Generation And Identification Of The MAbs Specific To Japanese Encephalitis Virus And West Nile Virus

Japanese Encephalitis Virus (JEV) and West Nile virus (WNV) are the members of the family Flaviviridae, genus Flavivirus, the Japanese Encephalitis Virus Serocomplex Group. These viruses are mosquito-borne flaviviruses, and maintained in mosquito-bird or mosquito-mammal cycle in nature, with humans and other vertebrate animals as accidental hosts. They cause a broad range of clinical syndromes, including fever, meningitis, encephalitis, and even death. Recently, the prevalence of West Nile fever has done great harm to people's health all over the world. It is also within the list of the potential emerging infectious diseases in our country. Early diagnosis and prevention are the major stratages which could be taken to prohibit the prevailing of mosquito-borne diseases. Rapid imuno-assay has notable advantage in the detection of the infection. And as a promising tool, it is time-, labor-, and cost-.economic.Flaviviruses are positive-sense single-stranded RNA viruses. As for the genome size of WNV, it is 10. 9 kb which is transcribed into a single polyprotein precursor encoding three structural proteins, the capsid (C), premembrane (prM), and envelope (E) proteins, and seven nonstructural proteins. Basically, M protein and E protein are the two major structural proteins of WNV and also primary antigens as well. There are, however, lots of similar antigenic epitopes among the members of flaviviridae family. Thereby, the cross-reaction is frequent in the serological detection of this family. Recently, it is reported that monoclonal antibody specific to prM of dengue viruses did not react with the serum taken from JEV infected patients, which suggesting that there are virus specific antigenic epitopes within prM protein.In current study, we aimed to producting the antibodies for the specific and rapid detection of JEV and WNV. First, we immunized BALB/c mice with JEV vaccine-strain.Through ELISA screening and 3 rounds of sub-cloning, V6B9 positive hybridoma cell line secreting monoclonal antibody against JEV was obtained. The titer of mAb in ascites was l:105 and the Ig subclass of the mAb was IgGl. ELISA, Western-blotting and immunohistochemistry assay were used to identify the specificity of mAb and the results shown that the obtained mAb could recognize JEV and WNV. This mAb can be used in initial screening the members of JEV serocomplex group.Meanwhile, the gene encoding JEV prM was isolated using RT-PCR from brain of JEV infected mouse and cloned into prokaryotic expression vector pET32a. After been verified by sequencing, the fusion protein was expressed under IPTG induction in the E.coli BL21 (DE3) LysS. The expression product was identified by SDS-PAGE and Western Blot assay. Ni-NTAcolume was included to purify the prM fusion protein and Western Blot was performed for the identification. The BALB/c mice were immunized with purified recombinant prM protein. ELISA results indicated that mouse developed strong immune responses to the antigen, and immune sera could specifically recognize the JEV. The positive hybridoma cell lines P1F2 secreting monoclonal antibodies against prM were established after cell fusion of mouse spleen cells and P3-X63-Ag8.653 cells. The titer of anti-prM mAbs in ascites was l:105, and the Ig subclass of the mAb was IgGl. ELISA, Western Blot and immunohistochemistry assays were used to identify the specificity of mAb. The results shown that the mAb we obtained could specificly recognize prM of JEV and did not react with prM of WNV.Moreover, prM gene of WNV was synthesized referring to the sequence from Genebank and cloned into prokaryotic expression vector pET-32a. WNV prM fusion protein was expressed and purified using the same method as described above. The BALB/c mice were immunized with purified prM protein and two hybridoma cell lines secreting monoclonal antibody against prM was established after cell fusion of mouse splenic cells with P3-X63-Ag8.653 cells. The titer of anti-prM mAb in ascites was l(T~10b respectively. The specificity of mAb was identified by ELISA, Western Blot assay, and the results shown that the obtained mAbs was able to recognize prM of WNV and WNVantigen specifically, and had no cross-reaction with JEV. These two anti-prM mAbs could be used for specificity virus screening.The obtained prM specific mAbs were favorable for the development of rapid descrimining detection, dignosing and hence, for the prevention of West Nile fever and Japanese encephalitis. Most importantly, the generation of mAbs specific to WNV or JEV not only made a foundation for the studing on the structure and function of prM, but also provide some experimental data for the designing the candidating antigens for the development of the vaccine.