CN103554235B - RBD (receptor binding domain) segment in MERS-CoV (Middle East respiratory syndrome coronavirus) membrane protein and coding gene and application thereof - Google Patents

RBD (receptor binding domain) segment in MERS-CoV (Middle East respiratory syndrome coronavirus) membrane protein and coding gene and application thereof Download PDF

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CN103554235B
CN103554235B CN201310461299.6A CN201310461299A CN103554235B CN 103554235 B CN103554235 B CN 103554235B CN 201310461299 A CN201310461299 A CN 201310461299A CN 103554235 B CN103554235 B CN 103554235B
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CN103554235A (en
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王新泉
张林琦
王年爽
史宣玲
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Tsinghua University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention discloses a RBD (receptor binding domain) segment in a MERS-CoV (Middle East respiratory syndrome coronavirus) membrane protein and a coding gene and application thereof. 1, A protein is as follows: (a) a protein comprising 4th to 243th-site amino acid residues from N terminal of a sequence 1 in a sequence table; or (b) a protein comprising 1th to 243th-site amino acid residues from the N terminal of the sequence 1 in the sequence table; or (c) a protein comprising 4 to 249-site amino acid residues from the N terminal of the sequence 1 in the sequence table; or (d) a protein shown as the sequence 1 of the sequence table. The invention finds that MERS-CoV may have a potential receptor binding domain (Receptor Binding Domain, RBD), and in-depth studies on the RBD help to discover a key target blocking the MERS-CoV invasion, and have important theory reference values to research and development of treatment drugs and preventive vaccines of the MERS-CoV.

Description

RBD fragment in MERS-CoV virus membrane antigen and encoding gene thereof and application
Technical field
The present invention relates to RBD fragment in a kind of MERS-CoV virus membrane antigen and encoding gene thereof and application.
Background technology
Middle East respiratory system syndrome coronavirus (Middle East respiratory syndrome coronavirus, be called for short MERS-CoV) be found in Middle East first in 2012 to infect the mankind, the disease that this virus infection causes subsequently successively appears at European several countries and regions again.The infected patient exceeding half all there will be serious respiratory tract disease, and its clinical symptom is closely similar with the disease caused by SARS-CoV of outburst in 2013.Because this disease people can be transmitted to people, therefore cause global showing great attention to.
MERS-CoV belongs to beta coronavirus, but its source and host still do not determine.Similar with other coronavirus, MERS-CoV utilizes the membranin S glycoprotein on its surface to enter permissive cell.S glycoprotein is made up of the S1 structural domain being positioned at N end and the S2 structural domain and membrane spaning domain being positioned at nearly film end.Wherein virus is determined by S1 structural domain cell susceptible.By utilizing MERS-CoV S1 structural domain to carry out copurification experiment, at the beginning of 2013, the research group of Raj determines the acceptor that dipeptideyl peptidase4 (dipeptideyl peptidase4, DPP4, also referred to as CD26) is MERS-CoV.
Summary of the invention
The object of this invention is to provide RBD fragment in a kind of MERS-CoV virus membrane antigen and encoding gene thereof and application.
Protein provided by the invention, available from MERS-CoV, is following (a) or (b) or (c) or (d) or (e) or (f):
A protein that the sequence 1 of () sequence table forms from N-terminal the 4 to 243 amino acids residue;
B protein that the sequence 1 of () sequence table forms from N-terminal the 1 to 243 amino acids residue;
C protein that the sequence 1 of () sequence table forms from N-terminal the 4 to 249 amino acids residue;
The protein shown in sequence 1 of (d) sequence table;
(e) by the protein described in (a) or (b) or (c) or (d) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by its derivative protein;
F () and the protein described in (a) or (b) or (c) or (d) have more than 70% homology and have the protein derived by it of identical function.
The gene of code for said proteins also belongs to protection scope of the present invention.
Described gene can be arbitrary described DNA molecular in (1) to (7) as follows:
(1) DNA molecular shown in sequence 2 in sequence table;
(2) DNA molecular shown in sequence 3 in sequence table;
(3) in sequence table sequence 5 from the DNA molecular shown in 5 ' end 1-843 position Nucleotide;
(4) in sequence table sequence 5 from the DNA molecular shown in 5 ' end 124-864 position Nucleotide;
(5) DNA molecular shown in sequence 5 in sequence table;
(6) DNA sequence dna limited with (1) is under strict conditions hybridized and the DNA molecular of code for said proteins;
(7) DNA sequence dna limited with (1) at least has the DNA molecular of more than 70% homology and code for said proteins.
Above-mentioned stringent condition can be in the solution of 6 × SSC, 0.5%SDS, hybridizes, then use 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively to wash film once at 65 DEG C.
Expression cassette containing described gene, recombinant vectors, transgenic cell line, recombinant virus or recombinant bacterium all belong to protection scope of the present invention.
Also protection scope of the present invention is belonged to the antibody that described protein obtains for immunogen.
The present invention also protects the application of described protein in preparing product; The function of described product is following (I) or (II): (I) treats and/or prevents MERS-CoV and infect; (II) treat and/or prevent MERS-CoV and contaminate mammalian cell.Described Mammals can be specifically people.Described mammalian cell specifically can be Huh7 cell.
The present invention also protects the application of described antibody in preparing product; The function of described product is following (I) or (II): (I) treats and/or prevents MERS-CoV and infect; (II) treat and/or prevent MERS-CoV and contaminate mammalian cell.Described Mammals can be specifically people.Described mammalian cell specifically can be Huh7 cell.
The present invention also protects a kind of product, and its activeconstituents is described protein or described antibody; The function of described product is following (I) or (II): (I) treats and/or prevents MERS-CoV and infect; (II) treat and/or prevent MERS-CoV and contaminate mammalian cell.Described Mammals can be specifically people.Described mammalian cell specifically can be Huh7 cell.
The present invention also protects described protein to have in conjunction with the application in the product of the function of DPP4 or DPP4 extracellular domain fragment in preparation.
The present invention also protects a kind of product with function in conjunction with DPP4 or DPP4 extracellular domain fragment, and its activeconstituents is described protein.
Described DPP4 extracellular domain fragment is following (g) or (h) or (i) or (j) or (k) or (l):
G protein that the sequence 6 of () sequence table forms from N-terminal the 4 to 731 amino acids residue;
H protein that the sequence 6 of () sequence table forms from N-terminal the 1 to 731 amino acids residue;
(i) the protein that forms from N-terminal the 4 to 737 amino acids residue of the sequence 6 of sequence table;
The protein shown in sequence 6 of (j) sequence table;
(k) by (g) or (h) or the protein (i) or described in (j) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by its derivative protein;
L () and (g) or (h) or the protein (i) or described in (j) have more than 70% homology and have the protein derived by it of identical function.
The present invention determines the position at receptor binding domains (Receptor Binding Domain, the RBD) place of the S glycoprotein of MERS-CoV, and RBD fragment can with DPP4 specific combination.The present invention, by building the pseudotype virus infection model of MERS-CoV, confirms RBD fragment, activity (the equal contestable of animal immune serum of additional RBD fragment or additional RBD fragment suppresses the combination of MERS-CoV and DPP) that the animal immune serum of RBD fragment all has good suppression MERS-CoV infected cell.RBD fragment as the target spot blocking MERS-CoV infected cell, can develop various newtype drug.The present invention has important theoretical direction to treatment and the disease that causes of prevention MERS-CoV and is worth and application prospect widely.
Accompanying drawing explanation
Fig. 1 is the result of pull-down experiment in embodiment 3.
Fig. 2 is the result of gel filtration experiment in embodiment 3.
Fig. 3 is the result of the Neutralization effect of RBD-His albumen in embodiment 5.
Fig. 4 is the result of the Neutralization effect of specific antibody solution in embodiment 5.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
PcDNA3.1(+): purchased from Invitrogen company.PFastBac tMdual vector:Invitrogen company, article No. is 10712-024.DH10Bac competent cell: Invitrogen company, article No. is 10361-012.Sf9 cell: Invitrogen company, article No. is 11496-015.293T cell: ATCC, article No. is CRL-1573.Huh7 cell: Chinese Academy of Sciences's cell bank, article No. is TCHu18.Insect cell medium (Insect-XpressTMMedia): Lonza Wokingham Ltd., article No. is 12-730F.Hang the affinity column material (affine beads) (Chelating SepharoseTM Fast Flow) of nickel: GE Healthcare, article No. is 17-0575-02.
At pcDNA3.1(+) HindIII and XhoI restriction enzyme site between the double chain DNA molecule shown in sequence 8 of insertion sequence table, obtain the eukaryon expression plasmid (called after recombinant plasmid penta) can expressing MERS-CoV total length membranin.
PNL4-3R-E-luciferase plasmid: reference: He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR.J Virol69:6705 – 6711,1995..
The preparation of embodiment 1, RBD fragment
One, the structure of recombinant plasmid
PFastBac tMdual vector itself is without the signal peptide of exocytosis, and RBD fragment itself is secreted protein, thus carrier needs the encoding sequence being connected into signal peptide in advance, albumen insect cell expression and secretion process in signal peptide by cut.
1, the double chain DNA molecule (coding of the double chain DNA molecule shown in the sequence 4 gp67 signal peptide of sequence table) shown in sequence 4 of composition sequence table.
2, with step 1 synthesize double chain DNA molecule for template, with F1 and R1 composition primer pair carry out pcr amplification, obtain pcr amplification product.
F1：5’-cgg AGATCTATGCTACTAGTAAATCAG-3’；
R1：5’-gcg GAATTC GC CGCAAAGGCAGAATGCGC-3’。
3, use the pcr amplification product of restriction enzyme BglII and EcoRI double digestion step 2, reclaim digestion products.
4, with restriction enzyme BamHI and EcoRI double digestion pFastBac tMdual vector, reclaims the carrier framework of about 5200bp.
5, the carrier framework that digestion products step 3 obtained and step 4 obtain is connected, and obtains recombinant plasmid first.
6, the double chain DNA molecule shown in sequence 2 of composition sequence table.
7, with step 6 synthesize double chain DNA molecule for template, with F2 and R2 composition primer pair carry out pcr amplification, obtain pcr amplification product.
F2：5’-cgc ggatccc GAGGCTAAGCCATCTGGCTCT-3’；
R2：5’-ATACGC gtcgacCTAatgatggtgatggtggtg GTATTCCACACAGTTGCC-3’。
8, use the pcr amplification product of restriction enzyme BamHI and SalI double digestion step 7, reclaim digestion products.
9, by the recombinant plasmid first that restriction enzyme BamHI and SalI double digestion step 5 obtain, the carrier framework of about 5300bp is reclaimed.
10, the digestion products of step 8 is connected with the carrier framework of step 9, obtains recombinant plasmid second.According to sequencing result, structrual description carries out to recombinant plasmid second as follows: at pFastBac tMthe double chain DNA molecule shown in sequence 5 of sequence table is inserted between BamHI and the SalI restriction enzyme site of dual vector.
The protein of recombinant plasmid second shown in the sequence 1 of expressed in insect cells sequence table (by its called after RBD-His albumen), wherein from N-terminal the 4 to 243 amino acids residue composition RBD fragment, the 244 to 249 amino acids residue composition His label.
Two, the preparation of restructuring Bacmid
1, recombinant plasmid second step one obtained adds in the DH10Bac competent cell just melted, and places 30min on ice; Then 42 DEG C of heat shock 75s, put back to 2min on ice; Then 500 μ l LB liquid nutrient mediums are added, in 37 DEG C of recovery 5h; Then draw the LB solid medium that 10 μ l coat containing 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins, 40 μ g/ml IPTG and 100 μ g/ml X-gal dull and stereotyped, lucifuge cultivates three days to growing blue hickie clearly.
2, the single bacterium colony of picking white, is inoculated in the LB liquid nutrient medium of 5mL containing 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins, 37 DEG C, 220rpm shaking culture 12 hours.
3, get the culture system that step 2 obtains, the little extraction reagent kit of plasmid (article No. is 27106 for QIAprep Spin Miniprep Kit, Qiagen company, wherein containing P1 reagent, P2 reagent and P3 reagent) of employing extracts plasmid, and concrete steps are as follows successively:
(1) by centrifugal for culture system 13000rpm 2min, bacterial sediment is collected, with the resuspended thalline of P1 reagent;
(2) add P2 reagent, slowly put upside down mixing 6-8 time;
(3) add P3 reagent, slowly put upside down mixing 6-8 time (visible white precipitation), the centrifugal 10min of 13000rpm, gets supernatant liquor;
(4) get the supernatant liquor that 600 μ l steps (3) obtain, add the Virahol of 800 μ l precoolings, place 10min, the then centrifugal 15min of 13000rpm, collecting precipitation for-20 DEG C;
(5) by the precipitation that the resuspended step of 70% aqueous ethanolic solution (4) of 500 μ l precoolings obtains, the centrifugal 5min of 13000rpm, collecting precipitation, after alcohol is dried up completely, with the ddH2O dissolution precipitation of 65 DEG C of preheatings, the centrifugal 5min of 13000rpm, draws supernatant, be the solution of restructuring Bacmid, be called for short Bacmid solution.
Three, the preparation of recombinant virus and amplification
1, get well-grown Sf9 cell, add in 10cm culture dish, leave standstill 10min, cell attachment, basis of microscopic observation, ensure about have 70%-80% to be covered by cell bottom culture dish.
2, Cellfectin II Reagent (Invitrogen company, article No. is 10362-100) 15 μ l are got, with 100 μ l insect cell medium dilutions.
3, the Bacmid solution that 15-20 μ l step 2 obtains is got, with 100 μ l insect cell medium dilutions.
4, liquid phase step 2 obtained slowly adds in the liquid phase that step 3 obtains, and slowly evenly, room temperature leaves standstill 30min, is diluted to 2ml with insect cell medium in piping and druming.
5, the culture dish of completing steps 1 is got, supernatant discarded, slowly uniform liquid phase step 4 obtained drops in culture dish, inhale after 27 DEG C of quiescent culture 5h and abandon supernatant, add the insect cell medium that 7ml is fresh, with the rear 27 DEG C of quiescent culture 8 days (in practical application 7-10 days) of sealed membrane sealing, collect nutrient solution, the centrifugal 6min of 600g, get supernatant liquor, add foetal calf serum and make its volumetric concentration be 2-5%, preserving for a long time, being the virus liquid (be called for short RBD-His-P0 for virus liquid) of P0 for recombinant virus.
6, get the virus liquid of P0 for recombinant virus, the cell concn adding shake-flask culture according to the volume ratio of 1:1000 is 2 × 10 6in the Sf9 enchylema of individual cell/mL, 27 DEG C, 110rpm cultivates 5 days, collect nutrient solution, the centrifugal 6min of 600g, gets supernatant liquor, is the virus liquid (be called for short RBD-His-P1 for virus liquid) of P1 for recombinant virus.
Four, the expression of albumen and extraction
1, get the virus liquid of P1 for recombinant virus, adding 1L cell concn according to the volume ratio of 1:100 is 2 × 10 6in the Sf9 enchylema of individual cell/mL, 27 DEG C, 110rpm cultivates 72 hours (in practical application 48-96 hour), the centrifugal 15min of 4000rpm, collects supernatant liquor.
2, the glass fibre membrane suction filtration that supernatant liquor step 1 obtained is double-deck 0.45 μm, collects filtrate.
3, by cross-flow ultrafiltration system (Masterflex PharMed BPT Tubing system, Cole-Parmer company, article No. is 06508-24) filtrate that step 2 obtains is concentrated, add pH7.2,10mM HEPAS damping fluid of Buffer1(containing 150mMNaCl simultaneously) constantly dilute, albumen is made to replace in Buffer1, then the centrifugal 30min of 13000rpm, collects supernatant liquor.
Five, the purifying of albumen
1, affinity chromatography
(1) add the affinity column material hanging nickel in the supernatant liquor obtained in 3 of step 4, hatch 3 hours (in practical application 2-5 hour) for 4 DEG C, the centrifugal 5min of 400rpm, gets precipitation.
(2) by the precipitation that the Buffer1 washing step (1) that 100mL contains 20mM imidazoles obtains, to remove foreign protein.
(3) by the precipitation that the Buffer1 washing step (2) that 20mL contains 500mM imidazoles obtains, solution is collected.
2, use 10kD evaporating pipe to concentrate the solution that step 1 obtains, obtain the protein concentrated solution of 1.2ml.
3, gel-filtration column (HiLoad Superdex75prep grade is used, column volume 120mL, GE Healthcare) protein concentrated solution that obtains of purification step 2, wash-out is carried out with Buffer1, flow velocity is 1mL/min, collecting retention volume is solution after the post excessively of 60-75mL, is RBD-His protein solution.
RBD-His protein solution is carried out electrophoresis, shows single band.This single band is carried out the order-checking of N end, N holds front 10 amino acids if the sequence 1 of sequence table is from shown in N-terminal the 1 to 10.
The preparation of embodiment 2, DPP4 extracellular domain fragment
One, the structure of recombinant plasmid
1, the double chain DNA molecule shown in sequence 7 of composition sequence table.
2, with step 7 synthesize double chain DNA molecule for template, with F3 and R3-1 composition primer pair carry out pcr amplification, obtain pcr amplification product.
F3：5’-cgc ggatcccAGTCGCAAAACTTACACTCT-3’；
R3-1：5’-ATACGC GTCGACCTAatggtggtgatggtggtg AGGTAAAGAGAAACATTGTTTTATG-3’。
3, with step 1 synthesize double chain DNA molecule for template, with F3 and R3-2 composition primer pair carry out pcr amplification, obtain pcr amplification product.
R3-2：5’-ATACGC GTCGACCTA AGGTAAAGAGAAACATTGTTTTATG-3’
4, use the pcr amplification product of restriction enzyme BamHI and SalI double digestion step 2, reclaim digestion products.
5, by the 5 recombinant plasmid first obtained of the step one of restriction enzyme BamHI and SalI double digestion embodiment 1, the carrier framework of about 5300bp is reclaimed.
6, the digestion products of step 4 is connected with the carrier framework of step 5, obtains recombinant plasmid third.The protein of recombinant plasmid third shown in the sequence 6 of expressed in insect cells sequence table (by its called after DPP4-His albumen), wherein from N-terminal the 4 to 731 amino acids residue composition DPP4 extracellular domain fragment, the 732 to 737 amino acids residue composition His label.
7, use the pcr amplification product of restriction enzyme BamHI and SalI double digestion step 3, reclaim digestion products.
8, by the 5 recombinant plasmid first obtained of the step one of restriction enzyme BamHI and SalI double digestion embodiment 1, the carrier framework of about 5300bp is reclaimed.
9, the digestion products of step 7 is connected with the carrier framework of step 8, obtains recombinant plasmid fourth.The protein (by its called after DPP4-WT albumen) that recombinant plasmid fourth forms from N-terminal the 1 to 731 amino acids residue in the sequence 6 of expressed in insect cells sequence table.
Two, the preparation of recombinant virus and the expression and purification of DPP4-His albumen
1, replace recombinant plasmid second with recombinant plasmid third, operate by the step 2 of embodiment 1, step 3 and step 4 successively.P0 is obtained successively for the virus liquid (be called for short DPP4-His-P0 for virus liquid) of recombinant virus, P1 for the virus liquid (being called for short DPP4-His-P1 for virus liquid) of recombinant virus and supernatant liquor in three steps.
2, affinity chromatography
(1) add the affinity column material hanging nickel in the supernatant liquor obtained in step 1, hatch 3 hours (in practical application 2-5 hour) for 4 DEG C, the centrifugal 5min of 400rpm, gets precipitation.
(2) by the precipitation that the Buffer1 washing step (1) that 100mL contains 20mM imidazoles obtains, to remove foreign protein.
(3) by the precipitation that the Buffer1 washing step (2) that 20mL contains 500mM imidazoles obtains, solution is collected.
3, use 30kD evaporating pipe to concentrate the solution that step 2 obtains, obtain the protein concentrated solution of 0.75ml.
4, Superdex20010/300GL(GE Healthcare is used, column volume 24mL, article No. is 17-5175-01) protein concentrated solution that obtains of purification step 3, adopt buffer2(pH8.8,25mM Tris damping fluid containing 30mM NaCl) carry out wash-out, flow velocity is 1mL/min, and collecting retention volume is solution after the post excessively of 12.5-15mL.
5, after the post excessively step 4 obtained, solution is splined on RESOURCE tMq ion exchange column (GE Healthcare, article No. is 17-1179-01, column volume is 6mL), first wash containing the buffer2 of 30mM NaCl with 10mL, then adopting and the concentration of NaCl linearly being risen to 1000mM(flow velocity by 30mM in 60 minutes is 2mL/min), solution after the post excessively obtained when collection NaCl concentration is 100-140mM.
6, use 30kD evaporating pipe to step 5 obtain cross post after solution concentrate, obtain the protein concentrated solution of 0.75ml.
7, Superdex20010/300GL(GE Healthcare is used, column volume 24mL, article No. is 17-5175-01) protein concentrated solution that obtains of purification step 6, wash-out is carried out with Buffer1, flow velocity is 1mL/min, collecting retention volume is solution after the post excessively of 12.5-15mL, is DPP4-His protein solution.
DPP4-His protein solution is carried out electrophoresis, shows single band.This single band is carried out the order-checking of N end, N holds front 10 amino acids if the sequence 6 of sequence table is from shown in N-terminal the 1 to 10.
Three, the restructuring preparation of Bacmid, the preparation of recombinant virus and amplification
Replace recombinant plasmid second by recombinant plasmid fourth, operate by the step 2 of embodiment 1 and step 3 successively.P0 is obtained successively for the virus liquid (be called for short DPP4-WT-P0 for virus liquid) of recombinant virus and the P1 virus liquid (abbreviation DPP4-WT-P1 is for virus liquid) for recombinant virus in two steps.
The binding experiment of embodiment 3, RBD and DPP4
One, coexpression and pull-down experiment
1, the RBD-His-P1 that DPP4-WT-P1 embodiment 2 prepared is prepared for virus liquid and embodiment 1 infects respectively for virus liquid or coinfection density is 2 × 10 6(when infecting respectively, the volume ratio of virus liquid and enchylema is 1:100 to the Sf9 enchylema of individual cell/mL; Two-strain liquid equal-volume mixing during coinfection, the volume ratio of hybrid virus liquid and enchylema is 1:100), 27 DEG C, 110rpm cultivates 72h, then the centrifugal 5min of 4000rpm, gets supernatant.
2, add the affinity column material hanging nickel in the supernatant liquor obtained in step 1,4 DEG C, 30rpm vibrates 3h, then the centrifugal 5min of 4000rpm, gets precipitation.
3, in the precipitation obtained to step 2, the Buffer1 added containing 20mM imidazoles washs, and then adds the Buffer1 containing 1M imidazoles, 4 DEG C, 30rpm hatches 10min, and the centrifugal 5min of 400rpm, gets supernatant liquor.
4, supernatant liquor step 3 obtained carries out SDS-PAGE electrophoresis, sees Fig. 1.
In Fig. 1, swimming lane 1 is the result of transfection RBD-His-P1 for virus liquid, and swimming lane 2 is the result of transfection DPP4-WT-P1 for virus liquid, and swimming lane 3 is the result of cotransfection.Transfection RBD-His-P1 is for after virus liquid, and can obtain RBD-His albumen by purifying, electrophoresis detection presents a band.Transfection DPP4-WT-P1, for after virus liquid, because DPP4-WT albumen does not have his-tag, is eliminated by washing, does not show any band.After cotransfection, due to DPP4-WT albumen can with RBD-His protein binding, thus display DPP4-WT albumen and RBD-His albumen two band.
Two, gel filtration experiment
Molecular sieve (Superdex200 post) is crossed after RBD-His protein solution mixing prepared by DPP4-His protein solution embodiment 2 prepared and embodiment 1, go out peak position more independent than two albumen to go out peak position all forward, define the larger mixture of molecular weight after proving two albumen mixing.Containing RBD and DPP4 two albumen in SDS-PAGE detection display mixture.The results are shown in Figure 2.RBD and DPP4 can form stable compound really
Three, the structure elucidation of RBD and DPP4
The DPP4-His albumen that RBD-His albumen embodiment 1 prepared and embodiment 2 prepare, according to mol ratio 1.2:1 mixing, hatches 1h on ice; Use 10kD super filter tube to be concentrated into 0.75ml, use Superdex200 to carry out purifying and obtain RBD-DPP4 mixture; By the protein concentration of RBD-DPP4 complex solution to 10mg/ml, sitting-drop methods screening crystal; Fusiformis monocrystalline is grown under the PEG1500 condition of 25%; Synchrotron radiation X-ray source is used to carry out Diffraction Data Collection; Use the software data processings such as HKL-2000, DPP4, Coot, phoenix, use molecular replacement technique to solve phase place, final analytic structure.
The preparation of embodiment 4, RBD rabbit immune serum and antibody purification
One, the preparation of antibody
Japan large ear rabbit purchased from company of dimension tonneau China is carried out following immunity:
Initial immunity: (protein concentration is 0.5mg/ml to the RBD-His protein solution 800 μ l embodiments 1 prepared, PBS damping fluid adjustment protein concentration with pH7.5) fully mix with 800 μ l complete Freund's adjuvants, after forming white emulsion, dorsal sc multi-point injection is carried out to single white rabbit.
Booster immunization, from initial immunity, carried out booster immunization once every two weeks, and the full freund's adjuvant that cannots be used up in booster immunization replaces complete Freund's adjuvant, other all same initial immunity.
Within after initial immunity the 13rd day, carry out auricular vein and get blood, within after booster immunization the 7th day, carry out getting blood, separation of serum.
Two, the purifying of specific antibody
Affinity column material: NHS Activated Sepharose4Fast Flow(GE company, 17-0906-01).Be coupled with albumen: RBD-His albumen prepared by embodiment 1.Be coupled damping fluid: the NaHCO of pH7.7,0.1M 3solution.Confining liquid: the Tris-HCl damping fluid of pH8.3,0.1M.Low ph value damping fluid: containing the 0.1M acetate buffer of 0.5M NaCl.
1, affinity column (carrying out following steps successively) is prepared
(1) get affinity column material 1.5ml, add 4.5ml1mM aqueous hydrochloric acid, mixing, the centrifugal 1min of 1500rpm, abandons supernatant; Add 4.5ml1mM aqueous hydrochloric acid, mixing, the centrifugal 1min of 1500rpm, abandons supernatant; Add 4.5ml1mM aqueous hydrochloric acid, the centrifugal 1min of room temperature activation 45min, 1500rpm, abandons supernatant.
(2) add 4.5ml and be coupled damping fluid, mixing, the centrifugal 1min of 1500rpm, abandons supernatant.
(3) RBD-His protein solution (protein concentration is 1mg/ml, the PBS damping fluid adjustment protein concentration with pH7.5) prepared by 1ml embodiment 1 is added, mixing, room temperature reaction 3h.
(4) add 4.5ml confining liquid, mixing, the centrifugal 1min of room temperature reaction 3h, 1500rpm, abandons supernatant.
(5) add 4.5ml low ph value damping fluid, mixing, the centrifugal 1min of 1500rpm, abandons supernatant, adds 4.5ml confining liquid, mixing, and the centrifugal 1min of 1500rpm, abandons supernatant; Add 4.5ml low ph value damping fluid, mixing, the centrifugal 1min of 1500rpm, abandons supernatant, adds 4.5ml confining liquid, mixing, and the centrifugal 1min of 1500rpm, abandons supernatant; Add 4.5ml low ph value damping fluid, mixing, the centrifugal 1min of 1500rpm, abandons supernatant, adds 4.5ml confining liquid, mixing, and the centrifugal 1min of 1500rpm, abandons supernatant; Add 4.5ml low ph value damping fluid, mixing, the centrifugal 1min of 1500rpm, abandons supernatant, adds 4.5ml confining liquid, and mixing, the centrifugal 1min of 1500rpm, abandons supernatant.
(6) with the post material that the PBS buffer preserving of the pH7.2 of sterilizing is coupled, 4 DEG C are stored in
2, aggressive pathogenic antibody (carrying out following steps successively)
(1) with affinity column prepared by the PBS damping fluid equilibrium step 1 of pH7.2.
(2) the PBS damping fluid of serum pH7.2 obtained for the 7th day after first time booster immunization in step one is diluted to triploid to amass, filters with the filter membrane of 0.45 μm, filtrate is added affinity column.
(3) with the PBS buffer solution of the pH7.2 of 15ml.
(4) with pH2.5,0.1M glycine buffer wash-out of 5ml, collected solution after post, adjust pH is to neutral (regulating pH with the Tris-HCl damping fluid of pH8.0,1M).
(5) that gets that step (4) obtains crosses solution after post, and dialyse with the PBS damping fluid of pH7.2, the solution obtained is specific antibody solution.
Embodiment 5, MERS-CoV pseudovirus infect experiment and Neutralization effect detects
One, the preparation of MERS-CoV pseudovirus
By recombinant plasmid penta and pNL4-3R-E-luciferase plasmid co-transfection 293T cell, 48 h before harvest cell conditioned mediums, are the solution containing MERS-CoV pseudotype virus, utilize ELISA kit (the HumanHIV p24ELISA Kit of p24 detection by quantitative; Beijing Quanto Biotechnology Co., Ltd., article No. 1950961) carry out the mensuration of virus titer.
Two, the Neutralization effect of RBD-His albumen detects
The PBS buffer gradient dilution of the RBD-His protein solution pH7.5 1, embodiment 1 prepared, the solution of MERS-CoV pseudotype virus of containing diluent and step one prepared mixes that (each mixed system is 200 μ L, and viral dosage is 300TCID 50, the peak concentration of RBD-His albumen is 50 μ g/mL), 37 DEG C of stationary incubation 1 hour.
2, the system 200 μ L steps 1 obtained and 100 μ L Huh7 enchylema are (containing 2 × 10 3individual cell) mixing, in 37 DEG C of stationary incubation 60 hours.
3, get the cell that step 2 obtains, adopt Luciferase Assay Reagent box (Renilla-Glo tMluciferaseAssay System; Promega company, article No.: E2750) and detect uciferase activity by test kit specification sheets lysing cell.
The blank group replacing diluent with the PBS damping fluid of pH7.5 is set.
With the PBS damping fluid of pH7.5 as solvent, prepare the BSA solution of each BSA concentration, replace RBD-His protein solution to carry out above-mentioned steps with BSA solution.
Fluorescence intensity × 100% of Neutralization effect=(fluorescence intensity of the fluorescence intensity-experimental group of blank group)/blank group.
Neutralization effect the results are shown in Figure 3.The IC50(of RBD-His albumen and Neutralization effect are the protein concentration of 50% correspondence) be 3.45 μ g/ml, the BSA of albumen does not have Neutralization effect in contrast.Result shows, RBD-His albumen has the ability that stronger suppression MERS-CoV infects Huh7 cell.
Three, the Neutralization effect of specific antibody solution detects
The specific antibody solution prepared by embodiment 4 replaces RBD-His protein solution, replaces BSA, other same step 2 with VRC01 antibody (control antibodies).
Neutralization effect the results are shown in Figure 4.The IC50(of specific antibody and Neutralization effect are the protein concentration of 50% correspondence) be 1.52 μ g/ml, control antibodies does not have Neutralization effect.Result shows, the serum that RBD-His immune rabbit obtains has the ability that stronger suppression MERS-CoV infects Huh7 cell.

Claims (13)

1. a protein, is following (a) or (b) or (c) or (d):
A protein that the sequence 1 of () sequence table forms from N-terminal the 4 to 243 amino acids residue;
B protein that the sequence 1 of () sequence table forms from N-terminal the 1 to 243 amino acids residue;
C protein that the sequence 1 of () sequence table forms from N-terminal the 4 to 249 amino acids residue;
The protein shown in sequence 1 of (d) sequence table.
2. the gene of protein described in coding claim 1.
3. gene as claimed in claim 2, is characterized in that: described gene is arbitrary described DNA molecular in following (1) to (5):
(1) DNA molecular shown in sequence 2 in sequence table;
(2) DNA molecular shown in sequence 3 in sequence table;
(3) in sequence table sequence 5 from the DNA molecular shown in 5 ' end 1-843 position Nucleotide;
(4) in sequence table sequence 5 from the DNA molecular shown in 5 ' end 124-864 position Nucleotide;
(5) DNA molecular shown in sequence 5 in sequence table.
4. the expression cassette containing gene described in Claims 2 or 3, recombinant vectors, transgenic cell line, recombinant virus or recombinant bacterium.
5. with the antibody that protein described in claim 1 obtains for immunogen.
6. protein described in claim 1 is preparing the application treated and/or prevented in the medicine of MERS-CoV.
7. protein described in claim 1 treats and/or prevents the application in the medicine of MERS-CoV dip-dye mammalian cell in preparation; Described Mammals is behaved.
8. antibody described in claim 5 is preparing the application treated and/or prevented in the medicine of MERS-CoV.
9. antibody described in claim 5 treats and/or prevents the application in the medicine of MERS-CoV dip-dye mammalian cell in preparation; Described Mammals is behaved.
10. treat and/or prevent a medicine of MERS-CoV, its activeconstituents is antibody described in protein described in claim 1 or claim 5.
11. 1 kinds treat and/or prevent the medicine that MERS-CoV contaminates mammalian cell, and its activeconstituents is described protein or described antibody; Described Mammals is behaved.
Protein described in 12. claims 1 has in conjunction with the application in the product of the function of DPP4 or DPP4 extracellular domain fragment in preparation.
13. 1 kinds of products with the function in conjunction with DPP4 or DPP4 extracellular domain fragment, its activeconstituents is protein described in claim 1.
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