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用PCR方法将编码人微小纤溶酶原 (microplasminogen ,mPlg)活性中心Ser的密码子置换为Ala密码子 ,突变体cDNA与分泌型酵母表达载体 pHIL D8重组 ,构成受醇氧化酶基因 (AOX1)的启动子与转录终止区控制的酵母表达质粒 ,转化GS115酵母菌 ,经表型筛选、PCR扩增筛选阳性克隆 ,以甲醇诱导表达 ,Mm Plg分泌至培养液 ,经Westernblot证实。培养液经离心 ,上清与重组葡激酶 (Staphylokinase,Sak)孵育后 ,以酪蛋白 琼脂糖平板溶圈法、发色底物法检测 ,MmPlg未显示纤溶活性。此研究为制备Sak·mPlg复合物的晶体提供了条件
The codon encoding Ser at the active site of microplasminogen (mPlg) was replaced by the Ala codon by PCR, and the mutant cDNA was recombined with the secretory yeast expression vector pHIL D8 to construct a recombinant plasmid that is regulated by the alcohol oxidase gene (AOX1) The promoter and transcription termination region controlled yeast expression plasmids were transformed into GS115 yeast. The phenotypes were screened and the positive clones were screened by PCR amplification. The positive clones were induced by methanol. Mm Plg was secreted into the culture medium and confirmed by Western blot. After the culture medium was centrifuged, the supernatant was incubated with Staphylokinase (Sak) and detected by casein agarose plate assay and chromogenic substrate method. MmPlg showed no fibrinolytic activity. This study provided the conditions for the preparation of crystals of the Sak · mPlg complex