CN102702349A - Bactrian camel VHH (variable domain of the heavy chain of HACbs) heavy-chain antibody for resisting foot-and-mouth disease AsiaI type viruses, preparation method and use thereof - Google Patents
Bactrian camel VHH (variable domain of the heavy chain of HACbs) heavy-chain antibody for resisting foot-and-mouth disease AsiaI type viruses, preparation method and use thereof Download PDFInfo
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Abstract
The invention discloses a bactrian camel VHH (variable domain of the heavy chain of HACbs) heavy-chain antibody for resisting foot-and-mouth disease AsiaI type viruses, a preparation method and use thereof, belonging to the field of biotechnology. In the method, recombinant AsiaI type virus-like particle antigens are used for immunizing bactrian camels; an AsiaI VHH immune antibody library is established with phage display technology; high-specificity single-domain antibodies having good affinity with foot-and-mouth disease AsiaI type virus antigens are screened; the antibody provided by the invention has amino acid sequence shown by SEQ ID NO.1 or amino acid sequence which has sequence homology more than 98% with the sequence shown by SEQ ID NO.1. The AsiaI VHH heavy-chain antibody provided by the invention has great importance for researching AsiaI type virus infection mechanisms and producing high-sensitive and specific antigen fast detection or diagnosis reagents.
Description
Technical field
The present invention relates to a kind of antibody, particularly a kind of two-humped camel VHH heavy chain antibody of anti-foot and mouth disease AsiaI C-type virus C.In addition, the invention still further relates to preparation method, Function Identification and the purposes of this antibody.The invention belongs to biological technical field.
Background technology
Foot and mouth disease (Foot-and-Mouth Disease; FMD) be by foot and mouth disease virus (Foot-and-Mouth Disease Virus; What FMDV) cause is master's a kind of acute, hot, height contact, strong zoonosis with the artiodactyl morbidity, is in the world wide animal husbandry development to be threatened maximum a kind of zoonosis.The foot and mouth disease infectivity is extremely strong, sickness rate is high, can reach 100% sometimes to the lethality rate of cub, so classified as first of 14 kinds of category-A transmissible diseases of animal by OIE (OIE), China animal health department also classifies it as type of transmissible disease.
FMDV is a Picornaviridae, and the member of foot and mouth disease virus book has been found that 7 serotypes at present, i.e. O, A, C, and SAT1, SAT2, SAT2 and AsiaI type do not exist cross reaction and protection between the different serotypes.Complete AsiaI C-type virus C particle comprises capsid and RNA two portions, and wherein structural protein VP0, VP3 and VP1 constitute the main part that the viral capsid capsid protein is a stimulation animal body generation protectiveness neutralizing antibody, also are the epitope places of distinguishing serotype.
At present, main the only resource that prevents the eqpidemic disease generation through the conduct of inoculation inactivated vaccine of popular foot and mouth disease country.Passed through high-density, on a large scale constantly vaccine inoculation, pestilence is effectively controlled, but the beastly domestic animal live body of artiodactyl band virus infection animal still is hidden in the drove, is the most dangerous potential contagium.Eliminate the developed country of foot and mouth disease, reach disease through vaccine immunity with the mode of catching and killing the positive animal of cause of disease and purify, but the frequent livestock product free trade of world wide Nellie makes these countries face the danger of eqpidemic disease outburst constantly.
In the face of so severe eqpidemic disease form, foundation is quick, responsive, special etiological diagnosis method just becomes particularly important.The diagnostic antigen method that OIE recommends; Comprise that virus is separated, polyclonal antiserum is basic U.S. linked immunosorbent adsorption test (ELISA), complement fixation test (CFT) and viral nucleic acid detection method RT-PCR; These methods need be operated under the condition with specialized equipment equipment, laboratory and professional, need use remaining serotype antibody as contrast simultaneously.If can obtain to have the antibody that a certain serotype has type specificity, so just can promote diagnosis speed and accuracy, thereby the control disease of taking measures early is popular for the foot and mouth disease epidemic situation, reduce the financial loss that causes.
1993, there is a kind of natural disappearance light chain in the body such as discovery camel such as Hamers and shark, the antibody of heavy chain is only arranged, be called heavy chain antibody (HCAbs).The antigen binding site of this antibody-like only is made up of variable region of heavy chain single-domain structure territory, be called the single domain heavy chain antibody (variable domain of the heavy chain of HCAbs, VHH).Though the disappearance variable region, VHH is still equally keeping good and special antigen binding capacity with common antibody, and it is present available minimum antibody molecule fragment with complete function; Molecular weight is merely 15kD; Be 1/10 of conventional antibody, 1/3 of monoclonal antibody, numberator height 4.8nm; Diameter 2.2 nanometers are also referred to as nano antibody (nanobody).This antibody-like has the complementary land of antigen of stretching, extension and higher tissue penetration property and littler molecule individuality, and the epitope that can combine some conventional antibodies to contact is given full play to the biological function of antibody.In addition, VHH has peculiar properties such as easy expression, good water solubility, high temperature resistant, the pH of anti-extreme value, and it has broad prospect of application as the miniaturized genetic engineering antibody in fundamental research, small molecular antibody medicine and highly sensitive diagnostic reagent research and development field.At present, the research of camel VHH single domain heavy chain antibody has become the research focus in American-European countries.Lorena utilizes rotavirus vp 6 protein immunization alpacas to obtain to have the active VHH of neutralization, can be used for treating diarrhea of mouse.It is the research focus and the difficult point of cancer diagnosis all the time that the early diagnosis and therapy effect is followed the tracks of.Cortez-Retamoz utilizes small molecular weight VHH to have good organization's perviousness, high-affinity and does not damage the characteristic of healthy tissues, can detect knub position with it in vivo fast and accurately as photographic developer.Because VHH all has high antigen-specific and avidity, can be used for the target tumor specific antigen, and anti-tumor medicine is directly acted on focus, brings into play effective killing tumor cells effect.Domestic research at camel VHH still is in the starting stage.Yan An etc. utilize H5N1 avian influenza virus antigen immunity alpaca, have made up anti-H5N1VHH immunity storehouse, and exist in the elementary storehouse and have the active anti-H5N1VHH antibody of potential neutralization, for early diagnosis and the clinical treatment of H5N1 lays the foundation.Utilize two-humped camel to prepare Asia I type immune antibody storehouse, thereby screening obtain and can combine the VHH heavy chain antibody with foot and mouth disease Asia I C-type virus C antigen-specific, develops the cause of disease fast diagnosis method and does not still have report at home and abroad.
Summary of the invention
One of the object of the invention provides a kind of two-humped camel VHH heavy chain antibody of anti-foot and mouth disease AsiaI C-type virus C;
Two of the object of the invention provides the nucleotide sequence of the said two-humped camel VHH heavy chain antibody of coding;
Three of the object of the invention provides described two-humped camel VHH heavy chain antibody or its encoding sequence to detect the application in the foot and mouth disease AsiaI C-type virus C reagent in preparation.
In order to reach described purpose, the present invention has adopted following technique means:
The present invention utilizes display technique of bacteriophage to set up AsiaI VHH immune antibody storehouse; Through three a large amount of connections and conversion; Select 15 cloning and sequencing results to show that 15 clones' fragment is the gene of the variable region of encoding heavy chain antibody at random, calculating storage capacity is 1.13 * 10
8Camel immunity single domain antibody storehouse, with this antibody library called after NA L-AsiaI, therefrom filter out single domain antibody with high specific and antigen binding capacity.
The two-humped camel VHH heavy chain antibody of a kind of foot-and-mouth disease virus resistant AsiaI type of the present invention, it is characterized in that described antibody have following (a) or (b) shown in aminoacid sequence:
(a) aminoacid sequence shown in the SEQ ID NO.1; Or
(b) with SEQ ID NO.1 shown in sequence homology at the aminoacid sequence more than 98%.
In the present invention, preferred, describedly comprise the sequence shown in SEQ ID NO.2 or the SEQ ID NO.3 in the sequence more than 98% with the sequence homology shown in the SEQ ID NO.1.
The invention provides the nucleotide sequence of the described two-humped camel VHH heavy chain antibody of coding.
In the present invention, preferred, described nucleotide sequence has the sequence shown in SEQ ID NO.4 or SEQ ID NO.5 or the SEQ ID NO.6.
Further, the present invention also provides a kind of recombinant expression vector, it is characterized in that containing above-described nucleotide sequence.
Further, the present invention also provides a kind of host cell, it is characterized in that described host cell is eucaryon or the prokaryotic cell prokaryocyte that contains above-described nucleotide sequence.
Preferably, described host cell is eucaryon or the prokaryotic cell prokaryocyte that contains above-described expression vector, and preferred described host cell is e. coli tg1 or intestinal bacteria HB2151.
Further again, described two-humped camel VHH heavy chain antibody infects the application in the reagent in preparation detection or diagnostics port fever aphthous AsiaI C-type virus C.And
Described nucleotides sequence is listed in the preparation detection or diagnostics port fever aphthous AsiaI C-type virus C infects the application in the reagent.
The present invention utilizes the AsiaI C-type virus C appearance particulate antigen immunity two-humped camel of reorganization first; Utilize display technique of bacteriophage to set up AsiaI VHH immune antibody storehouse; The high specific single domain antibody that screening and foot and mouth disease AsiaI C-type virus C antigen have good affinity, Asia I VHH heavy chain antibody of the present invention is significant for studying Asia I virus infection mechanism and setting up extremely sensitive and special antigen rapid detection or diagnostic reagent.
Description of drawings
Fig. 1 is that two-humped camel peripheral blood lymphocyte RNA extracts the result;
1: total RNA; The 2:DL2000 standard molecular weight
Fig. 2 is VHH gene first round pcr amplification result;
The M:DL2000 standard molecular weight; 1.PCR amplified production;
Fig. 3 takes turns the pcr amplification result for VHH gene second;
The 1:PCR amplified production; The M:DL2000 standard molecular weight
Fig. 4 is VHH gene third round pcr amplification result;
The 1:PCR amplified production; The M:DL2000 standard molecular weight
Fig. 5. be 4,5 and No. 6 clones' of two-humped camel VHH PCR electrophoresis result;
M：DL2000Marker
Fig. 6 is two-humped camel VHH 4,5 and 6 clones' SDS-PAGE result;
M:Protein Marker (50); IB: inclusion body; S: solubility; C1-2: empty carrier is expressed contrast
Fig. 7 is the carrier synoptic diagram of pE-Sumo.
Embodiment
Through experiment and combination embodiment the present invention is further specified below, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.Those of ordinary skills understand, and in spirit that claim of the present invention limited and scope, can carry out many changes to it, revise, even equivalence change, but all will fall in protection scope of the present invention.
1. materials and methods
1.1 material
1.1.1 experimental animal, antigen, adjuvant and AsiaI antibody assay kit
The male two-humped camel in 1 peak (1 one full year of life), the female two-humped camel in 1 peak (6 monthly age); Reorganization AsiaI/YNBS/58 strain VLPs antigen, AsiaI/YNBS/58 (analyze, and Chang Hui rues etc., viral journal, 2007 (5): 407-411) totivirus inactivation antigen by foot and mouth disease virus AsiaI/YNBS/58 strain whole genome sequence; 206 adjuvants; FMDV AsiaI type antibody liquid phase ELISA detection kit is provided by the development of Lanzhou veterinary institute.
1.1.2 bacterial strain, helper phage and reagent
E. coli tg1 and HB2151, helper phage M13K07, carrier pHEN2 are so kind as to give by doctor Wang Xiangbin.
Expression vector pE-Sumo (Kanr, LifeSensors, Inc, the carrier collection of illustrative plates is as shown in Figure 7.), restriction enzyme available from NEB, lymphocyte separation medium (1.077) available from Shanghai give birth to worker, Trizol, nickel affinity chromatography resin, dna fragmentation reclaim test kit and plasmid extraction kit be OMEGA product, PCR related reagent available from precious biotech firm, molecular biology reagent from Sigma company; Other biochemical reagents are homemade analytical pure.
1.2 method
1.2.1 the mensuration of inoculation of antigen dosage, immune programme for children and serum antibody titer
Neck two-humped camel is subcutaneous, the back is subcutaneous, the subcutaneous branch inoculation reorganization AsiaI/YNBS/58 strain VLPs antigen that carries out of subcutaneous abdomen and back leg; The virus-like particle antigen (VLPs) that reorganization VLPs antigen is formed after through self-assembly by VP1 antigen, VP3 antigen and the VP2-VP4 antigen of expressing; Wherein encode the antigenic nucleotide sequence of VP1 shown in SEQ ID NO.7; The antigenic nucleotide sequence of coding VP3 is shown in SEQ ID NO.8; The antigenic nucleotide sequence of coding VP2-VP4 is shown in SEQ ID NO.9, and each position is no less than 3 inoculation points.Gather venous blood with each immunity back before the first immunisation, detect serum antibody titer, back 5 days of the 4th immunity, venous blood collection with FMDVAsiaI type antibody liquid phase ELISA detection kit.Concrete animal immune program and inoculation of antigen dosage are seen table 1:
Table 1 two-humped camel immune programme for children and antigen dose
1.2.2 lymphocyte separates
Back 5 days of the 4th immunity; Vein is gathered anticoagulation, and use proportion is 1.077 lymphocyte separation medium, separates peripheral blood lymphocyte through density gradient centrifugation; Wash isolating lymphocyte with the MEM basic culture solution; Place 6 orifice plates to hatch 1 hour at 37 ℃ then, removal cell debris and most of monocyte are got the suspension cell supernatant and the counting back is subsequent use.
1.2.3 the extraction of cell total rna and RT-PCR
Utilize the total RNA of the isolating lymphocyte of Trizol extracting, carry out the synthetic cDNA of RT-PCR amplification.
1.2.4VHH gene amplification, Construction of Vector for Phage Display and storage capacity calculate
According to reference (structure of anti-H5N1 bird flu virus VHH antibody library, Yan An etc., 2011 the 27th volumes of Chinese Journal of Immunology; And A single-step procedure of recombinant library construction for the selection of efficiently produced llama VH binders directed against cancer markers; Damjana Kastelic et.al, Journal of Immunological Methods 350 (2009) 54-62; And Llama-Derived Single-Chain Antibody Fragments Directed to Rotavirus VP6 Protein Possess Broad Neutralizing Activity In Vitro and Confer Protection against Diarrhea in Mice; Lorena Garaicoechea et.al; JOURNAL OF VIROLOGY; Oct.2008, p.9753 – 9764) 3 pairs of primers of design, the cDNA that obtains with amplification is a template amplification VHH gene fragment; Carry out the three-wheel amplification, primer sequence is seen table 2.
Table 2VHH gene fragment amplification is used primer
With cDNA is template, and H1, G1 obtain 2 bands that size is respectively 600bp and 900bp for the upstream and downstream primer amplification, reclaims the 600bp band as template, carries out second and takes turns pcr amplification.Taking turns the PCR product with second is template, and H2, H3 obtain big or small approximately 450bp band for the upstream and downstream primer.Taking turns the PCR product with second is template, and with P1, TN obtains about 450bp band for the upstream and downstream primer carries out the third round amplification.Behind third round PCR product process SfiI/NotI double digestion, be connected into the pHEN2 carrier, utilize electric method for transformation will connect product and be transformed in the e. coli tg1; After electricity changes end; 37 ℃ of bacterium liquid, the 180r/min shaking table is cultivated 1h, is coated with dull and stereotyped (2% glucose+Amp of 2YTG of ammonia benzyl resistance after centrifugal the concentrating
r+ 2 * YT, 2YTG).Get 10 μ L simultaneously and be diluted to 1 * 10
4Doubly carry out coated plate, 37 ℃ of incubated overnight are used to calculate storage capacity.
Be inverted to cultivate after 16 hours for 37 ℃, the lawn that grows on the culture plate scraped wash clean, add the glycerine of final concentration 25% with 2 * YT substratum of 10mL, packing be kept at-70 ℃ subsequent use, this is the immune storehouse of the anti-FMDV AsiaI type antibody VHH heavy chain antibody that obtains.
Clone after random choose electricity after electricity transforms changes, the extracting plasmid checks order, and identifies the variety of antibody library, calculates storage capacity according to variety and clone's number.
1.2.5 preparation phage antibody library and preliminary screening
(1) get a frozen VHH heavy chain antibody storehouse, be inoculated in the 2YTG substratum of 500mL, 37 ℃, it is OD that the 250rpm/min concussion is cultured to cell concentration
600≈ 0.8-0.9.
(2) add helper phage M13K07 to final concentration 5 * 10
9Pfu/mL, 37 ℃ leave standstill 30min; Then under 37 ℃ of conditions, 200rpm/min, the gentle 60min that cultivates.
(3) 2200g, the centrifugal collection bacterial precipitation of 15min is used isopyknic 2YT-AK (100 μ g Amp again
r, 50 μ g Kan
r) substratum suspension deposition, 30 ℃, the 300rpm/min incubated overnight.
(4) 7000g, 4 ℃ of centrifugal 15min are transferred to supernatant in the triangular flask of 1L precooling, add the phage deposition of 0.3 volume, behind the gentle mixing, ice bath 1h.
(5) 7000g, 4 ℃ of centrifugal 15min abandon clean supernatant, with the resuspended deposition of 8mL PBS.
(6) 12000g is centrifugal 10 minutes, collects supernatant, titration antibody library titre then, and the phage that is divided into aliquot is kept at 4 ℃.
1.2.6 the elutriation of antibody library
(1) antigen coated: in 96 hole enzyme plates, encapsulate foot and mouth disease AsiaI totivirus inactivation antigen at 1: 4 with carbonate buffer solution (pH9.6), 100 μ L/ holes, 4 ℃ encapsulate and spend the night, and process antibody and eluriate plate.
(2) sealing: discard liquid in the hole, add 100 μ L/ hole confining liquids (PBST+5% skim-milk+5% sucrose), 37 ℃ of sealing 2h.
Mix phage antibody library and isopyknic confining liquid, (note: the quantity of first round elutriation phage particle should be higher 100 times than the library size, if for example the library size is 10 for 30 ℃ of preparatory sealing 30min
10Cfu preferably adds 10 during elutriation
12Cfu).
(3) the antibody library mixture 100 μ L/ holes after will sealing in advance add antibody and eluriate plate, incubated at room 60min, and with PBST washing 15 times, PBS washing 5 times, 3min/ time.
(4) wash-out: 1mL0.1M pH2.2HCl-Glycin (Glycocoll hydrochloride buffering system) wash-out, room temperature is shaken 10min, washes out elutriant, and 80 μ L left and right sides 1M Tris (Tutofusin tris) are neutralized to 7.4; With the effective XL1-Blue direct infection that 1.2mL is counted vegetative period of immunity, simultaneously HCl-Glycin (containing 4%BSA) wash-out and 9 times of volume logarithmic phases of neutral phage antibody XL1-Blue are infected.Totally 4 duplicate samples room temperatures infect 10min, change 37 ℃, 150rpm over to and cultivate 1h, get 1%, 0.1%, 0.01% coated plate calculating quantum of output respectively.Remainder mixes to be concentrated to after centrifugal and is 1mL, is applied to 2 12cm diameter LBATG respectively and (adds Amp in the LB substratum
r: 100 μ g/mL; Tet
r: 10 μ g/mL; Glucose:0.5%; ) the solid culture plate, 37 ℃ of overnight cultures (about 12h).
(5) appear: the bacterium colony on the overnight cultures culture plate is scraped, and part drops into the next round screening, and remainder is frozen.With part bacterium liquid join in the 100mL LBATG nutrient solution to bacteria concentration be OD
600About=0.35, cultivate about 1h to OD in 37 ℃
600=0.5 (bacterium amount is 1 * 10
8/ mL), adding 50:1 helper phage M13K07, room temperature leaves standstill 15min behind the mixing, centrifugal acquisition deposition after 37 ℃, 150rpm/min are cultivated 1h.LBATK at 90mL; Be divided into 3 parts behind the mixing: a copy of it add IPTG to final concentration be 0.1mM and Glucose to 0.25% (g/100mL) (6 μ L 0.5M IPTG, 300 Μ l25% glucose); A add IPTG to final concentration be 0.1mM; The 3rd part is blank; Three increments originally continue at 30 ℃, 180rpm/min, overnight cultures (about 12h).
(6) 4 ℃, 14000rpm/min, the centrifugal bacterial cultures of 30min is received culture supernatant; PEG/NaCl (20%PEG8000+2.5mol/L NaCl) buffered soln that adds 1/5 volume precooling, the preparatory 60min of ice behind the mixing, deposition phage; 4 ℃, the centrifugal 20min of 14000rpm/min is in the PBS damping fluid that contains 2%BSA and 15% glycerine of the resuspended 10mL of being deposited in; The centrifugal 10min of 10000rpm under the normal temperature removes deposition, obtains supernatant; 0.22 μ m membrane filtration degerming; Get 10 μ L and carry out the phage antibody library titer determination, all the other are distributed into aliquot, frozen-70 ℃ subsequent use.
Carry out " combination-washing-wash-out (infection) " by above step, carry out three-wheel repeatedly.
1.2.7 phage E LISA identifies positive colony
A) picking through the screening after the clone in 96 hole depth orifice plates, every hole 250 μ L2 * YTATG substratum, 37 ℃ of shaking tables are cultured to OD
600=0.5, according to MOI=20: 1 adds helper phage, and room temperature leaves standstill 15min, and 37 ℃, 150rpm/min cultivates 1h, adds equal-volume 2 * YTATG, and 30 ℃, shaking table 200rpm overnight cultures pact.
Next day, 10000rpm/min is centrifugal, and 10min collects supernatant, adds BSA to final concentration 2%, adds Tween-20 and leaves standstill 15min for 0.1%, 37 ℃ to final concentration, and is subsequent use.
B) encapsulate foot and mouth disease AsiaI totivirus inactivation antigen coating buffer and be diluted to 1: 4, add in the 96 hole enzyme plates, 100 μ L/ holes, 4 ℃ encapsulate and spend the night.
C) next day, abandon coating buffer, wash 3 times with DPBS solution 220 μ L/ holes, with 5% skim-milk+0.1%Tween-20 sealing, 100 μ L/ holes, 37 ℃ of sealing 2h.
D) discard confining liquid, add the mono-clonal phage antibody after sealing, 100 μ L/ holes, 37 ℃ leave standstill 2h.
E) discard liquid, wash 6 times, 220 μ l/ holes with DPBS.
F) mouse-anti M13-HRP antibody was diluted 100 μ L/ holes, 37 ℃ of effect 1h by 1: 5000 with the sealing damping fluid.
G) discard liquid, enzyme plate is washed 6 times with DPBS.
H) discard washings, add substrate OPD colour developing liquid, 100 μ L/ holes, room temperature leaves standstill colour developing.With 100 μ L/ hole 2molH
2SO
4Color development stopping.ELIASA is measured absorbance value.
1.2.8 the AsiaIVHH heavy chain antibody of screening is expressed in E.coli and is identified
Above-mentioned experiment obtains positive colony altogether, through sequential analysis relatively, screens specific anti-FMDV AsiaI type antibody VHH heavy chain antibody sequence.Utilize PCR with these sequence amplifications, glue reclaims fragment behind the pcr amplification, and the double digestion enzyme is cut then, and the purpose fragment after enzyme is cut is connected with E.coli expression vector pSMK, structure VHH heavy chain antibody recombinant expression vector, and be transformed among the E.coli and express.Utilize SDS-PAGE and Western blotting to identify the VHH antibody protein of expressing, utilize the double-antibody sandwich elisa test to identify the binding ability and the type specificity of expressing antibodies.
1.2.9 reorganization AsiaIVHH heavy chain antibody conjugated antigen ability and type specificity are identified
1.2.9.1 double-antibody sandwich elisa is identified VHH antibodies AsiaI/YNBS/58 totivirus antigenic capacity
(1) antibody sandwich: purifying VHH antibody sandwich enzyme plate (1 μ g/ hole); 100 μ L/ holes; The anti-FMDV AsiaI of rabbit totivirus antigen polyclonal antiserum is as positive control (1: 1000), and SUMO albumen is as negative control (1 μ g/ hole), and 4 ℃ are spent the night and encapsulate;
(2) adding antigen: 1 * PBST washing is 4 times, and liquid in the clear opening is abandoned in 220 μ L/ holes for the last time, claps and does, and according to 1: 5 adding AsiaI totivirus inactivation antigen, 37 ℃ of effect 60min washed plate;
(3) add cavy antibody: add the anti-AsiaI polyclonal antibody of cavy according to 1: 1000 ratio, 37 ℃ of effect 60min wash plate;
(4) add ELIAS secondary antibody: the anti-cavy HRP of adding rabbit mark two was anti-in 1: 1000, and 37 ℃ of effect 60min wash plate;
(5) colour developing: add 37 ℃ of effects of OPD substrate colour developing 15min;
(6) stop also reading: add 2Mol/L H
2SO
4Termination reaction, OD490 reads absorbance.
2. result
2.1 the extraction of the total RNA of complete lymphocyte
Total RNA of immunity back two-humped camel peripheral blood lymphocyte is sent in extraction outside, and total RNA that the electrophoretic analysis of condensing is extracted does not have genome and pollutes, and size conforms to expection, sees Fig. 1.
2.2VHH heavy chain antibody gene PCR amplification
With cDNA is template, and H1, G1 are the upstream and downstream primer, carry out first round pcr amplification, obtains 2 bands about big or small 600bp and 900bp, sees Fig. 2.Reclaim the 600bp band,, carry out second and take turns PCR as template.
With first round amplification PCR products is template, and H2, H3 see Fig. 3 for the upstream and downstream primer obtains about 450bp band.
Taking turns the PCR product with second is template, is that the upstream and downstream primer obtains about 450bp band with P1, TN, sees Fig. 4, and this moment, the SfiI/NotI restriction enzyme site was introduced at VHH gene two ends respectively.
2.3 confirming and storage capacity of the many variety in phage displaying antibody storehouse
Through three a large amount of connections and conversion, select 15 cloning and sequencing results to show that 15 clones' fragment is the gene of the variable region of encoding heavy chain antibody at random, calculating storage capacity is 1.13 * 10
8Camel immunity single domain antibody storehouse, with this antibody library called after NA L-AsiaI.
2.4 the elutriation of antibody library
Adopt phage library NAL-AsiaI eluriate can with AsiaI antigen-specific bonded phage particle; Measure every recovery of taking turns the phage of screening; Take turns elutriation through 3; The phagocytosis scale of construction that shows selective adsorption obviously increases (table 3), can with the enrichment that obtained of AsiaI antigen-specific bonded phage.
Table 3 three-wheel is eluriated phage enrichment result
Each is taken turns wash-out does not have the phage library that obtains of amplification and carries out the phage-ELISA monitoring; Identical in antigen coated concentration; Under the identical condition of phage input amount; The absorbance of measuring raises along with the increase of eluriating number of times, and explanation can obtain enrichment with target antigen bonded phage clone, and the result sees table 4:
Table 4 phage-ELISA is indirectly measured phage clone
| Round | AsiaI_ antigen 1: 4 | |
| 1 | 1.707 | 1.907 |
| 2 | 1.85 | 0.215 |
| 3 | 2.447 | 0.675 |
| M13KO7 | 0.1 | 0.063 |
| PBS | 0.069 | 0.052 |
2.5 mono-clonal phage-ELISA
Select 24 clones after third round is eluriated, save and purifying.Phage particle behind the purifying adopts AsiaI totivirus antigen indirect ELISA to identify (table 5); Through comparing with negative control; And identifying 3 behind the dna sequencing can clone with AsiaI/YNBS/58 totivirus antigen-specific bonded; Positive rate is 20.8%, the sequence called after Clone 4,5 and 6 of mensuration, and Nucleotide and amino acid whose sequence are seen sequence table; Clone 4,5 and 6 nucleotide sequence are respectively shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the coded aminoacid sequence of Clone 4,5 and 6 nucleotide sequence is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.The homology of nucleotide sequence of Clone 5 and 6 nucleotide sequence and Clone is all more than 98%.
Table 5 mono-clonal phage-ELISA result
2.6VHH expression and the purifying of heavy chain antibody in E.coli
Design 3 pairs and express primer; See table 6; Utilize PCR that specific VHH heavy chain antibody sequence is cloned (Fig. 5) from phage vector respectively, recombinate behind the double digestion with at pE-Sumo then, transform DH5a clone bacterium; Extract bacterium colony PCR and identify the male recombinant plasmid, its carrier is transformed into E.coli expresses bacterial classification BL21CondonPlus competent cell (Cm
r34 μ g/mL), extract mono-clonal in 2YT (Kan
r80 μ g/mL, Cm
r34 μ g/mL) in the substratum; Adding the IPTG final concentration is 0.5mmol/mL; Induced 12 hours under 20 ℃ of conditions, the bacterial sediment that ultrasonic degradation is collected under the condition of ice bath, SDS-PAGE are identified proteic expression and expression-form discovery; All with the formal representation in supernatant (Fig. 6) of solubility, the aminoacid sequence of 3 strain VHH antibody is respectively shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 for 3 strain VHH antibody.
Table 6 is expressed VHH antibody primer
* remarks: expression vector pE-Sumo (LifeSensors, Inc) in BsaI and BsmBI be isocaudarner because the restriction enzyme site of BsaI is arranged in the target gene sequences, replace BsaI with BsmBI.(the carrier collection of illustrative plates is as shown in Figure 7.）
Claims (9)
1. the two-humped camel VHH heavy chain antibody of anti-foot and mouth disease AsiaI C-type virus C, it is characterized in that described antibody have following (a) or (b) shown in aminoacid sequence:
(a) aminoacid sequence shown in the SEQ ID NO.1; Or
(b) with SEQ ID NO.1 shown in sequence homology at the aminoacid sequence more than 98%.
2. two-humped camel VHH heavy chain antibody as claimed in claim 1 is characterized in that the sequence homology shown in described and the SEQ ID NO.1 comprises the sequence shown in SEQ ID NO.2 or the SEQ ID NO.3 in the sequence more than 98%.
3. the nucleotide sequence of coding claim 1 or 2 described two-humped camel VHH heavy chain antibodies.
4. nucleotide sequence as claimed in claim 3 is characterized in that described nucleotide sequence has the sequence shown in SEQ ID NO.4 or SEQ ID NO.5 or the SEQ ID NO.6.
5. a recombinant expression vector is characterized in that containing claim 3 or 4 described nucleotide sequences.
6. a host cell is characterized in that described host cell is eucaryon or the prokaryotic cell prokaryocyte that contains claim 3 or 4 described nucleotide sequences.
7. host cell as claimed in claim 6 is characterized in that described host cell is eucaryon or the prokaryotic cell prokaryocyte that contains the described expression vector of claim 5.
8. claim 1 or 2 described two-humped camel VHH heavy chain antibodies infect the application in the reagent in preparation detection or diagnostics port fever aphthous AsiaI C-type virus C.
9. claim 3 or 4 described nucleotides sequences are listed in the application in preparation detection or the diagnostics port fever aphthous AsiaI C-type virus C infection reagent.
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| CN104086652A (en) * | 2014-06-30 | 2014-10-08 | 中国农业科学院兰州兽医研究所 | Anti-O type foot and mouth disease virus specific single-domain antibody and recombinant expression vector thereof |
| US10017560B1 (en) | 2017-11-16 | 2018-07-10 | King Saud University | Nanobody against begomoviruses |
| CN109438574A (en) * | 2015-12-25 | 2019-03-08 | 中国农业科学院兰州兽医研究所 | Porcine epidemic diarrhea virus M protein-specific heavy chain antibody |
| CN109709330A (en) * | 2018-12-25 | 2019-05-03 | 内蒙古必威安泰生物科技有限公司 | A kind of foot and mouth disease virus competitive ELISA detection kit |
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| CN102277332A (en) * | 2010-10-30 | 2011-12-14 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody for anti-foot and mouth disease virus and application thereof |
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| WO2011054011A2 (en) * | 2009-11-02 | 2011-05-05 | The Trustees Of The University Of Pennsylvania | Foot and mouth disease virus (fmdv) consensus proteins, coding sequences therefor and vaccines made therefrom |
| CN102277332A (en) * | 2010-10-30 | 2011-12-14 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody for anti-foot and mouth disease virus and application thereof |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104086652A (en) * | 2014-06-30 | 2014-10-08 | 中国农业科学院兰州兽医研究所 | Anti-O type foot and mouth disease virus specific single-domain antibody and recombinant expression vector thereof |
| CN104086652B (en) * | 2014-06-30 | 2016-06-22 | 中国农业科学院兰州兽医研究所 | Resisting O-type foot and mouth disease virus specificity single domain antibody and recombinant expression carrier thereof |
| CN109438574A (en) * | 2015-12-25 | 2019-03-08 | 中国农业科学院兰州兽医研究所 | Porcine epidemic diarrhea virus M protein-specific heavy chain antibody |
| CN109438573A (en) * | 2015-12-25 | 2019-03-08 | 中国农业科学院兰州兽医研究所 | Porcine epidemic diarrhea virus M protein-specific heavy chain antibody |
| CN109438574B (en) * | 2015-12-25 | 2020-10-27 | 中国农业科学院兰州兽医研究所 | Porcine epidemic diarrhea virus M protein specific heavy chain antibody |
| CN109438573B (en) * | 2015-12-25 | 2020-11-10 | 中国农业科学院兰州兽医研究所 | Porcine epidemic diarrhea virus M protein specific heavy chain antibody |
| US10017560B1 (en) | 2017-11-16 | 2018-07-10 | King Saud University | Nanobody against begomoviruses |
| CN109709330A (en) * | 2018-12-25 | 2019-05-03 | 内蒙古必威安泰生物科技有限公司 | A kind of foot and mouth disease virus competitive ELISA detection kit |
| CN109709330B (en) * | 2018-12-25 | 2021-11-09 | 内蒙古必威安泰生物科技有限公司 | Foot-and-mouth disease virus competition ELISA detection kit |
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