摘要
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.
作者
高洪波
袁超
周磊
韩一帆
陈佳琪
段志强
GAO Hongbo;YUAN Chao;ZHOU Lei;HAN Yifan;CHEN Jiaqi;DUAN Zhiqiang(Key Laboratory of Plateau Mountain Animal Genetics,Breeding and Reproduction,Ministry of Education,Guizhou University,Guiyang,Guizhou 550025;College of Animal Science,Guizhou University,Guiyang,Guizhou 550025)
出处
《中国家禽》
北大核心
2020年第8期7-11,共5页
China Poultry
基金
国家自然科学基金(31760732)
贵州省科技计划项目(黔科合平台人才[2017]5788号)。
关键词
鸡
膜联蛋白A2
原核表达
纯化
chicken
annexin A2
prokaryotic expression
purification