Generation And Application Of The Monoclonal Antibodies Against NS2B Protein Of Japanese Encephalitis Virus

Japanese encephalitis(JE) is a serious mosquito-borne disease caused by Japanese encephalitis virus, belonging to family Flaviviridae. JE is one of the leading causes of viral encephalitis and neurological infections in Asia. Patients may present multiple manifestations like fever, nausea, disturbance of consciousness etc. Survivors of JE often suffer neurological sequelae. And reproductive disorder has also been causing huge economic losses to pig industry. Accumulating evidences suggest the importance of these NS proteins in viral pathogenesis, however, the precise mechanism of each protein still needs to be fully explored. Till now, it is unclear about the functions of non-structural(NS) proteins and the roles of NS proteins in the pathogenesis of JEV. In this study, NS2B-specific monoclonal antibodies were produced and characterized that may be used as powerful tool for investigating the distribution of NS2 B protein at different times in different cell lines in both P3 strain and SA14-14-2 strain. It will be a new idea to reveal the function of JEV NS2 B proteins. The contents of this thesis are as follow: 1. Preparation of monoclonal antibodies(MAbs) against JEV NS2 B and epitope characterization of MAbs against NS2 B.The purified recombinant GST-NS2 B protein expressed by Escherichia coli was used to immunize BALB/c mice. After fusing with mouse myeloma cell line SP2/0, and screening by indirect enzyme-linked immunosorbent assay( ELISA), three clones of monoclonal antibodies(MAbs) against NS2 B protein, named 1B9, 3E12 and 4E6 were generated. Ig G subtypes are Ig G2 b, Ig G1, and Ig G1, respectively. The light chains of these antibodies are Kappa chain. The specificity and sensitivity of MAbs were demonstrated by indirect ELISA, indirect immunofluorescence assay, and Western blot.Based on this, the JEV NS2 B gene fragments were divided into two parts, and then cloned into the bacterial expression vector p GEX-KG, named p GEX-KG-NS2B-1(1-201bp) and p GEX-KG-NS2B-2(190-393bp). Induced expression of two mutant proteins with four overlapped amino acids to each other were generated, named GST-NS2B-1(1-67aa) and GST-NS2B-2(64-131aa). Indirect ELISA and Western blot results indicated that all clones of MAbs reacted with amino acids from 68 to 131 of NS2 B. 2. Compare NS2 B expression profiles in P3 and SA14-14-2 stains of JEV.The cell samples were collected, which were infected with the same MOI of JEV P3 strain and SA14-14-2 strain. The virus, RNA samples and protein samples were extracted from the cell samples at different time points in different cell lines. Comparison between two viral strains was done by viral titration and m RNA expression profiles. Further, protein samples were subjected to western blot analysis by NS2 B MAbs prepared in this study along with an already prepared E protein MAbs. The results of this study revealed that: SA14-14-2(which is an attenuated strain) viral titers were significantly higher than P3 virulent strain in the SK cell line. But P3 virulent strain was significantly higher than SA14-14-2 attenuated strain at the level of replication in both cell lines. The expression of E protein of SA14-14-2 strain was higher than P3 strain, but the contradicting to this expression of NS2 B protein was higher for P3 compared to SA14-14-2.