猪STAT-1α基因的原核表达及其多克隆抗体制备

[目的]通过制备STAT-1α多克隆抗体,为进一步研究STAT-1α蛋白的功能特性及探索STAT-1α与猪瘟病毒间的相互作用机制奠定基础.[方法]利用RT-PCR从猪肾PK-15细胞中克隆出STAT-1α基因保守片段,与pET-32a（＋）构建原核表达重组质粒,转化原核宿主菌（Rosetta）,进行IPTG诱导表达,表达的重组蛋白经纯化和复性浓缩后,免疫小鼠制备STAT-1α多克隆抗体.[结果]STAT-1α基因能与原核表达载体pET-32a（＋）构建原核表达重组质粒pET-STAT-1o,转化大肠杆菌Rosetta后的最佳体外诱导条件是IPTG 0.5 mmol/L、诱导时间6h,其重组蛋白...

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Published in南方农业学报 Vol. 45; no. 1; pp. 118 - 122
Main Author 李延生 刘诚 张珂 李晓宁 李晓泉 尹珊 谢琳娟 何晓霞 罗扬 钟桃珍 罗廷荣
Format Journal Article
LanguageChinese
Published 广西大学动物科学技术学院,南宁530005 2014
亚热带农业生物资源保护利用国家重点实验室,南宁530005
Subjects
STAT-1α基因
原核表达
多克隆抗体
猪
swine
原核表达
polyclonal antibody
STAT-1α基因
猪
prokaryotic expression
多克隆抗体
STAT-1α
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Summary:[目的]通过制备STAT-1α多克隆抗体,为进一步研究STAT-1α蛋白的功能特性及探索STAT-1α与猪瘟病毒间的相互作用机制奠定基础.[方法]利用RT-PCR从猪肾PK-15细胞中克隆出STAT-1α基因保守片段,与pET-32a（＋）构建原核表达重组质粒,转化原核宿主菌（Rosetta）,进行IPTG诱导表达,表达的重组蛋白经纯化和复性浓缩后,免疫小鼠制备STAT-1α多克隆抗体.[结果]STAT-1α基因能与原核表达载体pET-32a（＋）构建原核表达重组质粒pET-STAT-1o,转化大肠杆菌Rosetta后的最佳体外诱导条件是IPTG 0.5 mmol/L、诱导时间6h,其重组蛋白主要以包涵体的形式表达.STAT-1α多克隆抗体具有高效特异性,与真核细胞PK-15细胞作用后,在PK-15细胞内能检测到STAT-1α蛋白表达,可观察到大部分细胞胞浆内有绿色荧光,但胞核内几乎无荧光,即STAT-1α主要定位在正常PK-15细胞的细胞浆内表达.[结论]制备获得的猪STAT-1 α多克隆抗体特异性好,既能与原核细胞大肠杆菌（Rosetta）表达的STAT-1α反应,又能与真核细胞PK-15的STAT-1α发生反应.
Bibliography:[Objective]The polyclonal antibody STAT-1α was prepared to provide references for further research on functional characteristics of STAT-1α and interactive mechanism between hog cholera virus and STAT-1α. [Method]Using RT-PCR, STAT-1α gene conservative fragment was cloned from PK-15 cell in swine kidney, and together with pET-32a （＋）, prokaryotic expression recombinant plasmid was constructed. The STAT-1α polyclonal antibody was prepared after IPTG inducible expression of Rosetta, and purification and concentration of inclusion body protein. [Result]Prokaryotic ex- pression recombinant plasmid pET-STAT-1α was produced by STAT-1α gene and prokaryotic expression vector pET-32a （＋）. The optimal induction conditions for transforming E. coli prokaryotic cells Rosetta were IPTG concentration of 0.5 mmol/L and induction duration of 6 h. The recombinant protein must be expressed in the form of inclusion body. STAT-Iot polyclonal antibody showed the traits of high reactivity and specificity. Following cytosis with euk
ISSN:2095-1191
DOI:10.3969/j:issn.2095-1191.2014.1.118