摘要
克隆人心肌肌钙蛋白I (cTnI)基因全序列 ,并进行分泌表达。从人心肌组织提取总RNA ,经逆转录得到cDNA第一链 ,以此为模板 ,PCR扩增目的条带 ,重新设计上游引物 ,用相同PCR程序完成点突变 ;突变后PCR产物克隆入Pfd5载体 ,并用PCR、SpeI+XhoI双酶切、载体测序等方法鉴定 ;用ITPG诱导cTnI的表达。PCR扩增出 63 0bp左右条带 ,Pfd5 cTnI融合表达载体用PCR、SpeI +XhoI双酶切均可见 63 0bp左右条带 ,测序结果与预期序列一致 ;诱导后培养基及细胞周质均检测到cTnI蛋白免疫原活性。
Partial cDNA sequence of human cardiac tropon in gene w as cloned and secretivly expressed. The total RNA was abstracted from human card iac tissues and converted to the first chain cDNA. The cTnI cDNA band was obtain ed by the methods of PCR, the second PCR was performed by designing another upst ream primer with the templates of the first PCR product. PCR product after muta tion were cloned into pfd5 vector. The fused vector was identified by the method s of PCR, restrictive enzyme analysis with SpeI+XhoI, and sequencing. In the mea nwhile cTnI wa s expressed by ITPG induced. Partial cDNA sequence of cTnI gene was obtained and the dot mutation was also accomplished, 630bp band was showed by PCR and restri ctive enzyme analysis of the Pfd5-cTnI fused-expressed vector; Sequencing resu lt was accordinative with the the expected sequence. The activity of cTnI was dete rmined in the medium and cell periplasm after induced. cTnI was successfully exp ressed.
出处
《药物生物技术》
CAS
CSCD
2003年第2期65-67,共3页
Pharmaceutical Biotechnology
