具有生物学活性的hMSH2蛋白在昆虫sf9细胞中的表达

Expression of functional hMSH2 protein in baculocyte expression system

目的表达具有生物活性的hMSH2蛋白。方法用搭桥PCR获得hMSH2全长基因,构建pAcGP67B-hMSH2重组质粒,与AcNPV昆虫病毒DNA共转染昆虫细胞sf9,表达hMSH2蛋白(同时构建pET30a-hMSH2重组质粒,用大肠杆菌表达hMSH2蛋白);Western blot鉴定hMSH2蛋白;ELISA,流式细胞仪检测蛋白与OT3肽及TCRγδ的结合特异性;检测细胞增殖(MTT法)和细胞因子分泌,观察重组蛋白体外活化γδT细胞的生物学功能。结果克隆到正确的hMSH2基因,并将其插入到表达载体pAcGP67B和pET30a中的正确位置。经Western blot鉴定获得了hMSH2蛋白。用昆虫病毒表达载体系统表达的hMSH2蛋白能与其探针肽OT3及TCRγδ特异性结合,并能在体外刺激γδ细胞增殖及分泌IFN-γ,其活性更优于原核表达的蛋白。结论成功利用昆虫病毒载体表达系统得到了比原核大肠杆菌表达系统更具生物活性的hMSH2蛋白。

Objective Expression for functional human DNA mismatch repair protein hMSH2. Methods cDNA encoding hMSH2 was amplified with overlap-extension PCR, cloned into expression vector pAcGP67B and pET30a respectively. The recombinant vector was identified by restriction enzymes. The baculovirus DNA and pAcGP67B- hMSH2 were then cotransfected into si9 cells. Meanwhile, the recombinant pET30a-hMSH2 was expressed in E. coli. The recombinant hMSH2 protein was identified by Western blot. The binding specificity of hMSH2 protein to OT3 peptide and TCRγδ was detected by ELISA and FCM. Functions of hMSH2 protein were further evaluated by MTT colorimetric assays and cytokines secret assay in vitro. Results The full length of hMSH2 gene was obtained and cloned into pAcGP67B and pET30a, respectively. After 3 rounds of virus amplifying by re-infection, the hMSH2 was detected in the supernatant. The recombinant hMSH2 was expressed in E. coli as well. Purified hMSH2 expressed in baculovirus expression system not only binds specifically to OT3 peptide, but also to TCRγδ. Moreover, results indicated that the resultant protein can trigger the proliferation and IFN-γ secreting of γδT cells in vitro. Conclusion Functional hMSH2 recombinant protein was successfully expressed in baculovirus vector expression system holding more activity than that expressed in E. coll.