Establishment Of Stably Expressing NS1 Protein Cell Line Of Japanese Encephalitis Virus

Japanese encephalitis (JE) is a serious zoonosis caused by Japanese encephalitis virus (JEV). The Japanese encephalitis virus serocomplex of the family Flaviviridae includes West Nile virus (WNV), Saint Louis encephalitis virus (SLEV) and Murray Valley encephalitis virus (MVEV). JEV widely distributes in East and South Asia. As a membrane-associated glycoprotein, NS1 protein is one of the major components of the antigen, it is involved in viral early replication, RNA replication and assembly, and release of the virus. NS1 protein cannot be part of virus particles, so it shows no activity in neutralizing and hemagglutination inhibition. Additionally, for its binding activity for soluble complements, it can induce protective immunity without neutralization antibody, which depends on Fc fragments of the antibody. So far, there is no definite functional mechanism reported on NS1 protein, so it should perform in-depth studies on its structure, function and characteristics. However, NS1 protein expressed by prokaryotic cell cannot form the original molecular conformation, for this shotcoming, it is necessary to establish the eukaryotic stably expressing NS1 protein cell lines of JEV.In the present study, NS1 Gene from SA14-14-2 l strain was artificially synthesized after codon optimization. NS1 gene was subcloned into eukaryotic expression vector pCAGneo to construct the recombinant plasmids. Through restriction endonuclease digestive identification and sequencing analysis, the recombinant plasmid presented positive, then named as pCAGneo-opti-JEV-NS1. The positive recombinant plasmid was transformed into DH5αcompetent cells, and the single colony was picked up. After complification, the plasmid was extracted under aseptic condition. The recombinant plasmids were adjusted to a suitable concentration after linearization. The linearized recombinant plasmids were transfected into RK-13 cell, after G418 pressurized culture, a cell line expressed stably NS1 protein was screened with limited dilution method. The NS1 protein was demonstrated stably expressed in that cell line by RT-PCR, Western-blot, IFA and immunohistochemictry. The present study has initiated the research on definite machenisms of NS1 protein, laying the foundation for the investigations of the pathogenic and immunological mechanisms of JEV.At present, there are some limitations in clinical diagnosis methods for JE. Such as the principle of enzyme-linked immunosorbent assay (ELISA) in the detection of JEV antibody, based on the combination of the antibody and the antigen. This may lead to non-specific reactions, then with a result of detection failure of specific antibody or false positive results. In this study, a cell line was chosen as the target cells, a novel detective approach for anti-NS1 antibody has been established via complement-mediated cytotoxicity test (CDC). This method has higher sensitivity、specificity and better accuracy than ELISA method. Currently, the extensively applied inactivated vaccines cannot induce anti-NS1 antibody in the body, so our method can be seen as an ideal approach for distinguishing natural infection from artificial immunity. This method has great significance to epidemiological research of JEV.