文献类型：期刊文献

中文题名：鸡PD-1胞外区基因的克隆表达及其蛋白生物学活性鉴定

英文题名：Cloning,Prokaryotic Expression and Biologic Activity Identification of Chicken PD-1 Extracellular Region Gene

作者：李博文[1,2];孙国鹏[1];王爱国[1,2];岳锋[1];张艳芳[1];朱艳平[1];银梅[2];杨媛[1,2];郭东光[1];王选年[1,2]

第一作者：李博文

机构：[1]新乡学院生命科学与技术系/生物技术研究中心;[2]河南科技学院动物科学学院

第一机构：新乡学院生命科学技术学院

年份：2014

卷号：43

期号：10

起止页码：112-116

中文期刊名：河南农业科学

外文期刊名：Journal of Henan Agricultural Sciences

收录：CSTPCD;;北大核心:【北大核心2011】；CSCD:【CSCD_E2013_2014】；

基金：国家自然科学基金项目(31272539)

语种：中文

中文关键词：鸡;程序性细胞死亡因子;1;胞外区;克隆;原核表达;生物学活性

外文关键词：chicken; PD-1;extracelluar region;clone;prokaryotic expression;biological activity

摘要：为研究鸡程序性细胞死亡因子1(PD-1)胞外区的生物学活性,根据GenBank中的鸡PD-1基因序列,经生物信息学比对分析,设计鸡PD-1胞外区特异性克隆引物,扩增目的基因,连接原核表达载体pET-28a(+),构建重组表达载体pET-28a-PD-1,转化至Rosetta(DE3)大肠杆菌,并对IPTG浓度、诱导温度、诱导时间进行优化,对表达产物进行SDS-PAGE和Western blot分析,应用流式细胞术对所获得重组蛋白的生物学活性进行鉴定。结果显示,成功克隆了鸡PD-1胞外区基因;SDS-PAGE和Western blot分析结果表明,37℃、0.1mmol/L IPTG诱导4h时,PD-1胞外区融合蛋白表达量最高,以包涵体形式存在;流式细胞术分析结果表明,PD-1胞外区重组蛋白具有一定的生物学活性。
In order to study the biological activity of chicken PD-1 extracelluar region,the extracellular region primers were designed and the chicken PD-1 extracelluar region gene was cloned according to PD-1 gene in GenBank by bioinformatics comparation.The purified fragment was ligated into pET-28a vector,ligated DNA was transformed in the E.coli strain Rosetta（DE3） after identification by sequencing.The identification of expression product by SDS-PAGE,western blot and flow cytometry after optimizing the various expression conditions such as concentration of IPTG and its action time and temperature.The result showed that the chicken PD-1 extracelluar region gene was successfully cloned.PD-1 presented as a form of inclusion bodies and its experssion was highest when it was induced by addition of IPTG to a final concentration of 0.1 mmol/L for 4 h at 37 ℃.Meanwhile the result of flow cytometry showed that the recombinant protein of PD-1 extracelluar region retained some biologic activity.