC mark, incubated at room 30 minutes.PBS washed cell 2 times, then uses 500 μ l re-suspended cells, by flow cytometer detect antibody to RBD the restraining effect in conjunction with CD26.Wherein, positive control is the BHK21 cell of the dyed blended expression of MERS RBD-Fc fusion rotein and substratum DMEM CD26-GFP, and negative control is the BHK21 cell of the dyed blended expression of MERS RBD-Fc fusion rotein and substratum DMEM ACE2-GFP.Result shows: MERS RBD-Fc does not have ruddiness skew after dying the cell of expressing ACE2-GFP fusion rotein, and MERS RBD-Fc can not be in conjunction with ACE2-GFP; After MERS RBD-Fc dyes the cell of expressing CD26-GFP fusion rotein, ruddiness has skew, and MERS RBD-Fc can be in conjunction with CD26-GFP.After antibody mixes with MERS RBD-Fc, if antibody and RBD have in conjunction with the combination that can suppress RBD and CD26, ruddiness is without skew so; Otherwise, if antibody can not be combined with RBD ruddiness and be had skew.
After please testing as stated above on the cell of Elisa reacting positive, we find, after in monoclonal cell strain 4C2 supernatant, the antibody of secreting, expressing mixes with MERS RBD-Fc, ruddiness side-play amount is less, be that this antibody can effectively suppress MERS RBD albumen and is combined with CD26 (Fig. 2), inhibiting rate reaches 67%.
In Fig. 2, N-BHK21 is the BHK21 cell of untransfected plasmid, is the two negative controls of experiment; ACE2-GFP can express ACE2-GFP fusion rotein after BHK21 cell transfecting pEGFPN1-ACE2 plasmid, is the single positive control of experiment; ACE2-GFP+MERS RBD-Fc is that MERS RBD-Fc albumen dyes the BHK21 cell of expressing ACE2-GFP fusion rotein, is experiment negative control; CD26-GFP+MERS RBD-Fc is that MERS RBD-Fc albumen dyes the BHK21 cell of expressing CD26-GFP fusion rotein, is experiment positive control; Other is the BHK21 cell that corresponding antibodies and MERS RBD-Fc albumen mixing after stain are expressed CD26-GFP fusion rotein.
In embodiment 5 pseudoviruss and experiment
(1) pseudovirus packing
Utilize Lipofectamine2000 by plasmid pCAGGS-MERS S and HIV pNL4-3.luc.RE (Invitrogen) cotransfection 293T cell.After transfection 6 hours, PBS washed cell 2 times, is changed to serum-free DMEM.After 48 hours, collect cell conditioned medium, the centrifugal cell debris that removes, frozen-80 ℃ of packing.
(2) TCID50 measures
By 10 times of doubling dilutions of the virus liquid of collecting, be followed successively by 10
-1, 10
-2... 10
-10, join in 96-well, now Huh7 cell degree of converging is 80%-100%.Infect after 4 hours, discard virus liquid, PBS washed cell 2 times, is changed to the perfect medium DMEM containing 10% serum.After 48 hours, discard nutrient solution, PBS washed cell 2 times, adds cell pyrolysis liquid, utilizes GloMax96Microplate Luminometer (Promega) to detect fluorescein plum activity value.Calculate TCID50 by Reed-Muench method.
(3) neutralization experiment
By 2 times of doubling dilutions of the antibody of purifying, mix with 100TCID50 pseudovirus, antibody final concentration is followed successively by 25ug/ml, 12.5ug/ml ... 50ng/ml, hatches altogether 30 minutes, mixed solution is joined in the 96-well that is paved with Huh7 cell for 37 ℃.Hatch after 4 hours for 37 ℃, discard virus liquid, PBS washed cell 2 times, is changed to the perfect medium DMEM containing 10% serum.After 48 hours, discard nutrient solution, PBS washed cell 2 times, adds cell pyrolysis liquid, detects fluorescein plum activity value.
Result shows: during antibody 4C2 has, with pseudovirus activity, can suppress the combination of MERS virus S protein and cell surface receptor CD26, ND50 is 103ng/ml (Fig. 3).
Surface plasma resonance analysis Biacore3000(Biacore Inc.) carry out.Concrete steps are as follows:
First, antibody 4C2 is dissolved in to 10mMNaAc(pH5.0) be fixed on CM5 chip (GE), then the MERS RBD albumen that is 3.125nM-100nM by concentration gradient injects respectively chip, analyze and carry out 25 ℃ of constant temperature, use damping fluid be HEPES-Tween damping fluid (10mM HEPES[pH7.4], 150mM NaCl, 0.005%Tween-20), flow velocity is 30 μ l/min.That the regeneration of chip surface is used is 100mM H
3pO
4, binding curve as shown in Figure 4.In figure the antibody concentration of 5 curves be respectively from top to bottom 100,50,25,12.5,6.25,3.125nM, the curve of different concns forms illustrated kinetic curve.The calculating of binding kinetics constant is to utilize BIAevaluation software version3.2 (Biacore, Inc.) software to carry out.Result shows, the affinity constant of antibody 4C2 and MERS RBD albumen is 17.5nM.
Embodiment 7 antibody 4C2Fab sequencings
(1) antibody subtype is identified
The 4C2 antibody of purifying is carried out to SDS-PAGE, have heavy chain and light chain two bands at about 45kD and 30kD place respectively, cutting ddH2O washing by soaking 3 times.After dehydration and enzymolysis, identify, instrument is MALDI-TOF/TOF mass spectrograph.After data gathering, GPS3.6(Applied Biosystems is integrated and used to firsts and seconds mass-spectrometric data) and Mascot2.1 (Matrix Science) to mass spectrum data analysis and Identification of Fusion Protein.In NCBI, search for and compare, identifying antibody 4C2 hypotype is IgG1 (κ).
(2) antibody cloning
Collect monoclonal cell strain cell 1 × 10
6, Trizol method is extracted mRNA.First the primer that designs gene specific carries out reverse transcription (RT-PCR) and obtains cDNA, then utilizes one to take turns pcr amplification and obtain 4C2Fab(Fab) sequence.Order-checking obtains after main sequence, is compared and is obtained 4C2Fab complete sequence by NCBI.
4C2Fab V
h-C
h1 order-checking the primer and reaction conditions are as follows:
Primer: 4C2-IgHV-F1:TCTGGATTCACTTTCAGTAG
IgHG1-R3：GGATCCAGAGTTCCAGGTCA
IgHG1-R4：CACTGGCTCAGGGAAATAGC
RT-PCR：
Reaction conditions: 42 °, 90min-72 °, 7min
4C2Fab V
l-C
lorder-checking the primer and reaction conditions are as follows:
Primer: IgKC-R1:TGGGGTAGAAGTTGTTCA;
IgKC-R2：TGAGGCACCTCCAGATG；
4C2-IgKV-F2：TCCTGATCTACTACACATCAAG
RT-PCR：
PCR
4C2Fab V
h-C
h1 nucleotide sequence as shown in SEQ ID NO.6,4C2Fab V
h-C
h1 aminoacid sequence is as shown in SEQ ID NO.7; 4C2Fab V
l-C
lnucleotide sequence as shown in SEQ ID NO.8,4C2Fab V
l-C
laminoacid sequence as shown in SEQ ID NO.9.
Embodiment 8MERS-RBD and the screening of 4C2 crystal and structure elucidation
(1) Fab fragments preparation and purifying
Antibody employing test kit Mouse IgG1Fab and F (ab ')
2preparation Kits (Thermo Scientific), utilizes ficin that antibody is cut into Fab and Fc fragment, then separates Fc, purifying Fab fragment by ProteinA affinity column (GE).
(2) MERS RBD albumen and antibody 4C2Fab are hatched altogether
The MERS RBD albumen of purifying is mixed with antibody 4C2Fab, mix according to mol ratio 1:1, hatch altogether on ice after 2h, further with molecular exclusion chromatography purifying, use AKTA-purifier (GE) and superdex200Hiload16/60 pillar (GE), bufferA (20mM Tris, 150mM NaCl, pH8.0), monitor the ultraviolet absorption value of 280nm simultaneously, collect target protein, and identify purity of protein by SDS-PAGE.Molecular sieve collection of illustrative plates and the SDS-PAGE analysis chart of target protein are shown in Fig. 5.
(3) crystallization and the structure elucidation of neutralizing antibody 4C2Fab and MERS RBD protein complex
Crystallization adopts gas phase diffusion to sit the method for dripping and completes at 18 ℃, and further obtains the measured crystal of diffraction matter by condition optimizing, prepares liquid be protein concn 10mg/ml for the albumen of crystallization.Crystallization condition: 0.2M sodium thiocyanate, 20%w/v PEG3350(JCSG-plusTM Screen MD1-37).The molar concentration rate of MERS RBD and 4C2Fab is 1:1.
Data gathering is used Japanese KEK synchrotron radiation light source to complete.Data processing is used HKL2000 to complete.First structure elucidation obtains original structural models by molecular replacement technique, then utilizes Refmac5 (CCP4suite) and Phenix.refine to structural models refine, obtains final compound crystal structure.
Structure shows that 4C2 antibodies is exposed to the pleated sheet position (Fig. 6) in outside in MERS-RBD, this position is the site of MERS-RBD bind receptor exactly, and the neutralization activity that therefore can explain antibody is to realize by the receptor binding site of competition protein surface.
(4) molecular level analysis in conjunction with MERS RBD key amino acid to 4C2Fab
By the analysis to crystalline structure, we learn V in 4C2Fab
hchain and V
lin chain, all there is effect in CDR1, CDR2, CDR3 district with MERS RBD, and the amino acid wherein playing a crucial role is as follows:
4C2Fab V
h-C
h1(SEQ ID NO.7) key amino acid is (underscore mark):
DVKLVESGGGLVKPGGSLKLSCAASGFTFS
SYTMSWVRQTPEKRLEWVA
TI
SSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTR
DGNDY
DYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKI
4C2Fab V
l-C
l(SEQ ID NO.9) key amino acid is (underscore mark):
DIQMTQTTSSLSASLGDRVTISCRASQ
DISN
YLNWYQQKPDGTVK
LLI
YYTS
RL
HSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ
GNTLP
RTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNSNEC
Embodiment 9 antibody humanizations
(1) humanized antibody sequence
4C2Fab is compared with the human antibody of reporting, look for the highest sequence of homology with it, in reservation CDR district, with the protein bound key amino acid of MERS RBD, other amino acid substitution is the corresponding aminoacid sequence of human antibody, and humanization postorder is classified as:
Antibody 4C2 heavy chain amino acid sequence after optimization is as shown in SEQ ID NO.3, and after optimizing, antibody 4C2 light-chain amino acid sequence is as SEQ ID NO.4.
(2) in humanized antibody and active
After antibody sequence humanization, be inserted between pCAGGS carrier EcoR I and Xho I restriction enzyme site.After expressing in mammalian cell 293T, by proteinA affinity chromatography column purification.Adopt method similarly to Example 5 to measure in it and pseudovirus activity.Result shows: after humanization, antibody still has and neutralizes activity, can suppress equally the combination of MERS virus S protein and cell surface receptor CD26, and the active not decline of neutralization, and ND50 is 90ng/ml (Fig. 7).Substantially approaching with the mouse source antibody neutralization screening.
Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, therefore protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (10)
1. a Middle East respiration syndrome coronavirus antibody, is characterized in that, is mouse resource monoclonal neutralizing antibody, and the aminoacid sequence of its heavy chain, light chain is respectively as shown in SEQ ID NO.1, SEQ ID NO.2.
2. antibody according to claim 1, is characterized in that, described mouse resource monoclonal neutralizing antibody Fab V
h-C
h1 aminoacid sequence as shown in SEQ ID NO.7, Fab V
l-C
laminoacid sequence as shown in SEQ ID NO.9.
3. prepare the method for Middle East respiration syndrome coronavirus antibody described in claim 1 for one kind, first to prepare high-purity activity MERS-CoV virus receptor land albumen, then take the mouse resource monoclonal neutralizing antibody of this albumen anti-MERS-CoV virus as antigen selection obtains.
4. method according to claim 3, is characterized in that, concrete steps are:
(1) gene order in coding MERS-CoV virus surface S protein receptor binding district is connected in carrier pFastBac1, construction recombination plasmid pFastBac-MERS RBD, and transform DH10Bac competent cell, Screening and Identification positive colony, extract recombinant baculovirus plasmid, transfection sf9 cell, cultivates and obtains a large amount of infectious titers; Then infect Hi5 cell, the concentrated target protein MERS-RBD that obtains of purifying from cell culture supernatant;
(2) using MERS-RBD albumen as antigen immune Balb/c mouse, the splenocyte and the myeloma cell SP2/0 that get the mouse that serum antibody titer is the highest merge, and screening obtains hybridoma; And further detect the efficiently monoclonal cell strain of secretion MERS-RBD protein antibodies of screening acquisition with ELISA, and injecting mouse peritoneal and prepare ascites monoclonal antibody, purifying obtains mouse source antibody.
5. method according to claim 4, is characterized in that, also gained mouse source antibody is detected and is suppressed RBD in conjunction with the effect of CD26 by flow cytometer, and in confirming to be had by pseudovirus neutralization test with the antibody of pseudovirus activity.
6. a humanization MERS-CoV mouse resource monoclonal neutralizing antibody, is characterized in that, the aminoacid sequence of its heavy chain, light chain is respectively as shown in SEQ ID NO.3, SEQ ID NO.4.
7. prepare the method for humanization MERS-CoV mouse resource monoclonal neutralizing antibody described in claim 6 for one kind, first to prepare high-purity activity MERS-CoV virus receptor land albumen, take the mouse resource monoclonal neutralizing antibody of this albumen anti-MERS-CoV virus as antigen selection obtains; Then resist the crystalline structure of mouse resource monoclonal neutralizing antibody and MERS-RBD albumen and analyze and sequence alignment, obtain this mouse source antibody and the protein bound key amino acid of MERS-RBD, finally mouse source antibody is carried out to humanization modified design.
8. method according to claim 7, is characterized in that, concrete steps are:
(1) gene order in coding MERS-CoV virus surface S protein receptor binding district is connected in carrier pFastBac1, construction recombination plasmid pFastBac-MERS RBD, and transform DH10Bac competent cell, Screening and Identification positive colony, extract recombinant baculovirus plasmid, transfection sf9 cell, cultivates and obtains a large amount of infectious titers; Then infect Hi5 cell, the concentrated target protein MERS-RBD that obtains of purifying from cell culture supernatant;
(2) using MERS-RBD albumen as antigen immune Balb/c mouse, the splenocyte of getting the mouse that serum antibody titer is the highest merges by 5:1 ratio with myeloma cell SP2/0, obtains hybridoma by HAT screening culture medium and limiting dilution assay; And further detect the efficiently monoclonal cell strain of secretion MERS-RBD protein antibodies of screening acquisition with ELISA, and injecting mouse peritoneal and prepare ascites monoclonal antibody, purifying obtains mouse source antibody;
(3) compare by order-checking the Fab complete sequence that obtains described mouse source antibody, preparation and antibody purification Fab fragment; MERSRBD albumen and Fab fragments are hatched altogether, by the analysis to compound crystal structure, determined in Fab fragments for the amino acid playing a crucial role in conjunction with MERS RBD albumen; The Fab fragment of described mouse source antibody and the human antibody of reporting are compared, look for the highest sequence of homology with it, retain in CDR district for the amino acid playing a crucial role in conjunction with MERS RBD albumen, other amino acid substitution is the corresponding aminoacid sequence of human antibody, finally obtains humanization MERS-CoV mouse resource monoclonal neutralizing antibody.
9. the application of antibody in the antibody for the preparation of prevention or treatment Middle East respiration syndrome described in claim 1.
10. the application of antibody in the antibody for the preparation of prevention or treatment Middle East respiration syndrome described in claim 6.
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