Construction of dengue virus serotype2 NS1 eukaryotic expression vector and its significance

HE Li; YI Xiao-fang; LUO Chun-hua

doi:10.3969/j.issn.1002-266X.2015.32.003

Abstract：Objective To construct a eukaryotic expression vector of dengue virus serotype 2( DENV2) NS1 gene, and to lay the foundation for screening the protein which was interacting with NS 1 and for the mechanism of being against the dengue virus infection .Methods NS1-Flag sequence was amplified from the DENV 2-infected THP1 genomic DNA by RT-PCR and cloned into pSG5 plasmid to construct the pSG5-NS1-Flag eukaryotic expression vector .Positive clones were screened and then were identified by the enzyme digestion and sequencing .The eukaryotic expression vector was transfected into 293T cells and A549 cells by lipofection transfection .The expression of NS1 protein was detected by Western blotting . Results The full-length gene fragment size of NS 1after amplification which had the Flag label was 1 089 bp, and was con-sistent with the expected one .The carrier and NS1 gene both contained 1 of ECORⅠsite, the single enzyme fragment sizes were 463 and 4 722 bp, respectively;the amplified fragment size of NS 1 and the double enzyme fragment size of pSG 5 plas-mid were 1 089 and 4 100 bp, respectively .During the six positive clones , the target bands of clone 5 and clone 6 were consistent with the expected results and were identified by sequence identification .The NS1 fusion protein expression was extremely low after 293T cells and A549 cells were transfected by empty vector , after the transfection of eukaryotic expres-sion vector , there was the NS1 protein expression .Conclusion The pSG5-NS1-Flag eukaryotic expression vector is suc-cessfully constructed , which paves the way for studying interaction between NS 1 and immunomodulatory protein , post-trans-lational modification and the mechanism of the body′s immune system against dengue virus infection .