论文部分内容阅读
目的 从人正常肺组织中提取羧肽酶 A基因并进行定点突变 .方法 从人正常肺组织中提取 RNA,并进行逆转录得到人羧肽酶 A(h CPA) .以计算机模拟 h CPA的结构 ,寻找合适的突变位点 .用 PCR重叠延伸法 ,对羧肽酶 A进行定点突变 ,并在大肠杆菌中表达 .结果 序列分析表明 ,所获 h CPA的核苷酸序列为 12 5 1bp,编码 417个氨基酸 . h CPA突变体 (mh CPA)基因的112 6位核苷酸由 G变为 A,其余核苷酸序列均未发生变化 ,相应的此突变体蛋白质的 376位核苷酸残基由丙氨酸突变为苏氨酸 .将 mh CPA基因在 E.coli中诱导表达 3h后 ,表达量高达 30 %左右 .结论 成功地构建并表达了 m h CPA基因 ,为 h CPA在肿瘤的“抗体导向酶 -前药疗法” (ADEPT)中的应用奠定了基础 .
Objective To extract carboxypeptidase A gene from human normal lung tissue and perform site-directed mutagenesis.Methods RNA was extracted from human normal lung tissue and reverse transcribed to obtain human carboxypeptidase A (h CPA) , And search for suitable mutation sites.Microspeptidase A was subjected to site-directed mutagenesis by PCR overlap extension method and expressed in E.coli.Results Sequence analysis showed that the nucleotide sequence of hCPA obtained was 125 bp, encoding 417 amino acids h The 1126 nucleotide of the CPA mutant (mh CPA) gene changed from G to A, with no change in the remaining nucleotide sequences, corresponding to the 376 nucleotide residue of this mutant protein From alanine to threonine.The mh CPA gene was expressed in E.coli for 3h and the expression was up to about 30% .Conclusion The mh CPA gene was successfully constructed and expressed, Guide enzyme - prodrug therapy "(ADEPT) laid the foundation for the application.