大鼠gremlin1真核表达载体的构建及表达

Molecular Cloning and Expression of Rat Gremlin1 in HSC-T6 Cell

构建大鼠gremlin1的真核表达载体pcDNA-gremlin,并观察其在真核细胞中的表达。从大鼠脑组织中提取mR-NA通过逆转录聚合酶链反应(RT-PCR)及巢式聚合酶链反应(nest-PCR)获得编码gremlin1的cDNA,定向克隆至真核表达载体pcDNA3.1(+)中;经酶切和测序鉴定构建正确,应用脂质体法转染HSC-T6细胞,并通过蛋白免疫印迹法检测细胞内grem-lin1的表达。结果显示,构建了大鼠gremlin1的真核表达载体,转染HSC-T6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HSC-T6细胞内gremlin1的表达显著增高。成功构建了真核表达载体pcDNA-gremlin,为研究gremlin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。

The research was to construct a recombinant eukaryotic expression vector expressing the rat gremlin1 in rat hepatic satellite cell line（HSC-T6）.Total RNA was extracted from rat brain tissue,and reverse transcription-polymerase chain reaction（RT-PCR） and nest-polymerase chain reaction were performed to obtain the cDNA fragment encoding gremlin1,then the gremlin cDNA was inserted into the pcDNA3.1（＋） vector by Kpn I and Xho I cloning sites.The construct of pcDNA-gremlin was further conformed by restrictional enzyme digestion analysis and DNA sequencing.HSC-T6 cells were transfected with the pcDNA-gremlin vector and the empty vector,pcDNA3.1（＋） as negative control.The expression of rat gremlin1 was detected by Western blotting assay.Results showed that the eukaryotic expression vector pcDNA-gremlin was constructed correctly,and significant increase of gremlin1 was detected in the HSC-T6 cells 48 hours after transfection.This result would facilitate the future study on the function and mechanism of gremlin in liver fibrogenesis.