| Bovine ephemeral fever(BEF) is caused by the arthropod-borne bovine ephemeral fever virus(BEFV). Animals with BEF present with high fever, spontaneous abortion, and lameness or paralysis, which leads to decline of milk production and serious economic losses.Five structural proteins and other unknown function proteins of BEFV have been described. The G protein is the main protective antigen. Four distinct antigenic sites(G1, G2, G3 and G4) on the surface of the G protein have been identified, G1 reacts only with anti-BEFV antibodies, but the other antigenic sites show cross-reactivity with sera against other related viruses. In addition, there are no commercially available diagnostic kits for detection BEFV or the antibody in the world. Although RT-PCR, LAMP, real time RT-PCR and MNT assays to detect BEFV, few of them have been widely applied for field investigations. These developed techniques are confined by their requirement of costly equipment, reagents, and technical expertise. Therefore, these methods are not suitable for filed samples investigation and can not be widely used with grass and large scale epidemiological survey.In the present study, the gene fragment containing part of the G1 epitope was cloned, and expressed and purified the targeted protein, then the immunogenicity and specificity of the protein were analyzed, and finally the recombinant protein was used as coating antigen to develop indirect ELISA diagnostic method for detection of BEFV antibodies. And the indirect ELISA kit for detection BEFV was successfully developed, which was examined the epidemiological investigation for 2822 sera in some regions of China. At the same time, the monoclonal antibody was synthesized according to the amino acid sequence of the G1 epitope from BEFV(LYC11), used as capture antibody; anti-BEFV cattle hyper-immune serum(polyclonal antibody) used as tracing antibody. We developed the antigen-capture ELISA assay using the monoclonal antibody and polyclonal antibody, evaluating the method using the 200 clinical blood samples. The result of the study was as follows: 1. We used the plasmid of the G gene of BEFV(LYC11 strain) were stored at in the lab as the template of PCR amplification. Then the specific primers were designed based on the BEFV(LYC11 strain) gene sequence on the NCBI, amplifying the targeted gene containing G1 gene. After sequence and identification of the goal fragment, the target protein was expressed using prokaryotic expression system. The immunogenicity and specificity were analyzed, proved that the recombinant protein has good reactogenicity, and only react with anti-BEFV antibody. 2. The purified recombinant protein was used as coating antigen to develop the indirect ELISA method for detection of BEFV. The various conditions of the assay were optimized, then the indirect ELISA kit for detection BEFV antibody was developed. Individual indicators of the kit were optimized, so that it can be stably used in clinical samples. 3. Using the kit we successfully developed a large scale serological survey of bovine ephemeral fever virus was conducted with the 2822 serum samples collected from various cattle breeds in 26 provinces in China, and the seropositive rate for the BEFV ranged from 0% to 81% between regions and species. And detail conclusion of the epidemiological investigation was obtained. 4. The monoclonal antibody(mAb) was synthesized according to the amino acid sequence of the G1 epitope from BEFV(LYC11) according to the gene sequence on the GenBank/NCBI, used as capture antibody; then the specificity and reactivity of mAb was evaluated. The results demonstrated that the mAb has better specificity and reactivity. In addition, anti-BEFV cattle hyper-immune serum(polyclonal antibody) was developed, which was assessed using the micro-neutralization test. 5. We successfully developed the antigen-capture ELISA assay using the monoclonal antibody and polyclonal antibody, and optimized the every condition and requirement for the assay. At last, we further evaluated the method using the 200 clinical blood samples. |
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