鸡 PD 1胞外区基因的克隆表达及其蛋白生物学活性鉴定

摘要

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.%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℃、0．1 mmol/L IPTG 诱导4 h 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。