摘要
固醇调控元件结合蛋白1(Sterol regulatory element-binding protein 1,SREBP1)通过调控脂肪生成相关的酶基因的转录,在机体脂类代谢调控中起着重要作用.本研究以21日龄的鸡胚肝组织的cDNA为模板,通过PCR分段克隆Srebp1全长基因的编码区片段,采用生物信息学分析其蛋白质功能结构域,将SREBP1功能结构域的编码片段克隆入原核表达载体pColdⅢ中,在大肠杆菌BL21(DE3)进行该功能片段的诱导表达.结果表明利用该基因内的KpnⅠ和NotⅠ两个限制性酶切位点,成功将鸡Srebp1基因上分别长700,1300,1500 bp 3个片段依次插入pcDNA3.1(+)质粒的HindⅢ和XhoⅠ酶切位点之间,获得全长Srebp1基因编码片段;在15℃和异丙基硫代半乳糖苷(Isopropylβ-D-Thiogalactoside,IPTG)的诱导条件下,pColdⅢ-g Srebp1-1125重组表达质粒在大肠杆菌BL21(DE3)中成功表达出重组蛋白gSREBP1-1125;SDS-PAGE电泳结果显示,重组蛋白gSREBP1-1125部分呈可溶性表达,但主要以包涵体存在.镍离子一步亲和层析法从菌体的裂解上清中获得了高纯度的重组蛋白;结合镍离子亲和层析,对重组蛋白进行固-液两相法复性,成功从包涵体中纯化得到高纯度的可溶性gSREBP1-1125功能多肽.鸡Srebp1真核表达载体和功能片段重组蛋白的获得为进一步研究其结构、功能以及抗体的制备奠定了基础.
The Sterol regulatory element-binding protein 1(SREBP1) plays an important role in regulating lipid metabolism by regulating genes transcription of the lipogenic enzymes. In order to obtain the full-length Srebp1, its fragments of 700 bp, 1 300 bp, and 1 500 bp were amplified by PCR from the cDNA of 21-day-old chicken embryo liver tissue in the paper. Then they were inserted between Hind Ⅲ and Kpn Ⅰ, Kpn Ⅰ and Not Ⅰ, Not Ⅰ and Xho Ⅰ restriction sites of pcDNA3.1(+), respectively. According to the bioinformatics analysis of the protein functional domains, the fragment of this domain was cloned into prokaryotic expression vector pCold Ⅲ. The recombinant protein of the SREBP1 functional fragment was induced in BL21(DE3) transformed with the recombinant plasmid by isopropyl β-D-thiogalactoside(IPTG) in BL21(DE3)at 15℃. The results of SDS-PAGE electrophoresis showed that the recombinant protein of gSREBP1-1125 could be expressed as soluble, but it mainly formed the inclusion body. The recombinant proteins with high purity were obtained from the cells lysed supernatant by Ni2+ affinity chromatography. After the denaturation of the inclusion body, the soluble gSREBP1-1125 with high purity was also successfully purified from the inclusion body using the method combined with the Ni^2+ affinity chromatography and the solid-liquid-phase renaturation of the recombinant protein, which lays a foundation for a further study on the structure, function of SREBP1 and preparation for its antibodies.
作者
杨毅鸿
潘玲
陈昱希
张景
徐璐
郁建锋
顾志良
YANG Yihong;PAN Ling;CHEN Yuxi;ZHANG Jing;XU Lu;YU Jianfeng;GU Zhiliang(School of Biology and Food Engineering,Changshu Institute of Technology,Changshu 215500,China)
出处
《常熟理工学院学报》
2020年第5期73-79,共7页
Journal of Changshu Institute of Technology
