| Tick Borne Encephalitis, usually called Forest Encephalitis, is spreaded by insect vector Tick and is a zoonotic arbovirus disease caused by Tick-borne encephalitis virus (TBEV) which is pathogenic for humans as well as for many species of animals. Tick Borne Encephalitis is also an acute infectious central nervous system disease; its main signs are outbreak high fever, muscle paralysis, conscious disturbance, respiratory failure and meningismus. This disease affects severely animal and human health. In China, a natural focus of TBE was found in the virgin forest of Northeast and Southeast. In recent years, follow with forest industry production environment, the destruction of forest ecological environment, and large number of people and animal influx to the cultivation forest, Tick Borne Encephalitis endemic area gradually expanded, and the number of cases increased year by year, so the prevention and treatment of Tick Borne Encephalitis are necessary.Tick-borne encephalitis virus is an important member of Flavivirus in Flaviviridae, which exits a large similarity compared to the other virus in terms of the genome sequence homology. Therefore, in the detection period of Tick Borne Encephalitis, rapid and easy diagnosis of Tick Borne Encephalitis virus is an effective monitoring mean. But the serum diagnostic method of Flavivirus for detection of Tick Borne Encephalitis virus is currently charactered with time-consuming, cross reaction and potential safety hazard in the respects of virus culture and antigen purification. Therefore, to find suitable alternative antigens to prepare diagnostic antibodies of Tick Borne Encephalitis is the foundation to build the rapid, easy and sensitive detection method.prM protein and E protein were two envelope proteins of Tick Borne Encephalitis virus. E protein could induce protective antibody, which had the function of receptor-binding and cell-membrane fusion and blood coagulation activation. prM protein was the precursor of M protein, whose existence contributed to the stable construction of M protein. M protein was a kind of antigenic endometrial protein with immunogenicity; it participated in the assembly of virus. M protein had synergistic effect on E protein, and increased the immunogenicity of E protein. In the experiment, Tick Borne Encephalitis virus (TBEV) gene complete sequence was downloaded from GenBank database. By software DNA Star 7.0, gene sequence was analyzed. The sequences of prM-E gene, prM gene and E gene were studied. The well expressed antigen gene sequence was selected as target gene to clone and express. Based on above results, prM protein and E protein of Tick Borne Encephalitis virus were selected for initial investigation.By software Oligo 6.0, 3 pairs of primers were designed in order to amplify 3 target genes. Recombinant plasmids were constructed by the amplified target gene fragment inserted into pUC57 vector. The constructed plasmids were digested with EcoRâ… and Xhoâ… , the obtained fragments were ligated directly to the expression vector pET-30a(+)-prM-E which was digested with EcoRâ… and Xhoâ… . The recombinant vector was transformed into the expression competent cells Escherichia coli BL21. By PCR identification, recombinant vector digeston and sequence analysis, it showed that the recombinant vector was successfully constructed. Then the target protein expression was induced by the recombinant vector at different temperature, different time points, and different concentrations of IPTG.It showed that target proteins were largely expressed in E. coli by SDS-PAGE electrophoresis. The acquired proteins were eluted and purified by affinity chromatography with Ni-NTA. These antigens was inoculated to the rabbits, the polyclonal antibodies were obtained. Then, the antigens as envelope antigens, the polyclonal antibodies as primary antibodies, and GAR-IgG as secondary antibody marked with horse radish peroxidase (HRP), the titers were made by enzyme linked immunosorbent assay (ELISA) in order to find the probability of the target protein used as diagnostic protein. This experiment lays a foundation for finding the diagnostic protein of Tick Borne Encephalitis and establishing a rapid and sensitive detective method.
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