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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 expression plasmid of dengue virus (DENV2) nonstructural protein 3 (NS3) and affinity tag fusion protein and to obtain the protein that interacts with NS3 by tandem affinity purification (TAP). Methods The primers were designed according to the sequence of DENV2 gene. The NS3 gene was amplified by PCR using DENV2 c DNA as a template. After digestion, the NS3 gene was cloned into mammalian eukaryotic expression vector p CI-SF containing FLAG-StrepⅡ. The recombinant plasmid p CI-NS3-SF was obtained. The recombinant plasmid was transiently transfected into HEK293T cells by transfection reagent LipofectamineTM2000. The expression of NS3 fusion protein was verified by Western blot. The protein interacting with NS3 was isolated and purified by TAP method. Results The NS3 fusion protein expression vector was successfully constructed. TAP system was used to isolate the host protein that interacted with NS3 protein. Conclusions Tandem affinity purification can effectively separate the cellular proteins that interact with DENV2 NS3.