人源性羧肽酶 A突变体基因的构建及表达

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(hCPA)。以计算机模拟hCPA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hCPA的核苷酸序列为1251bp，编码417个氨基酸。hCPA突变体(mhCPA)基因的1126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mhCPA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mhCPA基因，为hCPA在肿瘤的“抗体导向酶前药疗法”(ADEPT)中的应用奠定了基础

Construction andexpression of a human carboxypeptidase A mutant gene

Aim To extract human carboxypeptidase A(hCPA)gene from pulmonary tissue and make point mutation to develop a new enzyme for antibody directed enzyme prodrug therapy(ADEPT). Methods The hCPA gene was amplified from human lung tissue by RT PCR. Based upon the computer model, we designed a mutant gene of hCPA and made point mutation by over lap extension PCR. The mutant gene was expressed in E.coli. Results sequencing indicated that the product of RT PCR consisted of 1251 bp encoding 417 amino acid residues. The mutant gene changed from G to A at 1 126 nucleotide, and the amino acid sequence was changed from alanine to threonine at relevant position. After induction for 3 hours, a new anticipatory protein band appeared on SDS PAGE gel and the expression amount of hCPA gene reached about 30% of total bacterial protein. Conclusion hCPA gene is constructed and expressed successfully in E.coli JM109. It might providea foundation for application of hCPA in ADEPT.