恒河猴tPA基因的克隆、测序与真核表达

Cloning of Rhesus Monkey tPA cDNA and Its Expression in CHO

目的 对恒河猴tPA编码区cDNA进行测序和表达。方法 采用RT PCR方法从恒河猴淋巴细胞中扩增tPA基因 ,将获得的cDNA克隆于T载体 ,序列确定后再克隆至真核表达载体。结果 测序结果表明恒河猴tPAcDNA编码区与人tPAcDNA编码区的核苷酸序列同源性为 96 % ,由此所推导的氨基酸序列的同源性为 97.5 %。随后将恒河猴tPAcDNA克隆于真核表达载体 ,转染CHO细胞后成功表达出了有活性的tPA。培养上清检测结果显示其活性约为 5 0U ml,略低于人tPA在CHO细胞中表达产物的活性。结论 本研究首次报道了恒河猴tPA基因编码区的全长cDNA序列并获得了有活性的恒河猴tPA真核表达产物。

Objective To sequence and express tissue type plasminogen activator (tPA) cDNA of rhesus monkey. Method\ The cDNA was obtained by RT PCR from the lymphocyte of rhesus monkey, of which the primers were designed according to human tPA cDNA sequence. The amplified fragment was then cloned into T vector. Results\ The result of nucleotide sequencing showed the coding region of rhesus monkey tPA to be 96% nucleotides and 97.5% amino acids identity with human tPA. In the subsequent experiment, the rhesus monkey tPA cDNA was cloned into eukaryotic expression vector, and tPA activity of 50 U/ml can be detected in the medium at the second day (48hours) after transfection of CHO cells. Conclusion\ The coding region cDNA sequence of rhesus monkey and its expression in mammalian cells are reported. It could be useful for the further study of tPA biological characters.