摘要
目的 :克隆与表达arresten ;方法 :从正常的肝组织提取总RNA ,逆转录成cDNA ,根据软件RNASTRUCTURE3.5设计两对引物 ,巢式PCR扩增arresten基因 ,其中每个内引物 5’端加上特殊核苷酸 ,PCR产物用T4DNAPolymerase处理后直接与用NdeI ,EcoRI酶切的表达质粒pTYB1连接 ,转化大肠杆菌ER2 5 6 6。筛选重组子 ,诱导表达。结果 :快速一步法克隆表达了arresten。讨论 :一步法克隆快速 。
Objective:To clone and express the new angiogenesis inhibitor,arresten.Methods:The total RNA was extracted from the normal human liver,and then was reversely transcribed mRNA to cDNA and arresten cDNA was amplified by nestle-PCR.After being treated by T4 DNA polymerase,the arresten cDNA was linked with the linear vector pTYB1 digested by NdeI and EcoRI.And then the complete ER2566 was transfected by recombinant vector,the recombination was screened and the expression of the interest protein was induced by IPTG.Results:The arresten was cloned by one-step cloning method with PCR product treated by T4 DNA polymerase.Conclusion:This method can be used to clone the interest gene more easily and quickly than T-A clone and others.
出处
《重庆医科大学学报》
CAS
CSCD
2004年第3期337-339,共3页
Journal of Chongqing Medical University
