幽门螺杆菌尿素通道蛋白基因(ureI)的克隆及其表达和纯化

目的　对幽门螺杆菌尿素通道蛋白基因ureI进行克隆、测序 ,并在昆虫细胞中表达及进行产物纯化。方法　克隆ureI基因 ,经测序正确后 ,酶切、连接到pFASTBACHb质粒上 ,与穿梭载体DH10BAC转座 ,获得Bacmid ureI质粒 ,转染Sf9细胞 ,采用Ni2 +螯合琼脂糖亲和层析纯化 ,经SDS PAGE和Westernblot鉴定。结果　克隆了ureI基因 ,并在昆虫细胞Sf9中表达纯化 ,蛋白纯度达 85 %以上 ,并与 6 His单抗特异结合。结论　表达及纯化的ureI蛋白为进一步研究打下了基础

Cloning and Expression of Urea Channel Protein Gene (ureI) of Helicobacter pylori and Purification of Expressed Product

Objective To clone and sequence the urea channel protein gene (ureI) of Helicobacter pylori ,express the gene in insect cells and purify the expressed proteins.Methods Isolate and sequence ureI gene of Helicobacter pylori .Sub clone the gene into plasmid pFASTBACHb and then transpose it into a shuttle vector DH10BAC.Transfect Sf9 cells with the recombinant plasmid Bacmid ureI.Purify the expressed proteins by Ni 2+ chelate sepharose affinity chromatography and identify by SDS PAGE and Western blot.Results The homologies of nucleotide and amino acid of the cloned ureI gene to those of international standard strain NCTC 11 637 were 97% and 98.5% respectively.SDS PAGE showed that (ureI) fusion protein,with a relative molecular weight of 26 000,were expressed.After being purified by Ni 2+ chelate sepharose affinity chromatography,the purity of expressed fusion protein reached above 85%.Western blot showed the specific binding of the purified fusion protein to McAb against 6 His.Conclusion ureI gene was cloned and expressed in insect cell Sf9.It laid a foundation of further study on application of ureI fusion protein.