Indirect-blocking ELISA For Detecting Antibodies Against GB Of PCMV And Epidemiological Investigation Of PCMV In Sichuan

The porcine cytomegalovirus belongs to the genus Cytomegalovirus, subfamily Betaherpesvirinae, family Herpesvirus. In1955, Done isolated the herpes virus-like particles of PCMV in England for the first time. Nowadays, PCMV is widely distributed around the world, with reported cases of the in Britain, Japan, Germany and the United States. PCMV are difficult to culture, there is no mature culture system, glycoprotein B (gB), one of the major structural proteins of PCMV, has good immunogenicity, and induces the production specific antibodies. An indirect-blocking enzyme-linked immunosorbent assay (ELISA) was developed using the expressed major gB epitope as a coating antigen for the detection of PCMV antibodies. This is a practical ELISA method that can be used to develop a new PCMV antibody detection ELISA kit that should aid in the routine detection of PCMV. Once a PCMV vaccine is developed, the assay might also aid in the evaluation of the vaccine. In this study, the results of the PCMV infection molecular epidemiological investigation provide a reference to the prevention and research of PCMV. This study has made the following findings:1. The epitope region of the gB gene was analyzed using molecular biology software, and the2305bp to2574bp major epitope of the PCMV gB gene in the region between was selected as the target fragment, the fragment containing the gB epitope was cloned into the pET-32a+vector, and the recombinant plasmid pET32-major epitope of gB was identified by sequencing. After induced by IPTG, the28.3kDa major gB epitope, was expressed in E. coli Rosetta (DE3), and purified successfully by HisTrap affinity columns as revealed by SDS-PAGE. The specific immune response between the purified gB epitope and the polyclonal antibody was confirmed with an agar gel precipitation test. The resulting28.3kDa band was identified by western blot analysis.2. An indirect-blocking enzyme-linked immunosorbent assay (ELISA) was developed using the expressed major gB epitope as a coating antigen and the polyclonal antibody as a blocking antibody for the detection of PCMV antibodies. The optimal conditions of ELISA were determinded:The0.05M carbonate buffer (pH9.6) was selected as the coating buffer, the optimal coating condition was4℃12h; whereas PBS containing3%BSA was selected as blocking solution of indirect-blocking ELISA, the optimal blocking condition was37℃1.5h; the optimal concentration of the purified gB epitope, used as the coating antigen, was1.94μg/mL, and the optimal dilution of the blocking antibodies was found to be1:1600, the optimal reaction time of blocking antibodies was1h; the optimal dilution of the IgG conjugate was1:10000, the optimal reaction time of the IgG conjugate was40min; the optimal dilution of the test serum was1:5, the reaction time was1.5h. The OD values of20negative control samples were also detected using the optimized indirect-blocking ELISA, the percent inhibition (PI) values were obtained, and the critical value was determined according to the following formula:Critical value Average PI of the negative control samples+3(or+2)×Standard deviation, the PCMV gB was antibody-positive when the PI of the serum sample was greater than24.5%, and it was antibody-negative when the PI of serum sample was less than19.8%. The indirect-blocking ELISA showed a specificity of95.5%, and a sensitivity of97.83%, and the compliance rate between the Western blot and indirect-blocking ELISA was high.3.257clinical serum samples were detected by PCR assay, the results showed the PCMV infection rate of the pigs in Sichuan region was78.2%, the newborn piglet and pregnant sows have the highest infection rate, and the PCMV infection rate in boar was low, this results was consistent with domestic and foreign reports.