Prokaryotic Expression And Monoclonal Antibody Preparation Of Zika Virus Nonstructural Protein 1

Zika virus(ZIKV)is a new arbovirus-borne virus that belongs to the genus Flavivirus and is mainly transmitted by Aedes aegypti and Aedes albopictus.ZIKV infection is generally self-limiting in humans,with a high prevalence of asymptomatic infections of up to 80%.While 20% of patients have mild symptoms,typically experience fever,maculopapular rash,arthralgia,conjunctivitis,and headache.Serious diseases of ZIKV include GuillainBarré syndrome and congenital microcephaly.The NS1 protein is the only nonstructural protein which can be secreted into the serum of the infected patients among the seven nonstructural proteins of flaviviruses.NS1 can be secreted extracellular in the form of soluble hexamer after virus infection,and can be detected in serum at the early stage during viral infection.Therefore,NS1 can be used as a marker for rapid clinical diagnosis of early viral infection.Previous studies have shown that NS1 protein carries several specific epitopes and can induce multiple specific antibodies that have no cross reaction with other flaviviruses.This study aimed to prepare monoclonal antibodies against the Zika virus NS1 protein.The NS1 fragment was amplified and cloned to the the expression vector pET-32 a.The recombinant plasmid was used as an expression vector for prokaryotic expression of ZIKV NS1.The expression of NS1 was identified by western blotting(WB)and enzyme-linked immunosorbence assay(ELISA).Recombinant proteins with high purity were obtained by inclusion body washing,dialysis and renaturation of the target protein,His-taq and molecular sieve chromatography purification successively.The purified recombinant protein was used as the antigen to immune BALB/c male mice within 6~8 weeks by subcutaneous injection.For each mice,100 ?g recombinant protein was injected for three rounds of immunization with a 14-day cycle.The indirect ELISA assay was performed to determine the titer of the antibody against the recombinant protein in the serum of the mice.After three times of immunizations,the titer of the antibody in the serum reached 1:100000 which is high enough for the monoclonal antibody screening assay.Three days later,the spleen cells of the immunized mice were isolated and fused with the myeloma cells SP2/6.The fused cells were cultured in the HAT-DMEM medium with 20% FBS,and the supernatants from the fused cells were subjected to WB and indirect ELISA to ensure whether the finally selected cell line can stably secrete anti-NS1 monoclonal antibody.In this study,the NS1 protein of ZIKV was expressed by E.coli prokaryotic expression system.The recombinant NS1 protein with high purity was obtaind and used as antigen for the monoclonal antibody screening assay.Three monoclonal antibodies with high activity and obvious immunoreactivity were selected.We will supply the materials for the establishment of double-antibody sandwich ELISA based ZIKV detection method and the related research about ZIKV NS1 protein.