摘要
目的 :克隆人羧肽酶A1 (carboxypeptidaseA1 ,CPA1 )活性中心基因 ,构建重组表达载体并对其进行诱导表达 .方法 :通过RT PCR方法扩增出正常胰腺组织中CPA1活性中心基因 ,克隆入载体 pGEM Teasy ,测序分析 .将该目的基因亚克隆于表达载体 pGEX 4T 1 ,测序证实序列正确后转化大肠杆菌DH5α ,经IPTG诱导表达重组蛋白 .结果 :获得了人CPA1活性中心基因 ,构建了重组表达质粒 ,表达的蛋白以SDS PAGE分析 ,在Mr 约 4 6 0 0 0处出现一条新生蛋白带 ,经凝胶薄层扫描检测 ,表达量约占菌体总蛋白的 2 2 .1 % .结论 :成功克隆人CPA1活性中心基因并得到了其原核表达产物 。
AIM To clone human carboxypeptidase A1 active center gene, to construct the recombinant vector and to express its products. METHODS: Human carboxypeptidase A1 active center gene was amplified by RT PCR from pancreas tissues and cloned into vector pGEM T easy. After sequencing, it was subcloned into prokaryotic expressive vector pGEX 4T 1 and transformed into E. coli DH5α. The recombinant protein expression was induced by IPTG. RESULTS: Human carboxypeptidase A1 active center gene was coloned and the expressed recombinant plasmid pGEX CPA1 was constructed. After induction, a new anticipatedprotein band of M r 46 000 appeared on SDS PAGE and amounted to about 22.1% of the total bacterial protein. CONCLUSION: The human carboxypeptidase A1 active center gene has been cloned and prokaryotic expression products obtained, which lays the foundation for obtaining active small molecular protein.
出处
《第四军医大学学报》
北大核心
2003年第23期2132-2135,共4页
Journal of the Fourth Military Medical University
基金
国家"十五"重大科技专项课题 (2 0 0 2AA2Z31 0 9)
国家"863"资助项目 (2 0 0 1AA2 1 532 )
国家教育部高等学校青年骨干教师资助计划
关键词
羧肽酶A1
活性中心
基因克隆
原核表达
carboxypeptidase A1
active center
gene cloning
prokaryotic expression
