10大鼠gremlin1真核表达载体的构建及表达.pdfVIP

生物技术通报 # # BIOTE CHN OLOG Y BULLET IN 2010 9 grem lin1 1 1 1, 2 1 刘艳玲 张贺吉 吴江锋 柳长柏 1 2 ( , 443002; , 443002) : 构建大鼠 gremlin1的真核表达载体 pcDNA2grem lin, 并观察其在真核细胞中的表达 大鼠脑组织中提取 mR2 NA通过逆转录聚合酶链反应 (RT2PCR)及巢式聚合酶链反应 ( nest2PCR )获得编码 gremlin1的 cDNA, 定向克隆至真核表达载 体 pcDNA31 1( + )中; 经酶切和测序鉴定构建正确,应用脂质体法转染H C2T6细胞, 并通过蛋白免疫印迹法检测细胞内 grem2 lin1的表达结果显示,构建了大鼠 grem lin1的真核表达载体, 转染H C2T6细胞培养 48 h后, 收集并裂解转染细胞,蛋白免疫 印迹法检测到转染H C2T6细胞内 grem lin1的表达显著增高成功构建了真核表达载体 pcDNA2grem lin, 为研究 grem lin在大 鼠肝纤维化发生发展过程中的作用及作用机制奠定了基础 : Grem lin1 肝纤维化 真核表达 M olecular Cloning and Expression ofRat Grem lin1 in HSC2T6 Cell 1 1 1, 2 1 L iu Yanling ZhangH eji Wu Jiangfeng Liu Changbai 1 2 ( Institute ofMoleculeBiology, ThreeGorgesUniversity, Yichang 443002; MedicalCollege of ThreeGorgesUniversity, Yichang 443002) Ab stra c:t The research was to construct a recomb inant eukaryotic expression vector expressing the rat gremlin1 in rathepatic sat2 ellite cell line (H C2T6). Total RNA was extracted from rat brain tissue, and reverse transcription2polymerase chain reaction ( RT2 PCR) and nest2 polymerase chain reaction were performed to obtain the cDNA fragment encoding gremlin1, then the gremlin cDNA was inserted into the pcDNA31 1( + ) vector byKpn I and ho I clon ing sites. The construct ofpcDNA2gremlin was further conformed by re2 strictional enzyme digestion analysis and DNA sequencing. H C2T6 cellswere transfectedwith the pcDNA2grem lin vector and the empty vector,