CN104086652A - Anti-O type foot and mouth disease virus specific single-domain antibody and recombinant expression vector thereof - Google Patents

Abstract

The invention discloses an anti-O type foot and mouth disease virus specific single-domain antibody and a recombinant expression vector thereof. A nucleotide sequence of the anti-O type foot and mouth disease virus specific single-domain antibody is shown in SEQIDNO1 or SEQIDNO2. The anti-O type foot and mouth disease virus specific single-domain antibody has the advantages that an O type foot and mouth disease virus inactivated vaccine immunes Bactrian camel, a phage display technology is utilized for establishing an anti-FMDVO (Foot-and-Mouth Disease Virus) type VHH (variable domain of heavy chain of heavy-chain antibody) immune antibody library, recombinant VP1 (virus polyprotein 1) protein is taken as an antigen used for screening, high-strength elutriation is carried out for three times, two specific anti-FMDV serum O type heavy chain antibody sequences with strong affinity with VP1 antigens are obtained and are respectively cloned into a prokaryotic expression vector by screening, soluble expression is completed in E.coli, affinity chromatographically purified VHH antibody protein can specifically interact with an O type FMDV totivirus antigen and recombinant VP1 protein, a cross reaction is not taken with FMD virus Asia1 and type A, and thus biological functions which are the same as those of the conventional IgG antibody are obtained.

Description

Resisting O-type foot and mouth disease virus specificity single domain antibody and recombinant expression vector thereof

Technical field

The present invention relates to biological technical field, specifically the specific resisting O-type foot and mouth disease virus single domain antibody of One serotype and recombinant expression vector thereof.

Background technology

Foot and mouth disease (FMD) is a kind of deadly infectious disease being caused by foot and mouth disease virus (FMDV), and main infection artiodactyl, comprises ox, the important domestic animals such as sheep and pig.The outburst of FMD often causes huge financial loss, is therefore classified as first of the most important transmissible disease that affects aquaculture safety by countries in the world.At present, in 7 serotypes of FMDV, O type is that clinical onset is maximum, one of serotype of destructive force maximum, and all there is the epidemic situation of O type FMD in tens countries of priority in this year.In the time of outburst FMD epidemic situation, developed country takes the mode of catching and killing thoroughly to eliminate all predisposing factors, reaches without FMD epidemic situation state, and developing country still takes means control FMD popular of vaccine compulsory immunization.In the structural protein of FMDV coding, VP1 can induce ox and pig to produce protectiveness neutralizing antibody, is viral immune protective antigen, is the candidate albumen of development recombinant vaccine.

Camelidae (comprises two-humped camel, dromedary, alpaca, camel horse, guanaco and llama) and the serum such as cartilage shark in not only there is conventional IgG antibody, also there is a kind of natural disappearance light chain simultaneously, only there is the IgG antibody of heavy chain, the antigen binding site of this antibody-like is only made up of the single structure territory of variable region of heavy chain, is named as heavy chain antibody (VHH) or single domain antibody (sdAb), and VHH has equally kept good, special antigen binding capacity with common IgG antibody molecule.It is the current known smallest molecule fragment with complete antibody function, and molecular weight is only 15kD, is called nano antibody (Nanobody, Nb).VHH antibody has the complementary land of longer antigen, and therefore tissue penetration sexuality is strong, can be applied to the antigen position that some common antibody molecules cannot contact, the function of performance antibody.In VHH amino acid, there is more hydrophilic amino acid, the antibody protein good water solubility of in-vitro recombination expression.Research finds, the VHH antibody that screening obtains also possesses the unique functions such as high temperature resistant, the pH of resistance to extreme value.Therefore, VHH antibody is the fundamental research at antigen as the genetic engineering antibody of miniaturization, and small molecular antibody medicine and highly sensitive diagnostic reagent research and development field have broad prospect of application.

Summary of the invention

The object of this invention is to provide a kind of resisting O-type foot and mouth disease virus specificity single domain antibody.

Another object of the present invention is to provide a kind of recombinant expression vector.

Technical solution of the present invention is as follows: a kind of resisting O-type foot and mouth disease virus specificity single domain antibody, is characterized in that described antibody has nucleotide sequence: SEQ ID NO 1 or SEQ ID NO 2.

A kind of recombinant expression vector, contains nucleotide sequence claimed in claim 1.

A kind of host cell, described host cell is the prokaryotic cell prokaryocyte that contains above-mentioned nucleotide sequence.

The present invention is with O type foot and mouth disease virus inactivated vaccine immunity two-humped camel, utilize display technique of bacteriophage to set up anti-FMDV O type VHH immune antibody library, using restructuring VP1 albumen as screening antigen, taking turns high strength by 3 eluriates, screening obtains the heavy chain antibody sequence of the 2 strain specificity anti-FMDV serotype O strong with VP1 antigen avidity, and it is cloned into respectively in prokaryotic expression carrier, e.coliin completed solubility expression, the VHH antibody protein of affinitive layer purification can interact with restructuring VP1 protein-specific by specific same O type FMDV totivirus antigen, there is not cross reaction with FMD virus Asia 1 and A type, possessed with the identical biological function of conventional IgG antibody.

Brief description of the drawings

Fig. 1 is mono-clonal Phage-ELISA result figure;

Fig. 2 is Multiple Sequence Alignment result figure;

Fig. 3 is the VHH antibody figure of D01 and A01 affinitive layer purification;

Fig. 4 is A01 and D01 Western blotting VHH histogram;

Fig. 5 is the type specificity qualification figure of two strain VHH antibody.

Embodiment

Experiment material used and related operating method are as follows:

1 laboratory animal and reagent

Domestic 1 female two-humped camel of age 2 peaks, ox lymphocyte separation medium, 96 hole enzyme plates (Costar); Restriction enzyme (NEB), DNA fragmentation reclaims test kit and is OMEGA product, plasmid extraction kit is purchased from Beijing Quanshijin Biotechnology Co., Ltd, primer is synthetic by Sangon Biotech (Shanghai) Co., Ltd., nickel affinity chromatography resin (Qiagen), nucleic acid Marker, pvdf membrane, the anti-His antibody of HRP mark is purchased from Beijing CoWin Bioscience Co., Ltd.; FMDV O type inactivated vaccine, VP1 recombinant antigen, the anti-FMDV A of standard, Asia1 and O type positive serum, without FMDV negative antibody serum and FMDV A, Asia1 and O type LPB-ELISA detection kit all from OIE/ country foot and mouth disease reference laboratory; Bacterial strain, helper phage and reagent e. coli tg1 and HB2151, helper phage M13K07, carrier pHEN2, purchased from Novagen company.Other reagent are domestic analytical pure.

2 methods

2.1 camel immunity and antibody tests

With the subcutaneous multiple spot immunity of the dosage O type FMD inactivated vaccine camel of 2 part oxen, inoculation position comprises back, back leg and both sides musculi colli.Before immunity and after vaccine inoculation 21,35,42 and 63 days, camel is carried out to booster immunization with identical dosage.Before first immunisation and after each booster immunization, all gather venous blood and be separated to serum, blocking Enzyme-linked Immunosorbent Assay method (LPB-ELISA) by liquid phase and detect antibody titers.

the separation of 2.2 peripheral bloods

Last immune latter 7 days (the 63rd day) by experiment the jugular vein collection anticoagulation 50mL(antithrombotics of animal is heparin sodium, 10IU/mL blood).Anticoagulation adds after lymphocyte separation medium, carries out density gradient centrifugation and separates outer total lymphocyte.After during isolated total lymphocyte is resuspended with MEM, be transferred in six orifice plates and cultivate (37 DEG C, 5%CO ₂) static 30 min, remove cell debris and scavenger cell, that again collects is stored in the extraction for cell RNA in liquid nitrogen for total lymphocyte.

3. total RNA extracts, and goal gene amplification and VHH antibody library build

Design primer ( table 1-vHH primer set for amplification )amplification VHH gene fragment, carries out three-wheel amplification.The total RNA of lymphocyte that utilizes Trizol extracting to separate, then use G1 as reverse transcription primer, synthetic the first chain cDNA, taking cDNA as template, H1, G1 are 2 DNA bands that upstream and downstream primer amplification obtains size and be respectively 600bp and 900bp, reclaim big or small 600bp band as template, carry out the 2nd and take turns pcr amplification.Take turns PCR product as template taking the 2nd, H2, H3 are that upstream and downstream primer obtains approximately big or small 450bp DNA band.Take turns PCR product as template taking 2, with P1, TN is that upstream and downstream primer carries out the 3rd and takes turns amplification, obtains about 450bp band.Take turns PCR product process by the 3rd sfii/ notafter I double digestion, be cloned on pHEN2 carrier, utilize electric method for transformation that connection product is transformed in e. coli tg1, electricity turns after end, 37 DEG C of bacterium liquid, and 180r/min shaking table is cultivated 1h, after centrifugal concentrating, be coated in 2 × YTG flat board (2% glucose, the 100 μ g/mL Amp of ammonia benzyl resistance ^r, 2 × YT).Get 10 μ L simultaneously and be diluted to 1 × 10 ⁴doubly carry out coated plate, 37 DEG C of incubated overnight, for calculating storage capacity.

Flat board after conversion is cultivated after 16 hours in 37 DEG C of incubators, the lawn growing on culture plate is scraped to wash clean with 2 × YT substratum of 10mL, add the glycerine of final concentration 25%, packing is kept at-70 DEG C of anti-FMDV serotype O antibody VHH heavy chain antibody immunity storehouses for subsequent use, this is acquisition.Random choose clone after electricity transforms, extracting plasmid checks order, and the diversity of qualification antibody library is calculated storage capacity according to diversity and clone's number.

4. the special resisting O-type FMDV VHH antibody of screening

O type foot and mouth disease restructuring VP1 antigen (20 μ g/mL) after the coated dilution of 100 μ L systems, utilizes display technique of bacteriophage to separate the heavy chain antibody of specific combination O type FMDV.Take turns high strength through 3 and screen, occur obvious enrichment effect.Pick out 131 clones are verified to wherein have 12 clones obviously positive with mono-clonal Phage-ELISA.Extract after plasmid, by life work biology (Shanghai), limited-liability company checks order.

5. the expression and purification of restructuring resisting O-type FMDV VHH

Extraction is accredited as positive clone's plasmid, and amplification VHH antibody variable gene (table 2), with containing bsaIwith bamHIthe primer of restriction enzyme site carries out after pcr amplification, after agarose gel electrophoresis qualification, under ultraviolet light conditions, observes amplification.By gel DNA fragmentation purification kit recovery object fragment, then bsaIwith bamHIcut rear enzyme and cut, the product after purifying enzyme is cut is subcloned on p-SMK expression vector, and transforms BL21-codon-Plus (DE3)-RIL competent cell.By cut the positive bacterium colony that qualification obtains with PCR by enzyme, at LB liquid nutrient medium (80 μ g/mL Kam ^r, 30 μ g/mL Cm ^r) in, 37 DEG C, when under 220rpm/min condition, concussion is cultured to OD value and reaches 0.6-0.8, adding final concentration is the IPTG of 1mM/L, 20 DEG C, 180rpm/min concussion was cultivated after 12 hours, centrifugal collection bacterium liquid, resolution of precipitate is in the Tris-HCl of 50mM/L damping fluid (pH8.0), (3 circulations, work 2 seconds ultrasonic degradation thalline under condition of ice bath, intermittently 2 seconds, 10min/ circulation), thoroughly cracking thalline.Liquid after ultrasonic is 4 ^oc, 8000rpm/min, 30min centrifugation supernatant.After the membrane filtration of supernatant with 0.45 μ m, add the target protein in the affine resin absorption supernatant of nickel, with washing foreign protein solution (30mM/L imidazoles, 150mM NaCl, 50mM/L Tris-HCl, 0.05% Tween-20, pH 8.0) abundant wash-out foreign protein, then use the elutriant (300mM/L imidazoles, 150mM NaCl, 50mM/L Tris-HCl, pH 8.0) of 5 mL to carry out wash-out to target protein.The target protein taking a morsel mixes with sample-loading buffer, boils after 10 minutes SDS-PAGE electrophoretic analysis target protein expression and purification result.Gel after SDS-PAGE electrophoresis finishes is washed twice (5min/ time) with PBST, with general-purpose equipment temperature formula transferring film system Transfer System(BIO-RAD) protein band is transferred on pvdf membrane.PBST washes film twice (5min/ time), and with 5% skim-milk sealing, 4 DEG C of placements are spent the night.Discard confining liquid, PBST washes film three times (5min/ time).Primary antibodie (mouse-anti His antibody) is diluted and is added on film by 1:1000, incubated at room 2h.Discard primary antibodie, PBST washes film three times (5min/ time).Two anti-(sheep anti mouses, and mark HRP) dilute by 1:100, are added on pvdf membrane incubated at room 2h.Discard two and resist, add nitrite ion (hydroperoxide dissolution of 30 μ l 30% is in 30mlPBS for 15mg cobalt chloride, 9mg DAB) dark room conditions to develop the color, in the time that object band occurs, at once film is transferred to termination reaction in ultrapure water.

6. the Property Identification of resisting O-type FMDV VHH antibody

6.1VHH anitibody type specificity analyses

Utilize double antibodies sandwich ELISA to detect the type specificity of single domain antibody.The VHH of the resisting O-type foot and mouth disease virus after purifying presses doubling dilution, and 100 μ l/ holes are coated with 96 hole enzyme plates, 4 DEG C of overnight incubation.Discard coated night, in 96 orifice plates, add respectively 1 × PBST(pH 7.4) the 1:5 Asia I type of dilution, A type, O type foot-and-mouth disease virus antigen (deactivation).To be coated with the positive contrast in sero-fast hole of the anti-different serotypes FMDV of cavy, be coated with the negative contrast in hole of camel negative serum respectively.The anti-FMDV serum of rabbit of different serotypes carries out adding in corresponding aperture as primary antibodie after 1:1000 dilution with 1 × PBST, hatches 1h for 37 DEG C.Discard primary antibodie, 1 × PBST washs 96 orifice plate 3 times, abandons liquid in clear opening.The goat-anti rabbit anteserum 1:1000 dilution of horseradish peroxidase (HRP) mark, as two anti-adding in 96 orifice plates, is hatched 1h for 37 DEG C.Discard two and resist, 1 × PBST solution washing, 96 orifice plate 3 times, dry.Add tetramethyl benzidine (TMB) as substrate colour developing, hatch after 15min for 37 DEG C, by the sulphuric acid soln termination reaction of 2M, read the absorbance in each hole in 450nm place by microplate reader.

the avidity analysis of 6.2VHH antibody and O type FMDV

Utilize Biacore 3000 to measure the affinity constant of VHH antibody and O type FMDV antigen.In experiment, with acetate buffer (pH5.0) dilution O type foot and mouth disease virus, be coated with CM5 chip (GE healthcare).With HBS-EP damping fluid (10mM hepes, 150 mM NaCl, 3mM EDTA, 0.005% P20 surfactant) single domain antibody is diluted to the solution of 5 concentration such as 7.5nM, 15nM, 32.5 nM, 75 nM and 150 nM, flow velocity is set as 10 μ l/min, after each sample detection, carry out the regeneration of chip with glycine hydrochloride (pH3.0), utilize software BIAevaluation to analyze each binding curve under different psdAb-AP protein concentrations, calculate the affinity costant of VHH antibody and O type FMDV antigen.

Embodiment

in camel serum, the antibody horizontal of resisting O-type FMDV detects:immunity through 4 O type FMD inactivated vaccines to camel and serum antibody statistics (table 3) result show, after the 2nd booster immunization, it is qualified that the antibody of 2 laboratory animal resisting O-type FMDV has reached immunity, when last immunity is surveyed antibody titer in latter 7 days, the antibody titer of 2 peak camels all reaches 1:1024, therefore can judge now in body that body fluid and cellular immune level are all in lasting ascent stage, meet the requirement of experiment that gathers peripheral blood lymphocyte.

embodiment 1: the structure in resisting O-type FMDV VHH library

In order to build the VHH library of resisting O-type FMDV, from 6 × 10 ⁸in individual peripheral blood mononuclear cell (Peripheral Blood Mononuclear Cell, PBMC), extract total RNA, and reverse transcription obtains cDNA.Pcr amplification can obtain two fragments of size in 600bp and 900bp left and right.And then taking 600bp size fragment as template, can increase and obtain the variable region of heavy chain of VHH, size is about 450bp.With expressing primer PCR amplification, purifying reclaims after object fragment double digestion, is cloned into phagemid vector pHEN2.Recombinant plasmid transformed, in e. coli tg1, builds the VHH antibody library of resisting O-type FMDV.

After the screening of four-wheel, comparatively significantly enrichment effect (table 4) can be detected.Mono-clonal Phage-ELISA experimental result has shown 12 positive colonies, each clone's OD ₄₅₀value is shown in Fig. 1.12 clones are carried out to DNA sequencing and obtain two different VHH gene orders, respectively called after A01 and D01.

A01 and two gene orders of D01 and several characteristic sequences are carried out to aminoacid sequence and compare, comparison result is shown in Fig. 2.

embodiment 2: resisting O-type FMDV VHH exists e.coliin expression and purification

With primer A01U, A01D and D01U, D01D is upstream and downstream primer, phage vector is that template amplification obtains A01 and D01 gene order.With glue reclaim method object nucleic acid fragment is collected.Restriction enzyme bsai and bamhI carries out after double digestion, is cloned in p-SMK carrier, transforms DH5 α competent cell.After the bacterium liquid PCR checking positive, extract positive plasmid and transform BL21-codon-Plus (DE3) competent cell.Picking list bacterium colony is at LB liquid nutrient medium (Kan ^r80 μ g/mL, Cm ^r34 μ g/mL) middle cultivation.In the time that OD value reaches 0.6-0.8, add IPTG to final concentration be 1mM/mL, 20 DEG C, 200rpm, abduction delivering 20h.Centrifugal after thalline ultrasonication, get cleer and peaceful precipitation and do the soluble analysis of albumen, SDS-PAGE shows that albumen expresses with soluble form, the VHH antibody (Fig. 3) of D01 and A01 affinitive layer purification.Affinity chromatography purifying target protein, the target protein of purifying is carried out to Western blotting analysis (A01 in Fig. 4 and D01), can be observed and A01 and the specificity object band that D01 size (approximately 33 kDa) conforms to, prove these two antibody successful expression purifying.

the type specificity qualification of embodiment 3:2 strain VHH antibody

With double antibodies sandwich ELISA(Double antibodies sandwich ELISA, Das ELISA) method detects the type specificity of two VHH antibody.Experimental result shows that this two strain VHH antibody can specificly be combined with O type FMDV, with A type and Asia 1 type FMDV do not exist cross reaction ( fig. 5).

4: two strain VHH antibody of embodiment is measured the affinity of O type FMDV antigen

The demonstration of surface plasma resonance (Surface Plasmon Resonance, SPR) experimental result (table 5), A01 and D01 reach 82.4nm and 62.3nm to the KD value of O type foot and mouth disease virus, demonstrate the affinity good to antigen.

From phage display library, 131 clones of picking carry out Phage-ELISA qualification, screen altogether 12 positive colonies, all send order-checking.In each mono-clonal Phage-ELISA, coated 10 μ g/L O type foot-and-mouth disease virus antigens in experimental group hole, negative control group is envelope antigen not, and all the other operations are identical.X-axis representative clone sequence number, the representative of y axle is at the light absorption value at 450nm place.

Sequence table

<110> Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120> resisting O-type foot and mouth disease virus specificity single domain antibody and recombinant expression vector thereof

<130> 2014

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 366

<212> DNA

<213> Bactrian camel

<400> 1

gatgtgcagc tggtggagtc tgggggaggc ttggttcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt caccttcagt aactactaca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtgtctagt atttatagtt acggtacatc cacgtactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240

ctgcaaatga acagcctgaa acctgaggac acggctgtgc attactgtgc ggcgtccgat 300

agtagctgga ggatcgtcat tgagcataac tactggggcc aggggaccca ggtcaccgtc 360

tcctca 366

<210> 2

<211> 381

<212> DNA

<213> Bactrian camel

<400> 2

gatgtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgacactc 60

tcctgtgcag cctctggcag caccgtcact atcggctcga tgggctggtt ccgccaggct 120

ccagggaagg agcgcgaggg ggtcgcgctt atttatactg gacgcggtag cacgaactat 180

gccgactccg tgaagggccg attcaccatc tcccaaggca acgccaagaa cacggtgtat 240

ctgcaaatga acagcctgaa acctgaggac actgccatat actactgtgc agcagccccc 300

gggcgcccca gtctgaagta cgagatgcga caggtggact ttcgttactg gggccagggg 360

acccaggtca ctgtctcctc a 381

Claims (4)

1. a resisting O-type foot and mouth disease virus specificity single domain antibody, is characterized in that described antibody has nucleotide sequence: SEQ ID NO 1 or SEQ ID NO 2.

2. a recombinant expression vector, is characterized in that containing nucleotide sequence claimed in claim 1.

3. a host cell, is characterized in that described host cell is the prokaryotic cell prokaryocyte that contains nucleotide sequence claimed in claim 1.

4. host cell as claimed in claim 3, is characterized in that described host cell is the prokaryotic cell prokaryocyte that contains expression vector claimed in claim 2.