TEM-1型β-内酰胺酶编码基因的克隆表达

Cloning and expression of TEM-1 gene

目的:构建TEM-l型β-内酰胺酶基因的表达载体。方法:通过PCR扩增TEM-l全编码基因,将其连接入pBK-CMV载体中,并在大肠杆菌JM109中表达。采用琼脂二倍稀释法对TEM-l克隆菌株进行MIC检测,双纸片法筛选及确证实验检测其表型,等电聚焦(IEF)电泳测定其等电点(pIs)。结果:经酶切测序鉴定TEM-l基因的表达载体构建正确。重组菌产pI为5.4的TEM-l广谱酶,仅对青霉素类耐药。结论:TEM-l基因原核表达载体正确构建,为进一步研究打下了基础。

Objective： To clone TEM-1 gene with full length open reading frame for a recombinant prokaryotic expression vector. Methods： TEM-1 gene was amplified by Polymerase chain reaction（PCR）, The target amplification fragment was cloned into pBK-CMV vector. The recombinant vector was sequenced by Sanger＇ s dideoxy chain termination composition method. Antimicrobial susceptibility of the clone strain was detected by the disc diffusion method and by the agar dilution method. Crude β-1actamases were extracted and then isoelectric point of TEM-1 was determinded by IEF. Results： Sequence and restriction anallysis revealed TEM-1 gene was cloned in frame into pBK-CMV. The clone strain producing TEM-1 with pl value of 5.4 was resistant to ampicillin and piperacillin, only. Conclusions： TEM-1 gene has been cloned and expressed, to prepare for the site-directed mutagenesis study.