2型登革病毒非结构蛋白NS3的表达及其相互作用蛋白的纯化

Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it

目的构建2型登革病毒(DENV2)非结构蛋白3(NS3)与亲和标签融合蛋白的表达质粒,串联亲和纯化(TAP)法获得与NS3相互作用的蛋白。方法根据DENV2基因序列,设计引物;以DENV2 c DNA为模板,PCR扩增出NS3基因,经酶切后克隆至含有串联亲和标签(FLAG-StrepⅡ)的哺乳真核表达载体p CI-SF,获得重组表达质粒p CI-NS3-SF;将上述重组质粒通过转染试剂LipofectamineTM2000瞬时转染进入HEK293T细胞,Western blot法验证NS3融合蛋白的表达;通过TAP法分离纯化与NS3相互作用的蛋白。结果成功构建了NS3融合蛋白表达载体,利用TAP系统分离得到与NS3蛋白相互作用的宿主蛋白。结论串联亲和纯化法可以有效的分离与DENV2 NS3相互作用的细胞蛋白。

Objective To construct the plasmid expressing the fusion protein of Dengue virus type 2（ DENV2） nonstructural protein 3（ NS3） with affinity tag,and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification（ TAP） assay. Methods Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments,after amplified by PCR with DENV2 c DNA as template,were digested and cloned into the mammalian eukaryotic expression vector p CI-SF with the tandem affinity tag（ FLAG-StrepⅡ）. The recombinant p CI-NS3-SF was transiently transformed by LipofectamineTM2000 into HEK293 T cells,and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay.Results The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system. Conclusion TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3.