pEGFP-C2-NLRC5重组质粒的构建及其表达  

Construction of recombinant plasmid pEGFP-C2-NLRC5 and its expression

作　　者：徐涛[1] 彭云云[1] 李琳[1] 黄成[1] 张磊[1] 金涌[1] 吕雄文[1] 李俊[1] 

机构地区：[1]安徽医科大学药学院,合肥230032

出　　处：《安徽医科大学学报》2014年第1期5-8,共4页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81273526);安徽省自然科学基金(编号:KJ2010A178)

摘　　要：目的构建含NLRC5蛋白功能区的cDNA序列的真核表达载体pEGFP-C2-NLRC5,并检测其在肾成纤维细胞(COS-7)中的表达。方法采用RT-PCR法从人肝星状细胞(LX-2)中获得NLRC5蛋白功能区的cDNA序列片段,用EcoRⅠ和BamHⅠ双切酶将片段和载体pEGFP-C2双酶切后,酶切产物加入T4连接酶16℃连接过夜,构建真核表达载体pEGFP-C2-NLRC5。将构建成功的pEGFP-C2-NLRC5重组质粒经PCR,限制性内切酶酶切和测序鉴定后用阳离子脂质体LipofectamineTM2000将其转染至COS-7细胞,荧光显微镜下观察绿色荧光蛋白表达,Western blot法鉴定融合蛋白表达。结果阳性克隆经双酶切法鉴定可见NLRC5基因片段。转染重组质粒后可观察到绿色荧光蛋白的表达,而且主要分布在细胞质中,Western blot法也可检测到在74 ku处有一明显条带,其大小符合EGFP-NLRC5表达的蛋白(NLRC5蛋白大小约为47 ku,EGFP蛋白大小约为27 ku),表明融合蛋白可在哺乳动物细胞中成功表达。结论成功构建pEGFP-C2-NLRC5质粒,NLRC5蛋白可在COS-7细胞中成功表达。Objective To construct pEGFP-C2-NLRC5 of expression plasmid, and observe its expression in COS-7 cells. Methods Get the cDNA sequence of NLRC5 protein from the LX-2 by RT-PCR. Then NLRC5 eDNA and the vector pEGFP-C2 were digested with restriction enzymes EcoR I and BamH I , and the digested productions were comlected by T4 enzyme at 16 ℃ , and then the eukaryotic vector of pEGFP-C2-NLRC5 was constructed. The recombinant vector was identified by the double digestion with restriction enzymes EcoR I and BamH I and DNA sequencing. After the analysis, pEGFP-C2-NLRC5 was transfected into renal fibroblasts COS-7 cells by Lipo- fectamine^TM12000, and the expression of pEGFP-C2-NLRC5 was monitored by fluorescence and confocal microscope and Western blot. Results The NLRC5 fragment was contained in the positive recombination by identification of restriction enzymes. After transfectting recombinant plasmid, green fluorescent protein could be observed, and they were mainly distributed in the cytoplasm. A stripe of 74 ku protein could be detected by Western blot, whose size was accord with EGFP-NLRC5 of protein expression （NLRC5 protein：47 ku, EGFP protein： 27 ku）. Therefbre, fusion protein could be successfully expressed in mammalian cell. Conclusion The eukaiyotic expression vector of pEGFP-C2-NLRC5 is constructed successfully,and the fusion expression of NLRC5 protein and GFP can be detected in COS-7.

关 键 词：NLRC5 COS-7 基因表达 重组质粒 转染 

分 类 号：R349.64[医药卫生—基础医学]