Screening, Identification And Application Of Epitope In McAb Against Glycoprotein E Of Japanese Encephalitis Virus

Japanese encephalitis (JE) is one of the most important endemic encephalitis in theworld especially in Eastern and Southeastern Asia. JE virus is a single stranded positivesense RNA virus belonging to family flaviviridae, it has3structural proteins, capsid protein(C), precursor to the membrane protein (PrM), and envelope protein (E), and7nonstructuralproteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The structural proteins are the maincomponent of JEV virus particles; they are essential parts to maintain the virus particlemorphology.E protein is the major antigen component of JEV, it contains antigenic epitope whichcan stimulate the body to produce specific neutralizing antibodies and agglutinate red bloodcells. The mutation of E gene can often lead to the neurotoxicity or invasiveness loss. Thestudy found that107,138,176,177,264,279,315and439on E genes are the key aminoacids, especially E138and E176; they played important roles on virulence weaken.We have developed two anti-monoclonal antibodies3E9and6A8of JEV E protein byusing JEV less strain SA-14-14-2. The McAb have been purified by acid-sulfuric acidmethod. The result of ELISA suggested that the titer of3E9is1:1.2×105, and6A8is1:8.2×104. Both of them are subtyping IgG1.To further the identification, we used phage display technology to screen the epitopethat can be founded by McAb3E9on JEV protein E. After4rounds of screening,20plaqueswere picked out to extract single-stranded for DNA sequencing analysis. At last the specificepitope MEPPL（/F） that can be recognized by McAb3E9were found out, it has a highfrequency up to85%. Then, that epitope were expressed into pGEX-KG expression systemand purified. The result of ELISA and Western blotting suggested that the fusion protein hasa good antigenicity.In subsequent experiments, we used HE staining and immunohistochemical methods toidentify the antigen of JEV in mice brain and the conditions were optimized. It shows thatthe optimized immunohistochemical method has a high specificity and sensitivity.