Expression And Mono Clonal Antibody Preparation Of VP1 Protein Of Foot-and-mouth Disease Virus

Foot-and-mouth disease(FMD)is a kind of acute animalinfectious disease caused by foot-and-mouth disease virus(FMDV).FMD was listed at the first class of animal infectious disease by China because it has serious damage to the healthy development of animal husbandry and public health security.The combination of antibody detection and combined vaccine is the main method to control the FMD.FMDV has seven serotypes,and different serotypes of FMDV have no cross protection.The main serotypes causing FMD in China are O,A,Asia ?types,and the O serotype is the most popular.O serotype FMDV at least contains five neutralizing antigenic sites which can stimulate organisms to produce neutralizing antibody.The VP1 protein,as the main structural protein of FMDV,contains three sites,which are respectively located on the linear epitope of amino acid residues at positions 200-213 of the carboxyl terminal,the B-C ring and the G-H ring.Thus VP1 antibody as the main neutralizing antibody of FMDV,is a important basis in evaluating vaccine immune effect.In this study,E.coli JM109 Fused with VP1 gene was induced by IPTG and expressed in the optimized condition;The titer of anti-VP1 protein antibody was tested by indirectly ELISA based on VP1 fusion protein(r VP1 protein);The spleen cells of immunized mice and SP2/0 myelomas cells were fused under the working of 50 %PEG 2000 and the positive clone was screened by limiting dilution method;The McAbs were prepared and identificated.The results showed that:1.Cloning and prokaryotic expression of the O serotype FMDVVP1 geneThe specific primers were designed for the O serotype FMDV VP1 gene according to the sequence published on GeneBank.The PCR amplification product of VP1 gene was cloned into the expression vector pQE-30.The fusion vector was transformed into E.coli JM109 and induced by IPTG,and the expression product was identified by SDS-PAGE.The results showed that the size of VP1 gene amplification product was 560 bp,consistent with the expected size of the amplified fragment.The expressed rVP1 protein band was about 26 kDa and no other bound was found,which showed that the rVP1 protein was efficiently expressed.The western-blot result showed that the expressed rVP1 protein has good reactogenicity.The rVP1 protein was successfully expressed and laid the foundations for developing PRV serological detection methods.2.Establishment and application of an indirect ELISA for the detection of O serotype FMDVVP1 protein antibodiesThe titer was used to screen the antigen concentration,the coating condition,the serum dilution concentration,the dilution concentration of the enzyme secondary antibody and the optimal blocking agent,then toperform a specific experiment.The results show that the optimum coating concentration of rVP1 protein was 2.0 ?g/mL;The optimum coating temperature and time was 4? for a night;The optimum blocking solution was 1% BSA-PBST;HRP-labelled goat anti mouse IgG was choosed as secondary antibody in l?3000 dilution.There was no evidence of crossreactivity with known positive sera to ?.The method did not react with A serotype FMDV,Asia?serotype FMDV,SVDV positive serum,only reacted with Oserotype FMDV positive serum,indicating good specificity.The indirect ELISA established in this study provided a simple and rapid method for the detection of Oserotype FMDV VP1 antibody in China.3.Preparation and identification of monoclonal antibodies against O serotype FMDV VP1 proteinThe female BALB/c mice were immunized with high concentration of O serotype FMDV inactivated vaccine,and the spleen cells of immunized mice were fused with SP2/0 myeloma cell.The hybridoma cell lines which could stably secrete MAbs against O serotype FMDV VP1 protein were screened after four subcloningand identified by indirect ELISA method.The hybridoma cell lines were injected into the abdominal cavity of mice to prepare ascites,and then to identify the subclass and purify of the ascites which were selected according to the highest titer.The hybridoma cells chromosome number,relative affinity constant,specificity,stability and other biological characteristics of the purified MAbs were identified.The blocking ELISA method was initially used to indentify the applicability of MAbs.The results showed that,a hybridoma cell line(1C7)which stably secreted MAbs against O serotype FMDV VP1 protein was screened out,the number of 1C7 was higher than the number of every parental cell chromosome;the ascites MAbs titer was 1:105 and the concentration was 4.7 mg/mL.The subtype of MAbs was identified as IgG2b;the relative affinity index of MAbs was 1.5mol/L.The indirect ELISA test result showed that the MAbs could specifically react to O serotype FMDV,but not cross react to A serotype FMDV,Asia? serotype FMDV and SVDV.The results of Western-blotexperiments showed that the MAbs could specifically react to O serotype FMDV recombinant VP1 protein;the hybridoma cell wasalways secreted antibody stablywhich was passaged to 30 generations and revived after frozen to 12 months.The blocking ELISA assay showed that 1C7 MAbs could be specifically blocked by antibody in O serotype FMDV serum samples.This study laid a foundation on detection of pathogen and antibodies of O serotype FMD using the blocking ELISA and colloidal gold rapid detection of immune chromatography method based on 1C7 MAbs.