人蛋白C cDNA突变体的设计、克隆与序列分析  

Design, Cloning and Sequencing of cDNA Mutant of Human Protein C

作　　者：贾莉玮[1] 吕茂民[1] 徐斯日古愣 沈永才[1] 赵保全[1] 章金刚[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《中国生物制品学杂志》2003年第5期257-260,共4页Chinese Journal of Biologicals

基　　金：军事医学科学院创新基金

摘　　要：目的 通过对人蛋白C cDNA的定点突变,构建人蛋白C突变体cDNA,以期实现从哺乳动物细胞中直接表达出具有生物活性的人蛋白C产物。方法 针对人蛋白C cDNA序列设计引物以引入突变序列,采用逆转录聚合酶链反应(RT-PCR)和重组PCR方法,从人胎肝总RNA中分别钓取人蛋白C的重链和轻链,经重组PCR反应将两者连接,然后将其克隆入pGEM-T载体中,经酶切和PCR鉴定后进行测序。结果 经RT-PCR和重组PCR扩增获得大小为1374 hp的cDNA片段,并成功构建了人蛋白C cDNA突变体的克隆质粒pGEM-T/mhPC,序列分析证实所引入的编码8个氨基酸短肽的核苷酸序列突变位点正确取代了人蛋白C的活性肽,获得人蛋白C突变体cD-NA的克隆。结论 已成功进行了人蛋白C cDNA的突变,获得了人蛋白c cDNA突变体的克隆,为进一步进行人蛋白C突变体cDNA的表达和活性鉴定奠定了基础。Objective To construct human protein C cDNA mutant by point mutation and directly express the human protein C with biological activity in mammal cells. Methods Design primers according to the cDNA sequence of human protein C, introduce point mutation and extract the heavy and light chains of human protein C from the total RNA of human fetal liver by RT-PCR and recombinant PCR respectively. Link the 2 chains by recombinant PCR and clone into pGEM-T vector.The recombinant plasmid was digested with restrict -tion endonuclease, identified by PCR and sequenced. Results A cDNA fragment with a length of 1374 bp was amplified by RT-PCR and recombinant PCR, and a recombinant plasmid pGEM-T/mbPC for cloning human protein C cDNA mutant was successfully constructed. The result of sequencing proved that the active peptide of human protein C was substituted with the introduced, nucleotide sequence encoding 8 amino acids. Conclusion A clone of human protein C cDNA mutant was obtained by point mutation. It laid a foundation of further study on expression and activity of human protein C cDNA mutant.

关 键 词：人蛋白C CDNA 突变体 设计 克隆 序列分析 定点突变 

分 类 号：Q754[生物学—分子生物学] Q51