重组人α-半乳糖苷酶A在巴氏毕赤酵母中的表达及鉴定

Expression and identification of recombinant human alpha-galactosidase A in Pichia pastoris

目的:克隆人α-半乳糖苷酶A(α-GalA)cDNA,并在毕赤酵母中表达重组的人α-GalA。方法:利用RT-PCR方法从人肝癌细胞中克隆人α-半乳糖苷酶A cDNA并构建到可用甲醇诱导的分泌型巴氏毕赤酵母表达载体pPICZαA中。将重组质粒导入毕赤酵母并用Ze ion抗生素筛选转化细胞。在甲醇诱导后,利用SDS-PAGE、W estern印迹及酶活测定方法在酵母上清中鉴定重组的人α-GalA。结果:获得人-αGalA cDNA,构建酵母表达质粒pHGCZɑA,将重组表达载体导入到酵母细胞,并在上清中检测到人α-GalA蛋白。结论:获得了人α-GalA cDNA及具有生物活性的重组人α-GalA,为临床应用于法布莱氏病的治疗奠定了基础。。

Objective： To clone human alpha-galactosidase A（α-GalA ）cDNA and express recombinant human α-Gal A protein in P. pastoris. Methods：Human α-Gal A cDNA was cloned from human liver cancer cell by RT-PCR and subcloned into secretion type P. pastoris expression vector pPICZαA with methanol-inducible promoter. The recombinant plasmid was transformed into P. pastoris and the transformants were selected by Zeion. After P. pastoris was.induced by methanol, the supematant was collected and identified by SDS-PAGE , Western blot as well as enzymatic analysis. Results：The human α-GalA cDNA was obtained and subcloned into pPICZαA. The recombinant vector pHGCZoA was transformed into P. pastoris and recombinant human α-GalA protein was detected in supematant of transformant by Western blotting and enzymatic analysis. Conclusion：The human α-GalA cDNA cloned and recombinant human α-GalA with biological activity were obtained. The recombinant human α-GalA can be used for Fabry＇s disease treatment in the future.