蓝氏贾第鞭毛虫α-4贾第素的原核表达及纯化  

Prokaryotic expression and purification of α-4 giardin of Giardia lamblia

作　　者：王洋[1] 余源[1] 王卫亮[1] 陈阳[1] 慈雅丽[1] 田喜凤[1] 郑佳[2] 杨志宏[2] 

机构地区：[1]河北联合大学生命科学学院,唐山063000 [2]唐山市职业技术学校,唐山063000

出　　处：《中国人兽共患病学报》2011年第6期465-469,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.30970313);河北省科技厅项目(No.07276101D-68)联合资助

摘　　要：目的原核表达并纯化蓝氏贾第鞭毛虫的α-4贾第素蛋白。方法经RT-PCR获得α-4贾第素基因片段,经双酶切连入原核表达载体pET-28a(+),构建成为重组表达载体pET-28a(+)-α-4,并转化大肠杆菌Rosetta(DE3)。IPTG诱导后,收集菌体,裂解后进行SDS-PAGE及Western blot检测,并用Ni-NTA亲和层析柱纯化融合蛋白。结果成功构建了原核表达载体pET-28a(+)-α-4,经IPTG诱导后,在大肠杆菌中高效表达,目的蛋白以包涵体形式存在;SDS-PAGE及Western blot分析显示,在相对分子量约34 kD的位置出现目的蛋白条带,与理论值相符;经Ni-NTA亲和层析柱纯化获得了高纯度的重组的α-4贾第素融合蛋白。结论成功克隆、表达并纯化了α-4贾第素蛋白,为α-4贾第素的细胞定位及功能研究提供了必要条件。In order to express α-4 giardin protein in E. coli, the full-length eDNA encoding α-4 giardin was amplified by RT-PCR. The PCR product about 900 bp long was cloned into prokaryotie expression vector pET-28a（＋） with restriction enzymes Nco Ⅰ and Xho Ⅰ. The recombinant vector pET-28a（＋ ）-α-4 was transformed into E. coli Rosetta（DE3）, then the α-4 giardin fusion protein was expressed by IPTG induction and the expression conditions were optimized. SDS-PAGE and western blot showed that the expressed product was a fusion protein about 34 kD. Highly purified recombinant α-4 giardin was obtained hy Ni-affinity chromatography. The present study might provide the foundation for the further study of α-4 giardin.

关 键 词：蓝氏贾第鞭毛虫 α-4贾第素 原核表达 

分 类 号：R383.1[医药卫生—医学寄生虫学]