有机磷农药降解酶的基因克隆与重组表达

Molecular cloning and recombinant expression of organophosphate hydrolase gene

目的:旨在从活性污泥中直接克隆有机磷农药降解酶基因,使其在大肠杆菌中大量表达。方法:利用土壤DNA提取技术从山东某农药厂活性污泥中提取DNA,根据有机磷农药降解酶基因设计引物,用巢式PCR从活性污泥DNA中扩增特异性片段,将该基因片段连接至表达载体pET-30 a(+)中,并转化感受态细胞BL21(DE3),经IPTG诱导表达重组蛋白,并对诱导剂浓度和诱导时间进行优化。结果:利用巢式PCR从活性污泥DNA中扩增出长度为1003 bp的特异性片段,将该片段测序并进行序列比对,结果显示该片段与甲基对硫磷降解酶基因的同源性为99%,且含有完整开放阅读框,长度为993 bp;IPTG诱导后获得分子量为41.7 kD的重组表达蛋白;经优化,IPTG的最佳诱导浓度为0.1 mmol/L,最佳诱导时间为5 h。结论:成功克隆出有机磷农药降解基因并获得高效重组表达蛋白。

Objective： To clone the organophosphate hydrolase gene （OPH） from activated sludge and express in E. coll. Methods：DNA was purified by soil DNA isolation kit from of a pesticide factory in Shandong Province. A DNA fragment was amplified by nested PCR with the primers designed according to organophosphate hydrolase genes. The open reading frame was subcloned into pET - 30a（ ＋ ） plasmid and transferred into E. coli BL21 （ DE3 ）. After being induced by IPTG, a recombinant protein protein was largely expressed in the cells, and recombinant expressing conditions （ such as concentration of IPTG and inducing period） was studied and optimized. Results：A 1003 bp DNA fragment was amplified by nested PCR and sequencing result indicated that the fragment contained a 993 bp full open reading frame. Blast analysis showed that the homology between the fragment and methyl parathion hydrolase gene was high to 99%. After being induced by IPTG, a protein with molecular weight of 41.7 kD was largely expressed in the cells. The result of optimization of recombinant expressing conditions showed that the optimal concentration of IPTG was 0. 1 mmol,/L and the optimal inducing period was 5 hours. Conclusion： OPH has been successfully cloned and largely expressed in E. coli.