鸡膜联蛋白A2的原核表达与纯化

Prokaryotic Expression and Purification of Chicken Annexin A2

试验旨在对鸡膜联蛋白A2(ANXA2)基因进行原核表达与纯化。利用鸡ANXA2基因特异性引物从鸡卵泡细胞cDNA中扩增ANXA2基因,亚克隆至原核表达载体p ET-32a(+)构建重组原核表达载体pET-32a-chANXA2。将其转化到大肠杆菌BL21(DE3)进行诱导表达,并分析重组蛋白的表达形式;利用含Ni2+琼脂球对重组蛋白进行纯化,并利用Western blot进行鉴定。结果显示:成功构建重组原核表达载体pET-32a-chANXA2,重组蛋白His-chANXA2在0.1 mmol/L IPTG、诱导表达5 h和30℃诱导温度条件下表达量高,其在上清和包涵体中均有表达,但主要以包涵体形式存在。利用含Ni2+琼脂球从诱导菌裂解物上清中得到了纯度较高的重组蛋白,Western blot检测到与预期大小相符的重组蛋白条带。研究结果为后续开展ANXA2调控鸡卵泡发育的分子机制研究奠定基础。

Aimed to perform the prokaryotic expression and purification of chicken annexin A2 gene.The chANXA2 gene was amplified from the cDNA of chicken follicular cells and then was subcloned into pET-32 a(+)to construct pET-32 a-chANXA2.The expression of recombinant protein His-chANXA2 was induced in E.coli BL21(DE3)and its expression forms were analyzed.In addition,the purification and identification of His-chANXA2 were carried out.The results showed that the plasmid pET-32 a-chANXA2 was successfully constructed.The higher expression levels of His-chANXA2 were obtained under the condition of 0.1 mmoL/L IPTG,5 h and 30℃.Although His-chANXA2 existed in the supernatant and inclusion body,there was much higher expression in inclusion body.In addition,the His-chANXA2 was purified from the supernatant of induced bacteria lysates using Ni2+Agarose Beads.Meanwhile,the desired size of His-chANXA2 was detected by Western blot analysis.These results provided theoretical foundation for further studying the role of ANXA2 in the development of chicken follicular cells.