猪EP1基因的克隆与原核表达

Cloning and prokaryotic expression of porcine epithelial progenitor 1 gene

为进一步体外研究EP1基因的生物学功能,本试验对EP1基因进行了组织分布分析。以猪骨髓cDNA为模板克隆了猪EP1基因的开放阅读框,对克隆的基因序列进行了序列分析,用非酶连接技术将此基因克隆至丙酸诱导型原核表达载体pBbB3a-His6-MBP-LIC中。用菌液PCR进行阳性克隆鉴定并测序,将测序鉴定正确的克隆菌液提取质粒,转化至E.coli BL21(DE3)中。丙酸钠诱导表达His6-MBP-EP1融合蛋白,并用Western blot进行鉴定。结果显示:本试验成功克隆了猪EP1基因,长度为651bp,猪EP1基因在骨髓中的表达量很高;构建了EP1丙酸诱导型原核表达载体pBbB3a-His6-MBP-EP1;His6-MBP-EP1融合蛋白在裂解菌液的上清中表达,相对分子质量为65560。结果表明,运用大肠杆菌表达系统成功表达EP1基因融合蛋白,为进一步在体外开展猪EP1基因生物学功能的研究提供基础。

Epithelial progenitor is a kind of cellular protein expressed by epithelial cells, which is correlated with tubuar deformation. To further explore the biological functions of EP1 gene, we an- alyzed the mRNA distribution of EP1 gene in tissue by real-time PCR,amplified the open reading frame （ORF） of EP1 gene from the cDNA of porcine thymus,then analyzed the sequence of the cloned gene, and cloned it into propionate inducible plasmid pBbB3a-His6-MBP-LIC by ligation-in- dependent cloning （LIC） technology. Identification of individual clones was performed by bacteria liquid PCR followed by DNA sequencing. Plasmids were extracted from the confirmed bacteria and transformed into E. coli BL21 （DE3）. The His6-MBP-EP1 fusion protein was induced by sodium propionate and identified by Western blot. Our results showed that： （1） EP1 gene,which is 651 bp in length,was successfully cloned;the expression of EP1 gene in the bone marrow of pig was very high. （2） The propionate inducible plasmid, pBbB3a-His6-MBP-EP1, was constructed; （3） His6- MBP-EP1 fusion protein was efficiently expressed in soluble form,with a molecular weight of a- bout 65 560. These results indicate that EP1 fusion protein was successfully expressed in E. coli BL21 （DE3） and this will facilitate the exploration of EP1 function in vitro.