CAJ | 학술논문

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 Cel s，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-PAGE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。
목적구건α-희순화매（alpha enolase，ENO1）중조표체질립，병연구기표체형식。방법이인제정맥내피세포（Human Umbilical Vein Endothelial Cel s，HUVEC）총RNA위모판합성cDNA；근거ENO1기인서렬，설계상하유인물，이cDNA위모판，경다취매련식반응（Polymerase Chain Reaction，PCR）확증득도목적기인편단ENO1；용T4DNA련접매련접매절산물화원핵표체재체pET28(a)，획득중조표체질립pET28(a)-ENO1；장pET28(a)-ENO1재BL21(DE3) E.Coli.대장간균중유도표체，분별취균액상청화침정진행SDS-PAGE전영，학정중조질립적표체형식。결과중조표체질립pET28(a)-ENO1중적ENO1측서결과여GeneBank상적서렬일치；pET28(a)-ENO1이가용성표체위주。결론ENO1중조질립적성공구건급기가용성위주적표체형식적학정，위연구ENO1적생물학활성급공능전정료기출。
Objective To construct an alpha enolase (ENO1) recombinant expression plasmid, and study its expression form. Methods The cDNA was synthesized using total RNA of Human Umbilical Vein Endothelial Cel (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 SDS-PAGE electrophoresis, respectively, to determine the expression form of the recombinant plasmid. Results The 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. Conclusion A kind of recombinant expression vector, pET28 (a) - ENO1, for expressing ENO1 protein was successful y constructed, and its expression form in E.Coli was also confirmed. The studies here wil lay the foundation for further research on biological activity and function of ENO1 .