目的 利用Bac-to-Bac杆状病毒表达系统在Sf9昆虫细胞中实现人类错配修复基因hMLH1及其错义突变体的高效表达.方法 分别用Bam HI和Not I双酶切构建于pcDNA载体中的全长野生型hMLHI基因及T117M和V384D错义突变型DNA片段,回收后定向克隆至pFastBac1转座载体中,经酶切鉴定后转化DH10Bac感受态细胞,PCR方法筛选阳性菌落,提取重组Bacmid,反复感染Sf9昆虫细胞,用SDS-PAGE和Western blot方法鉴定目的蛋白的表达.结果 提取转染72h后的Sf9细胞总蛋白,用7％的SDS-聚丙烯酰胺凝胶电泳,可见分子量为85kDa清晰的蛋白条带,经Western blot方法证实该条带为hMLH1基因的表达产物.结论 利用Bac-to-Bac杆状病毒表达系统在Sf9昆虫细胞中大量表达出hMLH1野生型和T117M及V384D错义突变体蛋白,为进一步开展体外错配修复实验奠定了良好的基础.%Objective To investigate high-level expression of human mismatch repair gene hMLH1 and its missense variants using Bac-to-Bac baculovirus expression system.Methods The full-length wild type hMLH1 gene as well as T117M and V384D missense mutations which constructed in pcDNA3.1 vectors were sub-cloned into the Bam HI and Not I sites of pFastBac1 donor vector.The recombinant plasmid was then transformed into E.coli DH10Bac competent cells.The recombinant bacmid was extracted from white colonies and confirmed by PCR,then transfected into St9 insect cells to express hMLH1 protein and missense variants.The expressed protein was tested using SDS-PAGE and Western blot.Results Lysis of Sf9 cells which infected with baculovirus after 72h was analyzed by SDS-PAGE and Western blot and the size of expressed protein was estimated to be 85KDa,which consistent with the expected molecular mass of hMLH1 protein.Conclusion Baculovirus expression system has been used successfully to over-express wild type hMLH1 protein as well as hMLH1 T117M and V384D missense variants in Sf9 insect cells,which lay a good foundation for further in vitro mismatch repair assay.
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