In Vitro Expression Of NS1 Gene Of Japanese Encephalitis Virus

A pair of primers P1 and P2 were designed and synthesized according to the NS1 sequence of vaccine strain SA14-14-2 of Japanese encephalitis virus. A 1087bp of target fragment amplified by RT-PCR from extracted RNA of SA-14-14-2, and cloned into cloning vector pMD18-T. The pMD18-T-NS1 containing NS1 gene was subjected to sequencing after identification by restrict digestion and PCR. Sequence analysis indicated that the opening reading frame of NS1 gene is 1056nt in length, encoding 352 amino acids, identical to the sequence of strain SA 14-14-2 deposited in GenBank. NS1 gene in the pMD18-T-NS1 was cut by BamHI and HindIII, was cloned into the expression plasmid pET-30b (+) treated with the same enzymes, resulting in a recombinant plasmid pET-30b-NS1. The pET-30b-NS1 was identified by PCR, restriction digestion, and sequencing. The pET-30b-NS1 was used to transform Escherichia coli BE-21 (DE3). High-level expression of the target protein was obtained after induction of the transformed cells with IPIG. The molecular weight of the recombinant NS1 protein was 46kDa whenanalyzed in SDS-PAGE, suggesting the protein was fused with a His-tag. The recombinant NS1 protein accounts for 30.3% of the total cell lyses. The recombinant NS1 protein showed immunologic activity specific for JEV as indicated by Western blotting. NS1 gene of SA14-14-2 was sequenced and expressed in Escherichia coli. High-level expression of NS1 protein will surely facilitate the preparation of recombinant NS1 antigens, the production of monoclonal antibody directed to NS1, the development of subunit vaccine against JEV, and the investigation of molecular epidemiology of JEV.