TaqMan PCR For Detection Of Caprine Arthritis Encephalitis Virus&Eukaryotic Expression Of Recombinant SU

Caprine arthritis and encephalitis (CAE) is a viral disease of goats caused bycaprine arthritis encephalitis virus (CAEV). CAEV are member of the genusLentivirus of the family Retroviridae. CAEV infections are prevalent worldwide indairy goat, and a higher incidence of CAE was noted in many industrialized nations.Economic loss of CAEV infection are considerably adverse in countries withintensive animal husbandry. So a sensitive and special assay based on TaqManreal-time PCR amplification has been developed for detecting CAEV proviral DNA.The complete env gene was obtained by overlap PCR method, and then the surfaceenvelope glycoprotein(SU) gene was cloned into eukaryotic expression vectorpCDNA4.0.In this study, primers and TaqMan probe, targetted for the highly conservedsequences(CA), was designed for a specifc and sensitive two-step real-time PCR.According to the standard curve, CAEV can be detected quantitatively. The specifcityresults did not produce any cross reactivity with some animal viruses (Swineinfluenza virus, goat pox virus, bovine leukemia virus,bovine mucosal disease virusand Nipah virus), indicating the high specifcity of the primers and probe used. Thesensitivity of the assay which depend on the recombinant plasmid pGEM T-CA wasapproximately102copies/ul.308specimens were tested using TaqMan qPCR andconventional PCR assay, and the positive rates were7.8%and7.5%, respectively. Allthe PCR-negative cases are TaqMan-negative, only one case showed a discrepancy(16/48vs.17/48). In conclusion, the TaqMan qPCR assays have proven advantageousin terms of rapidity, specifcity and sensitivity for the CAEV genomic DNA of wholeblood and peripheral blood lymphocyte in goats.According to the env sequence of pGEM T-CA4and pGEM T-CA5, thecomplete env gene was cloned into pUC-T vector based on overlap PCR method.Thenthe eukaryotic expression vector which consisted of the surface envelopeglycoprotein(SU) gene and plasmid pCDNA4.0was finally constructed. Theeukaryotic expression of recombinant SU protein was obtained by transfectedBHK-21cell lines, and was identificated by Indirect Immunofluorescence Assay.