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.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
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
