Preparation Of Monoclonal Antibodies Against PrM/ M Protein Of JEV And Proteomic Analysis Of U251 Cells Infected With JEV P3

Japanese encephalitis(JE), is a serious impact on the central nervous system function Arbovirus of zoonotic diseases, cause by Japanese encephalitis virus(JEV). As a structural protein of JEV, Pr M mainly presence the immature virus particles in the infected cells. The Pr M protein gets cleaved form M protein and M protein involved in constituting the viral envelope, interacts with the C-terminal E protein and the E protein folding, modification and assembly in cells. Also, M protein can induce mild neutralizing antibodies, and compose the infectious virus with the virus cored. Meanwhile, JEV targeted neurons in the central nervous system and can activate astrocytes and microglia signal to induce a large number of pro-inflammatory cytokines and chemokines and cause brain inflammation storm eventually. However, the function of Pr M in viral replication, infection and induction of neutralizing antibody activity is unknown. While, how to E protein folding, modification and assembly with Pr M protein is unclear. In addition, how many potential proteins have an effect in the brain inflammation storm pathway caused by JEV. There is no answer.In this study,one clone of monoclonal antibodies against Pr M / M protein was screened. And the biology properties of the MAbs were verified by indirect ELISA, IFA and Western Blot. In addition, U251 cells infected JEV P3 were quantitative proteomics analyzed by i TRAQ technology.The content of this study is as follow: 1. Preparation of JEV Pr M / M monoclonal antibodiesJEV M protein in prokaryotic expression plasmid p28a-sumo-M is constructed and mice were immunized with the purified protein of p28a-sumo-M. One clone of monoclonal antibodies(6C11) was screened after cell fusion and indirect ELISA, and four subclones strains(E2,G3,D4 and E4) were got who can stably secrete monoclonal. After identification, the subtype of MAbs is Ig G2 b, and the light chain is Kappa. By indirect ELISA, IFA, Western Blot to detect, these four MAbs have good specificity, and can identify natural Pr M / M protein. Meanwhile, the titer of ascites can up to 105. According DNAStar software predicts the M protein antigenic potential sites, two prokaryotic expression plasmids p KG-M1 and p KG-M2 were constructed. The M protein antigenic potential sites may exist in the first half, namely 1-126 base area. 2. U251 cells infected JEV P3 were quantitative proteomics analyzed by i TRAQ technology.U251 cells infected JEV P3 were collected to quantitative proteomics analyze by i TRAQ technology. 1780 kinds of proteins were identified in total. Most of them involved in cell and organelle composition, some of them involved in metabolic process, biological regulation, and response to stimulus, as well as a small number of them involved in the regulation of catalytic activity, molecular transducer activity and transporter activity. After proteins were quantitative analyzed, compared with the control group, there were had 54 up-regulated proteins and 35 down-regulated proteins in U251 cells infected JEV P3. Using the relative fluorescence quantitative techniques to detect eight kinds of m RNA in up-regulated proteins and one kind of m RNA in down-regulated proteins, the results were the same with i TRAQ technical analysis.