Prokaryotic Expression The Major Dominant Antigenic Epitopes Of Torque Teno Suis Virus2ORF1and Establishment Of Indirect-blocking ELISA Of Torque Teno Suis Virus2

Torque teno suis virus(TTSuV) is a small circular single-stranded DNA virus, belongs to Anelloviridae.TTSuV includes two genotypes,TTSuV1and TTSuV2.Since TTSuVs first detected from pigs in2002, many countries around the world have reported TTSuV infection.Until now,there are no evidences to confirm that TTSuV could directly lead to clinical disease,but according to statistics, TTSuV may take synergy pathogenic reaction with PRRSV and PCV2,often characterized by mixed infection. TTSuVs infection were significantly higher than healthy pigs when pigs were suffer from PMWS. TTSuV2and PCV2virus mixed infection positive rate were higher than TTSuV1and PCV2virus mixed infection according to the reports.Till now, we haven’t find TTSuV cultured cell lines, TTSuVs were difficult to culture. Current studies of TTSuV more focused on its epidemiological surveillance.In our country, most areas have reported TTSuV infection, so the TTSuV epidemiological monitoring is very important.ORF1protein is the major capsid protein of TTSuV2, it has good antigenicity and can induce the body to produce ORF1antibodies. Therefore,ORF1protein could be used as a kind of detection antigen,through the detection of ORF1antibody to diagnose TTSuV2.According to the published genomic seqenence of TTSuV2ORF1,1523bp-2272bp of TTSuV2was selected to be the target gene. A pair of primers was designed to amplify the target gene by PCR and cloned into the prokaryotic expression vector pET32a(+). After induced by IPTG, the expressed recombinant protein was detected by SDS-PAGE, and the protein was45Kda in size consistent with the expected. The purified recombinant protein was used to abtain polyclonal antibody and the polyclonal antibody has specific antigen-antibody reaction with TTSuV2ORF1protein according to the agar gel precipitation test.In this study, we use TTSuV2ORF1recombinant protein as antigen, rabbit serum was used as anti-blocking antibodies TTSuV2ORF1established TTSuV2ORF1indirect antibody blocking ELISA methods, and optimize the reaction conditions, and ultimately determine the optimal reaction conditions were as follows:carbon acetate buffer (pH=9.6,0.05mol/L) as antigen coating buffer,4℃coated overnight; then with5%skim latex at37℃for2h; optimal concentration of antigen was1.81μg/mL, resistance optimal working concentration of antibody-off1:1600dilution effect of blocking antibodies optimum conditions were37℃for1h; optimal dilution of the sample to be tested is1:10serum samples optimal incubation time1h; HRP most good working concentration of1:10000, the best color condition is37℃chromogenic20min.Twenty negative control samples was detected by the optimized indirect blocking ELISA, the results revealed the average percent inhibition(PI) rate of the20samples was10.27%, the standard deviation was3.49%.The critical value was determined according to the formula: critical value=average PI of the negative control samples+3(or2)×the standard deviation.The results revealed that:TTSuV2was antibody-positive when the PI value of the tested sample was Greater than or equal to20.7%; TTSuV2was antibody-negative when the PI value of the tested sample was less than17.3%. Furthermore, when the PI value was between20.%and17.3%, the tested sample needs to detect once again. According to the sensitivity test, specificity test and compared with Western blot,the established indirect blocking ELISA has high sensitivity and specificity,and it can rapidly and effectively achieve detection of TTSuV2antibodies.The established TTSuV2-ORF1indirect blocking ELISA method in this study could detect the TTSuV2ORF1antibody, and provided technical support for the effective monitoring of of TTSuV2infection.at the same time, this method could support related vaccines assessment tools and lay the fouldation for the development of TTSuV2ELISA Kit.