Prokaryotic Expression Of P28 Protein Of CAEV And The Research Of CAEV Infecting GC

Caprine Arthritis Encephalitis (CAE) was a kind of chronic and transmissible disease in goats caused by CAEV which belonged to lentivirus genus of retrovirus family. Typical clinical symptoms of CAEV infection may appear as leukoencephalomyelitis in young kids and arthritis and mastitis and interstitial pneumonia in adult goats. The high prevalence of CAEV infection is a concern in many regions of the world particularly in industrialized countries like England and America. CAEV was transmitted into China in 1982 from imported goats. The disease cause great economic loss to goat's husbandry. But at present, it was still a difficult issue for all countries in the world to prevent and control CAE. In order to better control spread and epidemicity of CAE, and develop a rapid, simple, suitable diagnostic test to CAEV, we induced and expressed CAEV specific protein p28 in E.coli. Then p28 protein was used as antigen of an indirect ELISA for anti-p28,which was used to detect 100 samples of goats' sera from farms.At first, goat synovial membrane(GSM) was cultured and used for the propagation of CAEV.Apair of primes with digestion site was designed according to the p28 sequence in Genbank. The p28gene was amplified by PCR and cloned into the plasmid pET30a, and then was expressed in E. coli.There is a 6 histidines tag at the recombinant p28 protein's N-terminal, so it is a fusion proteinactually. It is purified by immobilized metal ion affinity chromatography under native conditions.p28 protein shows reactivity to CAEV positive serum samples and no reactivity to normal goat serain indirect enzyme-linked immunosorbent assay (ELISA) and immunoblot assay. Whichdemonstrates that the recombinant p28 protein has very good antigenicity and specificity. Anindirect ELISA for anti-p28 antibody has been developed by coating plate with the recombinantp28 protein. The optimum conditions were that the fold of the p28 protein coated is 0.1 μg, thepositive sera are 1:50, the rabbit anti-goat IGg conjugated with HRP is 1:1000, and the reactiontime of the rabbit anti-goat IgG is 2 hour. A positive/negative(P/N) value higher than 2.0 and ODvalue about 1.0 was determined as comprehensive positive standard for the ELISA. At the sametime we inoculated mice with purified p28 protein to prepare positive sera of goat. It was found thatall of 3 goats inoculated with CAEV induced the antibody against p28 2 weeks post-inoculationwith the recombinant p28 protein as antigen. And the p28-specific antibody titer eventually reachesa plateau and remains for period of time. The results of AGID and ELISA with the recombinantp28 protein as antigen were compared for detecting 3 goats' sera inoculated CAEV. Both AGIDand indirect ELISA could detect the antibody against p28 protein 5 weeks post-inoculation, but theearliest time to detect the antibody with AGID was later 3 weeks than that with indirect ELISA.The results also showed that the positive result of 6 months post-inoculation detected with indirectELISA was negative detected with AGID. The results demonstrated that expressed p28 proteinhave good antigenicity and the indirect ELISA have higher sensitivity than AGID with p28 proteinas antigen to detect CAEV. In addition, 100 sera samples detected by p28 as antigen for ELISAshowed the positive rate was 67%, indicated the result was credible.Recent reports demonstrated the susceptibility of epithelial cells from different organs to caprine arthritis-encephalitis virus (CAEV) both in vitro and in vivo. Since granulosa cells (GC) are of epithelial origin and currently used for in vitro oocyte maturation, we addressed the question whether these cells are susceptible or resistant to CAEV infection. Furthermore, given the high serprevalence of CAEV in the all industrialized countries and the large number of ovaries derived from unknown serological status animals used for in vitro goat embryo production, one can conclude that these feeder cell cultures might be a potential source of early transmission of CAEV to goat embryos. So we did experiments to detect the susceptibility of GC to CAEV. Isolated granulosa cell(GC) was incubated at 37°C,5% CO2 in a humidified atmosphere for 48 h. After two to three passages of cell cultures, cells were inoculated with CAEV-Gansu and cytopathic effects(CPE) were observed. Proviral DNA was extracted from GC monolayer infected by CAEV. The p28 gene was amplified by PCR and a 660bp brand was obtained. Sequence analysis demonstrated the gene was p28 gene. Expression of CAEV p28 protein on GC was shown by immunobiotting using CAEV-positive serum from mice inoculated purified p28 protein. Supernatant of infected cells were shown to contain high titers (ranging 105 tissue culture infectious doses 50 per ml: TCID50 per ml) of infectious cytopathic viruses when assayed onto the indicator goat synovial membrane (GSM) cells. These results demonstrate that CAEV provirus genome was present in infected GC and expressed into proteins that were correctly processed and assembled into infectious particles released out of these cells.Therefore, we succeeded in expressing CAEV p28 gene and developing a diagnostic test to p28-specific antibody for ELISA. The result showed that the assay had such high sensitivity, specificity and repeatability that can be used practically. It was shown that the ELISA was specific and suitable for large-scale sero-epidemiological investigation for CAEV infection. In this study, the results show clearly that the granulosa cells derived from goat ovarian follicles that are commonly used as a feeder layer in the in vitro caprine embryo production procedures for commercialization of high-value goats are fully susceptible to infection by the widespread lentivirus CAEV. The susceptibility of granulosa cells to CAEV infection represents a potential danger to in vitro fertilisation and embryo transfer procedures used for the expansion of high-value breeding animals. So periodically detecting the serum of goats in farms was very essential.