结核分枝杆菌热休克蛋白基因真核表达载体的构建及表达

Construction of eukaryotic expression vector for Mycobacterium tuberculosis heat shock protein X gene

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 a eukaryotic expression vector for fusion protein of Mycobacterium tuberculosis（Mtb）heat shock protein X（HspX）and enhanced green fluorescent protein（EGFP）, and express in RAW264. 7 cells. Methods HspX gene was amplified by PCR using the genome of Mtb H37 Rv as a template, and inserted into a multiple cloning site of eukaryotic expression vector p EGFP-N1. The constructed recombinant plasmid p EGFP-N1-HspX were identified by restriction analysis and sequencing, and transfected to RAW264. 7 cells. The expression of GFP was observed by fluorescence microscopy, while that of HspX by real-time PCR and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid p EGFP-N1-HspX was constructed correctly. The expression of GFP was detected in RAW264. 7 cells 24 h after transfection. The relative molecular mass of expressed Flag-HspX fusion protein was about 43 000. The HspX m RNA level in RAW264. 7 cells transfected with recombinant plasmid p EGFP-N1-HspX was significantly higher than that with empty vector（P〈0. 01）. Conclusion Eukaryotic expression vector p EGFP-N1-HspX was constructed successfully, and Flag-HspX fusion protein was effectively expressed in RAW264. 7 cells.