Establishment Of Indirect ELISA Diagnose Based On The VP1 Protein Of Foot And Mouth Disease Virus Serotype A

Foot and mouth disease(FMD)is caused by foot-and-mouth disease virus(FMDV)which could induce an acute,febrile,highly contagious disease for cloven hoofed animals.Because of the extensive route of transmission and the highly infectivity,FMD prevalents frequently in many regions and countries and brings about serious pandemic threat of development for animal husbandry.FMDV has 7 serotypes,respectively:O,A,C,Asia 1,SAT 1-3.Each type includes a multiple subtypes with no cross reactivity between them.Furthermore,A type,O type and Asial type shows alternating prevalent trend in China.2009-2013,Akesu area in Xinjiang Province,Shanghai City,Wuhan city in Hubei Province in China,the neighboring South Korea outbroke Foot and Mouth Disease serotype A in seccession which caused significant economic losses.The objective of this research is to establish an evaluation procedure based on the VP1 protein of FMDV serotype A which claims to obtain the protein with high activity.The recombinant expression vector pET-A-vp1 was constructed by cloning vpl gene which was amplified by PCR into the prokaryotic expression plasmid pET-28a.Expressed the E.coli BL21(DE3)bacteria which contained recombinant plasmid pET-A-vpl and induced by IPTG,SDS-PAGE and Western-Blot showed that A-VP1 protein was expressed rightly with molecular weight is about 29 ku and it can be specific recognited by positive serum of FMDV serotype A.Based on optimizing OD600 value,IPTG concentration and expression time,the largest amount of expression of A-VP1 protein was induced at OD600 0.6,0.3 mmol/L IPTG for 6 hours at 37℃.ELISA test showed that A-VP1 with high activity after purified by washing inclusion and refolded by urea concentration gradient dialysis.Expression of high activity A-VP1 protein is foundation for further development of genetic engineering vaccine and diagnosis reagent of foot-and-mouth disease.The VP1 protein of FMDV serotype A was prokaryotic expressed and purified to replace the traditional virus antigen for establishing a ast,safe,effective indirect ELISA kits which were used to detect antibody of foot-and-mouth disease virus serotype A.Western-Blot test showed that the A-VP1 recombinant protein could be used as detective antigen as it can be specific recognition by bovine positive serum of FMDV serotype A.In order to prepare and assess the ELISA method for detecting antibodies against foot-and-mouth disease virus(FMDV)type A,the optimal mass concentration of A-VP1 protein as coating antigen(1 mg/L),the best dilution of serum(1:100)and enzyme combined antibodies(1:2000)were determined by matrix titration method.The results showed that the sensitivity and specificity of this method were 94.32%and 99.09%,intra-assay and inter-assay reproducibility test of the method was below 10%.Comparing this assay with the liquid phase blocking ELISA kits by detecting 201 serum samples,results showed that the agreement of them was 92.54%.The A-VP1-ELISA kit is specific,sensitive,stable and simple operation which can be used to monitor the antibody level of FMD serotype A.