Preparation Of Monoclonal Antibodies Against 2A And VP1 Of EV71 And Preliminary Test Of The Antibodies For Their Effects On Viral Replication

Enterovirus 71(EV71)is the main pathogen causing hand,foot and mouth disease(HFMD).HFMD mainly affects infants and young children.People with mild symptoms may have rashes and herpes and severe patients may have neurological system diseases such as viral meningitis,encephalitis,brainstem encephalitis and acute flaccid paralysis.It had posed a serious threat to public safety.In this thesis,a series of monoclonal antibodies against EV71 non-structural protein 2A and surface structure protein VP1 were prepared and screened using classical monoclonal antibody technology.These antibodies were preliminary identified and analyzed.1.Firstly,using prokaryotic expressed 2A protein to immunize mice and screened hybridoma cell lines secreting 2A-specific IgG by cell fusion.According to the antibody class switch mechanism,the 2A-specific IgA-producing hybridoma cell line was screened by limiting dilution method.2A5 and 5F11 IgA monoclonal antibodies were prepared from mouse ascites.After that the transport activity of IgA antibodies through epithelial cells and their effects on viral replication of EV71 in Transwell system in vitro were determined.The results showed that both 2A-specific antibodies 2A5 IgA and 5F11 IgA could transport through epithelial cells and effectively inhibit viral replication,and the inhibitory effect was dependent on the concentration of IgA antibodies.2.At the same time,to screen 9 strains of VP1 specific monoclonal antibodies with classical monoclonal antibody technology and purify IgG monoclonal antibody from mouse ascites.The results in vitro neutralization experiments in Vero cells and THP-1 cells showed that these 9 IgGs had no significant neutralizing activity in Vero but had the effect of enhancing virus replication in THP-1.Two strains of 9 VP1 IgGs C1 IgG and F4 IgG significantly enhanced the replication of the virus.Then the antigen-recognizing epitopes regions of the 9 antibodies were preliminarily identified by construction of VP1 protein truncated clones,suggesting the correlation between VP1 protein and antibody-enhanced infection and possible VP1 protein-associated regions,to lay a certain foundation to further study of the phenomenon and mechanism of antibody-enhanced EV71 infection.