【摘要】 目的:构建pET-PEP重组原核表达载体,并对表达产物进行鉴定。方法:采用RT-PCR技术从黑曲霉(Aspergillus niger)W3350中扩增出脯氨酰内肽酶(PEP)的基因,插入表达载体pET-22b(+),经EcoRⅠ和HindⅢ双酶切和DNA序列测定验证,将正确的重组质粒转入大肠杆茵BL21(DE3)中,IPTG(终浓度1mmol/L)诱导获得表达。结果:表达产物进行超声破碎,经SDS-PAGE电泳检测,PEP分子质量大约为57kDa,与理论值相符,通过酶活方法测定脯氨酰内肽酶酶活为0.656U/ml,约是出发菌株的5倍。结论:首次成功构建表达载体pET-PEP并在原核细胞中表达,为进一步研究PEP的生物学功能奠定了基础。更多还原

【Abstract】 Objective:To construct the prokaryotic expression vector of the pET-PEP,and identify the expressed production.Method:The PEP gene were amplified from Aspergillus niger W3350 cells by RT-PCR,and inserted into the prokaryotic expression vector pET-22b(+) and verified by EcoRⅠ and HindⅢ digestion and DNA sequencing.The recombinant plasmid was expressed in E.coli BL21(DE3) by inducing of IPTG(1 mmol/L) and identified by SDS-PAGE.Result:After sonication,SDS-PAGE analysis indicated that the relative molecular mass(M)of the protein was about 57kDa,and it was equivalent to the expected value.the activity of PEP was 0.656U/ml by determination,and it is about as five times as the original strain.Conclusion:To construct the recombinant expression vector and to express the protein in E.coli successfully and to further the study of the biological function of PEP.更多还原