Screening The Monoclonal Antibodies Of Japanese Encephalitis Virus E Protein And Identification Of Its Epitopes

Epidemic encephalitis B was a mosquito-borne viral human zoonotic diseases that caused by the Japanese encephalitis virus. This study was major based on the E protein and its eiptopes of Japanese encephalitis virus as coating antigen, screening and identification the epitope region on E protein, using the indirect-ELISA analyze method that based on the JEV monoclonal antibodies.1. Cloning and expression E protein of JEVThis study amplified the JEV E gene by PCR method using pMD19T-ME template, E gene is 1500 bp and was cloned into pMD19-T vector. Then, the E gene was subcloned onto pET28a (+) vector though double enzyme method, after the identification of PCR and restriction enzyme digestion the recombinant plasmid was transformed into BL21 E.coli expression host. The recombinant plasmid expressed the E protein under the inducer of IPTG, the recombinant E protein was analyzed by the SDS-PAGE electrophoreses and the weight of the protein was about 55KDa the same as we predicted by DNAman prediction software. The results showed that the prokaryotic recombinant ecpression vector pET28a (+)-E was successfully constructed and expressed. This result showed that the pET28a (+)-E vector could express E protein successfully in BL21 E.coli expression host. Cloning and expressing the JEV E protein was the foundation of identifying the JEV monoclonal antibodies in the whole study.2. Screening the monoclonal antibodies of JEV E proteinIn the study, the purified E protein was gradienting diluted and coated in the ELISA plates. We used the anti-JEV pig serum to determine the best concentration of E protein, the results showed that the concentration of E protein achieved to 200ug/ml, the P/N value was the largest one; the positive OD450 value was closest to 1 and the negative OD450 value was lass than 0.2. This experiment closen the concentration 200ug/ml to coated the ELISA plate, and then the monoclonal antibodies supernatant sample was used to screening and identifies the epitopes on the E protein. The results showed 13 MAbs could recognize the epitopes on the E protein, another three MAbs cannot specificly recognize the E protein epitopes. The results revealed that using the E protein as antigen to screen the JEV monoclonal antibodies had high success screening rate and this study was also the foundation of further research of accurately analysis of MAbs against JEV E protein.3. Cloning and expression of the major antigen proteins on JEV E proteinIn this study, the E protein was cutted into six overlapping fragments based on the prediction software. These six fragments conteins three domains on E protein and they were the major antigenic epitopes on E protein. Firstly, we using the pMD19T-E template respectively cloned the six genes on E by PCR method (el is 186bp, encoding 62 amino acid residues; e2 is 249bp, encoding 83 amino acid residues; e3 is 213bp, code 71 amino acid residues; e4 is 345bp, encoding 115 amino acid residues; e5 is 336bp, encoding 112 amino acid residues; e6 is 327bp, encoding 109 amino acid residues). Then, the six section genes were cloned into pMD19-T vector. Thirdly, these genes were subcloned into PRSET (a) vector respectively. Afterly, we the recombinant plasmid was transformed into BL21 E. coli expression host, and then the recombinant epitope proteins were expressed under inducer of IPTG. The expressed proteins were analyzied by SDS-PAGE and its size is the same as we predicted. This study was a foundation of the further epitope localization on E protein by the JEV E protein MAbs. 4. Identification of the epitopes on JEV E protein by the MAbs of JEVThis study based on previous studies we used thirteen E protein MAbs to further identify the epitope regions on the E protein. Firstly, we used the purified epitope fragments to coat the ELIS A plates and determined the concentration of these proteins, and then we successfully constructed the indirect-ELISA method. The results showed that thirteen MAbs could recognize the different epitope on E protein. Among them, the MAb 7B5,10G3 and 15B5 could recognizes epitope on the e5 and the MAb 9C5 and 12F3 could recognizes epitope on the e6, and these two epitopes are included in the domain III of E protein which also included the neutralization epitope of JEV. Therefore, the results of the present study offered the relevant new ideas to the follow-up of JEV monoclonal antibody screening and identification and the passive treatment of JE disease.