CUEDC2突变体的构建及在原核和昆虫表达系统中的表达

目的：构建CUEDC2的突变体并在原核和昆虫表达系统中表达验证。方法：根据人cuedc2序列设计引物，以其突变体质粒为模板PCR扩增目的片段，酶切后连接至pET28a原核表达载体以及pFastBac1-Flag-N和pFastBac-HTA昆虫表达系统的表达载体，转化至DH5α后提取质粒，经菌液PCR、测序鉴定后，转入大肠杆菌BL21(DE3)及DH10Bac感受态细胞中。原核表达载体在E.coli中表达后用Ni-NTA亲和纯化，SDS-PAGE检测。昆虫表达载体提取杆粒，转染至昆虫细胞Sf9中表达，最后用western 进行鉴定。结果：成功构建了CUEDC2的突变体，并在原核和昆虫表达系统中表达。结论：在原核和昆虫表达系统中，成功表达的CUEDC2突变体蛋白为CUEDC2的结构和功能研究奠定了基础。

Construction and expression of CUEDC2 mutants in BL21 (DE3) and Sf9 cells

To generate a serious of mutated CUEDC2 expression clones,and to testify the expression of them in BL21(DE3) and Sf9 cells,the primers were designed according to the CDS of CUEDC2.A serious of CUEDC2 mutants were cloned to expression vectors such as pFastBac1-Flag-N,pFastBac-HTA and pET28a and transformed into the host cells E.coli BL21(DE3) and DH10Bac respectively.The expressed CUEDC2 mutant proteins in BL21(DE3) were purified by the His-tag affinity chromatography and tested by SDS-PAGE.After isolation o...