摘要
目的本文旨在构建11β-羟基类固醇脱氢酶2（11β-HSD2）基因的重组真核表达载体，并检测其转染人哺乳动物细胞后对白介素6（interleukin-6，IL-6）表达水平的影响。方法用PCR方法扩增11β-HSD2基因的全长cDNA，先亚克隆至pGMT载体，测序正确后再亚克隆至pcDNA3．1myc-hisC以构建其重组真核表达载体。将重组的真核表达载体瞬时转染人肺腺癌细胞系A549，用Western blot方法鉴定表达情况，并用ELISA方法检测IL-6表达水平。结果测序表明11β-HSD2的重组真核表达载体构建成功，Western blot显示真核表达载体能够在A549细胞中成功表达，并可使IL-6表达水平升高。结论成功构建了11β-HSD2基因的重组真核表达载体，初步验证了该基因的促炎效应。
Objective To establish recombinant eukaryotic vector of 11β-hydroxysteroid dehydrogenase 2 gene in mammalian cell and to detect the expression of interleukin-6 after the vector transfection. Methods The cDNA of 11β-hydroxysteroid dehydrogenase 2 gene was amplified by PCR. After subcloned with pGMT vector and sequenced, the gene was inserted into the eukaryotic vector pcDNA3,lmyc-hisC. The expression vector was transiently transfected in human lung adenocarcinoma cell line A549 cells, and then the protein was detected using Western blot,interleukin-6 was measured by EI.ISA. Results Sequencing revealed 11β- hydroxysteroid dehydrogenase 2 recombinant eukaryotic expression vector was constructed successfully, and Western blot indicated that eukaryotic expression vector expressed successfully in A549 cells. Transfection with recombinant eukaryotic vector of 11β-hydroxysteroid dehydrogenase 2 gene significantly increased interleukin-6 expression. Conclusions The recombinant eukaryotic vector of 11β-hydroxysteroid dehydrogenase 2 gene is established successfully and could be highly overexpressed in A549 cells. The increase level of interleukin-6 suggests the proinflammatory effect of 11β-hydroxysteroid dehydrogenase 2.
出处
《国际呼吸杂志》
2013年第12期906-910,共5页
International Journal of Respiration
基金
基金项目：军队医药卫生“十一五”科技攻关项目（08G006）
