α烯醇化酶重组质粒构建及其表达形式研究.docVIP

α烯醇化酶重组质粒构建及其表达形式研究 　　摘要：目的 构建α-烯醇化酶（alpha enolase，ENO1）重组表达质粒，并研究其表达形式。方法 以人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells，HUVEC）总RNA为模板合成cDNA；根据ENO1基因序列，设计上下游引物，以cDNA为模板，经多聚酶链式反应（Polymerase Chain Reaction，PCR）扩增得到目的基因片段ENO1；用T4DNA连接酶连接酶切产物和原核表达载体pET28(a)，获得重组表达质粒pET28(a)-ENO1；将pET28(a)-ENO1在BL21(DE3) E.Coli.大肠杆菌中诱导表达，分别取菌液上清和沉淀进行SDS电泳，确定重组质粒的表达形式。结果 重组表达质粒pET28(a)-ENO1中的ENO1测序结果与GeneBank上的序列一致；pET28(a)-ENO1以可溶性表达为主。结论 ENO1重组质粒的成功构建及其可溶性为主的表达形式的确定，为研究ENO1的生物学活性及功能奠定了基础。 　　关键词：图书馆 　　Construction and Expression of α-enol Form of the Enzyme in Clinical Study of Recombinant Plasmi 　　NING Xing-wang1,XIE Xiao-bing1,ZHU Hui-bin1,WANG Shu-xiang1,GE Jin-wen2 　　(1.Medical Examination Center,The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,Hunan,China;2.Institute of Integrative Medicine of Hunan University of Chinese Medicine,Changsha 410208,Hunan,China) 　　Abstract：ObjectiveTo construct an alpha enolase (ENO1) recombinant expression plasmid, and study its expression form. MethodsThe cDNA was synthesized using total RNA of Human Umbilical Vein Endothelial Cell (HUVEC) as the template. ENO1 gene was amplified from cDNA using specific forward and reverse primers by polymerase chain reaction (PCR). The recombinant expression vector was obtained by ligating ENO1 gene and pET28(a) using T4 ligase. ENO1 protein was produced by transfecting expression vector pET28(a)-ENO1 into BL21(DE3) E.Coli. and subsequent IPTG induction. The supernatant and bacteria precipitates were collected and separated by SDSelectrophoresis, respectively, to determine the expression form of the recombinant plasmid. ResultsThe sequence of cloned gene of ENO1 in the recombinant expression plasmid pET28 (a) - ENO1 was consistent with the sequence from the GeneBank; the expression form of PET28 (a) - ENO1 is soluble expression. ConclusionA kind of recombinant expression vector, pET28 (a) - ENO1, for expressing ENO1 protein was successfully constructed, and its expressi