人组织型纤溶酶原激活剂cDNA糖基化位点突变体的构建

MUTAGENESIS OF t-PA cDNA CLONE AT GLYCOSYLATION SITES

构建人组织型纤溶酶原激活剂（ｔｉｓｕｅｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ，ｔ－ＰＡ）ｃＤＮＡ糖基化位点突变体，为ｔ－ＰＡｃＤＮＡ在Ｐｉｃｈｉａｐａｓｔｏｒｉｓ酵母表达系统中的表达提供条件。方法运用ＤＮＡ重组技术，构建ｐＡＨＲ、ｐＡＲＨ质粒，然后应用改良的寡核苷酸诱导定位突变技术，构建ｐＭＡＨＲ、ｐＭＡＲＨ质粒，并用限制性核酸内切酶鉴定、核酸序列分析证实。结果琼脂糖凝胶电泳显示了特异的片段，序列分析证实获得的ｐＭＡＨＲ为Ａｓｎ１１７和Ａｓｎ１８４位点突变的阳性克隆，ｐＭＡＲＨ为Ａｓｎ４４８位点突变的阳性克隆。结论成功地构建了ｔ－ＰＡｃＤＮＡ糖基化位点突变体。

PURPOSE To construct mutants of t-PA cDNA clone at glycosylation sites for expression in methylotrophic yeast Pichia pastoris . METHODS According to the method of oligonucleotidemediated site directed mutagenesis modified by Promega Corporation, positive clones have been selected with restriction enzyme fragment analysis at mutagenic sites and confirmed by nucleotide sequencing. RESULTS The specific fragments appeared on agarose gel of electrophoresis and mutagenic sites have been confirmed by sequence analysis. CONCLUSIONS The mutants of t-PA cDNA clone at glycosylation sites are successfully constructed.