Study Of Differential Diagnosis Of ELISA By Tandem Epitopes Antigen Of Nonstructural Protein3A And3B

Foot-and-mouth disease（FMD）is caused by foot-and-mouth disease virus（FMDV）is a acute,thermal and highly contagious disease of cloven-hoofed animals. The disease seriously limitedanimal susceptibility of international trade and could bring huge economic losses when it occurred.The world organization for animal health (OIE) will it as a must notified one of the many kinds ofanimals were suffering from infectious diseases Therefore, how to quickly identified naturalinfection from vaccinated animals（DIVA）is a very important part of prevention and control of thedisease. Several studies indicated that a reliable DIVA measure to detected the non-structuralprotein3AB and3ABC antibody of FMD is the most effective method.We selected two cell linear epitopes of non-structural protein3A which was identified fromAsia1type1strain of FMDV in this study was processed and modified to a short peptide. Thenconnected with the non-structural protein3B synthesis short peptide and continuous repeated. Thereconstructed sequence was transformed into pET-32a(+) and expressed in E. coli Rosetta(DE3).The resultant five tandem repeat mul-timer（ABF，2ABF，4ABF，2BF-2AF，(2BF-2AF)2）wereexpressed as soluble fusion proteins E. coli. The results of SDS-PAGE and Western blot showedthat the recombinant proteins was recognized specifically by anti-FMDV positive serum. Anindirect ELISA was developed based on the recombinant (2BF-2AF)2protein.Of184known negative bovine serum was detected against the antibody of nonstructural ofFMDV by using indirect ELISA and calculated the antibody titer. With "negative control serummean value+3SD" as a cut-off value criterion, the S/P>0.31for positive, and the suspiciousrange was set: negative control+2SD≤suspicious<positive control+3SD, the0.23≤suspicious<0.31, S/P<0.23is considered to be negative.Of40known positive bovine serum was detectedagainst the antibody of nonstructural of FMDV showed that100%of sensitivity for it by usingindirect ELISA.The ELISA specificity cross experiment results showed that the antigen is notcross-reacted with4common cow disease positive serum. Has good specificity and repeatabilitytest between batch and batch of variation coefficient is less than10%. The performance of theELISA was compared with three commercial NSP ELISA kits. The results showed the coincidencerate with Ceditest FMDV-NS ELISA,3ABC-I-ELISA and Blocking-ELISA were96.25%，93.48%and84.35%, respectively. In different parts of the5cow farms in829serum samples weredetected against antibody of non-structural protein of FMDV, the positive rate range from8.02% to47.75%by using this ELISA method. The establishment of r3AB-ELISA can be used for theDIVA of the disease, it provides technical support and material basis for the comprehensiveprevention and control of FMD.In this study, the (2BF2AF)2for serological ELISA as the detection antigen to established theIndirect ELISA for detected milk samples and established threshold criteria: S/P≥0.2as positive,S/P≤0.2as negative. The3ABC indirect ELISA kit serum as the reference standard, thecorresponding milk ELISA test, in line with86.36%;522milk samples were detected from threedairy farms in different parts by use of the milk ELISA, the positive rate between10%-16.58%;monitoring of mixed milk the initial discovery, can be detected in1of the16negative milk weaklypositive milk samples, and provides a theoretical basis for the monitoring of bovinefoot-and-mouth disease epidemic in groups.