C端带Myc和多聚组氨酸双标记的重组人β-防御素-2在COS-7细胞的转染表达

Expression of Recombinant Human β-Defensin-2 Gene with C Terminal of Double Marks of Myc and Poly-Histidine in Transfected COS-7 Cells

本研究旨在探索建立能有效表达和分泌人类β-防御素 - 2 (h BD- 2 ) ,其表达产物又易于被检测和分离纯化的哺乳类动物细胞表达系统的可能性及技术路线。将 h BD- 2的全长 c DNA片段插入编码双重报告基因 Myc和 6个多聚组氨酸 (His)的真核表达质粒 pc DNA3.1/Myc- His(+) ,构建出 C端带 Myc和 6× His的 rpc DNA3.1/Myc-His(+) /h BD- 2。用脂质体转染法将此重组真核表达载体导入 COS- 7细胞 ,分别从 m RNA和蛋白质水平分析 h BD-2的表达情况并同步检测细胞可溶性蛋白及培养上清的抗菌活性。采用 RT- PCR法从被转染的细胞中扩增出一条约 2 4 0 bp的片段 ,其大小与预测相符。采用 Western blot法 ,用抗 His抗体检测到细胞可溶性蛋白在约 10 Kd处有强反应条带显示 ,其大小与该重组质粒结构中由 p CMV启动子驱动的基因片段有可能表达成肽链的分子量 (10 .1)相符。双层肉汤琼脂平板扩散法结果显示 :分别与正常细胞可溶性蛋白和培养上清比较 ,转染重组质粒的 COS- 7细胞的可溶性蛋白及培养上清在金黄色葡萄球菌的平板上均形成明显抑菌环。这些结果提示 :所建立的 h BD- 2哺乳类动物细胞表达系统能有效表达和分泌活性 h BD- 2。

The aim of this study is to explore the possibility and technical itinerary of establishing an mammal engineering cell line in which hBD 2 can be effectively expressed, secreted, detected, separated and purified. The full hBD2 cDNA was inserted into the multi clone site of a eukaryotic expressive plasmid pcDNA3.1/Myc His(+) and located closely at the upstream of two tag gene (myc and 6 Poly histidines ) so as to construct another recombinant eukaryotic expressive vector of hBD 2 gene: rpcDNA3.1/Myc His/hBD 2. By the use of RT PCR with special primers, a band of 240bp was amplified from COS 7 cells transfected by this recombinant plasmid, which matched full length of cDNA coding hBD2 plus myc epitope and 6 poly histidines tags. Western blot analysis with specific anti histidines antibody revealed that the lysate of COS 7 cells transfected by rpcDNA3.1/Myc His/hBD 2 had a strong band with molecular weight of about 10 Kd that was approximate to the size of chiasmic peptide. Antibacterial activity assay showed that obvious bacterial inhibition occurred in both lysate and supernatant of COS 7 cells transfected by rpcDNA3.1/Myc His/hBD 2.