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目的构建结核分枝杆菌热休克蛋白X(heat shock protein X,HspX)基因与EGFP融合基因的真核表达载体p EGFP-N1-HspX,并在小鼠单核巨噬细胞RAW 264.7中表达。方法以结核分枝杆菌(H37Rv)基因组DNA为模板,PCR扩增HspX基因,定向插入至质粒p EGFP-N1的多克隆位点,经酶切及测序鉴定正确后,将质粒p EGFP-N1-HspX转染RAW264.7细胞,荧光显微镜下观察GFP的表达,Real-time PCR及Western blot法鉴定HspX基因的表达。结果质粒p EGFP-N1-HspX经酶切及测序鉴定,证明构建正确;质粒p EGFP-N1-HspX转染24 h后,荧光显微镜下可见GFP表达,表达的重组融合蛋白Flag-HspX相对分子质量约43 000;转染组细胞中HspX基因m RNA水平明显高于空载体组(P<0.01)。结论成功构建了p EGFP-N1-HspX融合基因真核表达载体,并在RAW264.7细胞中获得表达。
Objective To construct the eukaryotic expression vector p EGFP-N1-HspX of Mycobacterium tuberculosis heat shock protein X (HspX) gene and EGFP fusion gene and express it in RAW 264.7 mouse monocyte-macrophage cells. Methods The genomic DNA of Mycobacterium tuberculosis (H37Rv) was used as a template to amplify the HspX gene by PCR. The HspX gene was inserted into the multiple cloning site of plasmid pEGFP-N1. After identification by restriction enzyme and sequencing, the plasmid p EGFP-N1- HspX was transfected into RAW264.7 cells. The expression of GFP was observed under a fluorescence microscope. The expression of HspX gene was identified by Real-time PCR and Western blot. Results The plasmid p EGFP-N1-HspX was identified by restriction enzyme digestion and sequencing. The plasmid p EGFP-N1-HspX was constructed correctly. GFP expression was observed under a fluorescence microscope 24 h after plasmid p EGFP-N1-HspX transfection. The relative molecular mass of the recombinant fusion protein Flag- About 43 000; The mRNA level of HspX gene in transfected cells was significantly higher than that in empty vector group (P <0.01). Conclusion The eukaryotic expression vector p EGFP-N1-HspX fusion gene was successfully constructed and expressed in RAW264.7 cells.