人β肌动蛋白基因原核表达载体的构建及其表达和纯化  

Prokaryotic Expression Vector Construction, Expression and Purification of Human β-Actin Protein

作　　者：宋烨琼 郭靖[2] 徐小洁[2] 李玲[2] 梁迎春[2] 洪甜[2] 纪贝贝 叶棋浓[2] 吕朝晖[1] 

机构地区：[1]解放军总医院内分泌科,北京100853 [2]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2015年第6期783-785,共3页Letters in Biotechnology

基　　金：国家自然科学基金(81372161;81472589;31100604);北京市科技新星计划(Z141102001814055);北京市自然科学基金(7132155);军事医学科学院创新基金转化医学项目(ZHYX003)

摘　　要：目的:构建带GST标签的人β肌动蛋白(β-actin)基因的原核表达产物,纯化出GST-β-actin融合蛋白,为探究β-actin的各项生理功能做准备。方法:以人乳腺文库为模板,利用PCR技术扩增β-actin基因,将其连接到带有GST标签的载体上,经鉴定正确的重组质粒转化大肠杆菌Rossate感受态细胞,小量诱导表达后,利用GST-Sepha-rose 4B亲和珠纯化GST-β-actin融合蛋白,经SDS-PAGE和Western印迹检测。结果:目的基因经PCR技术得以扩增,将其与带GST标签的载体连接后再经双酶切鉴定及测序后确认构建成功;转化大肠杆菌Rossate感受态后获得小量诱导表达,纯化出GST-β-actin融合蛋白,并证实其有生物活性。结论:构建了人β-actin的原核表达载体,并获得了GST-β-actin融合蛋白。Objective：To construct the prokaryotic expression vector of human β-actin gene and to express and purify GST-β-actin fusion protein.Methods：Human β-actin coding region was amplified from human mammary gland cDNA by PCR,and was inserted into the prokaryotic expression vector of pGEX-KG.The recombinant plasmid GST-β-actin was transformed into E.coli Rossate.The expressed product was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis.Results：The DNA fragment of about 1100 bp was successfully amplified by PCR,and inserted into vector of pGEX- KG correctly.The results of double digestion and sequencing indicated that the GST-β-actin recombinant plasmid was successfully obtained.The GST-β-actin fusion protein of about M,40 000 was successfully induced,and identified by SDS-PAGE and Western blot analysis.Conclusion：The prokaryotic expression vector of GST- β-actin was constructed successfully,which lay the foundation for further research.

关 键 词：人β肌动蛋白 原核表达 纯化 

分 类 号：Q78[生物学—分子生物学]