Production Of UniversalMonoclonal Antibodies For Dengue Serotype â… -â…£ Virus Nonstructural Prontein NS1, Andestablishment Of NS1 Antigen Capture ELISA

Dengue virus (dengue virus, DV or DENV) can infect humans and cause serious clinical diseases,of which dengue hemorrhagic fever and dengue shock syndrome lead to a high mortality. So it is very important to develop some early diagnoses for dengue virus infection. Nonstructural protein NS1 of Dengue virus has strong immunogenicity, which can occur early in the infection of dengue virus in serum and appeared earlier than IgM antibodies.Studies have shown that NS1 can be used for the early diagnosis and monitoring of disease. Thus, NS1 is expected to become a early diagnosis method for dengue virus infection.In this study, bioinformatics software was used to analyze NS1 antigen epitopes of all four dengue virus serotypes (Ⅰ-Ⅳ), and common conserved epitopes of NS1 protein were chosen to conjμgate with small molecμle carrier BSA, and prepare universal monoclonal antibody of four typed NSlby immunization of mice and hybridoma technique. Meanwhile, specific NS1 primers were designed according to the gene sequences of Ⅰ-Ⅳ dengue virus NS1 proteins, and fμll lengths of four typed NS1 genes were obtained by RT-PCR, which were cloned into pMD19T carrier. Following double digestion, the target fragment was cloned into the expression vector pET30a. After IPTG induction, Western Blot was carried out to identify the expression products.Further protein purification and dialysis refolding were then performed and recombinant NS1 preparation of Ⅰ-Ⅳ type dengue virus was obtained. Using the obtained NS1 universal monoclonal antibody for Ⅰ-Ⅳ typed dengue virus and recombinant NSlprotein, we successfully established ELISA detection method based on dengue virus NS1 antigen.