甲基对硫磷水解酶高活筛选及荧光纳米分子生物传感器的研究
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摘要
本研究从E.coli DH5a/pET5a-mph出发,PCR扩增出甲基对硫磷水解酶MPH结构基因,将其克隆进宿主大肠杆菌系统E.coli BL21(DE3)中表达,获得E.coli BL21 (DE3)/pET20b(+)-mph重组系统,经金属螯合一步亲和纯化后,即得纯度在90%以上的目的蛋白,从而发展了一种方便、快速、高效生产和纯化甲基对硫磷水解酶的方法。通过采用易错PCR的方法,在大肠杆菌系统中,对MPH进行了酶活力筛选研究。经一轮易错PCR,产生了一个库容量约为10800的易错PCR突变体库,经酶活测定后,筛选得到2株具有高活力的突变株A7(R41H)和E6(R49P／G117D),其对底物甲基对硫磷MP催化活性分别是野生型的约2倍和3倍。
本研究同时还以甲基对硫磷水解酶MPH为识别元件,对pH值敏感的E2GFP为换能元件构建了一个检测有机磷农药的荧光生物分子传感器E2GFP-MPH。为了进一步提高该生物传感器的检测灵敏度,将酵母淀粉样蛋白Sup351-61的基因与E2GFP-MPH融合,得到一种能自组装成蛋白纳米线的生物传感器Sup351-61-E2GFP-MPH。实验表明,此纳米线生物传感器的检测灵敏度在荧光生物分子传感器E2GFP-MPH的基础上提高了约10,000倍,对甲基对硫磷的检测下限为1pmol/mL。
A mature methyl parathion hydrolase gene from E.coli DH5a/pET5a-mph, an existing strain for methyl parathion hydrolase expression, was cloned into the plasmid pET20b (+) and transformed into E.coli BL21 (DE3).Thus an overexpression strain was obtained. The recombinantlipase was purified in a one-step procedure of His-tag affinity chromatography. Through one cycle of error-prone PCR coupled with a sensitive screening method, two mutants of MPH were obtained. The catalytic activity of one mutant, which named A7, was 2-fold higher than that of wild MPH, while the other, which named E6, with 3-fold higher catalytic activity than that of wild MPH.
In this study, combining molecular recognition, MPH with a fluorophore, which acts as a signal transducer, enables a fluorescent molecular biosensor to be constructed based on a pH-sensitive fluorescence probe, E GFP. For a highly sensitive assay, E2GFP-MPH was further attached to a yeast prion protein Sup351-61 through gene fusion. The resultant nanowire fluorescent molecular biosensor could detect as low as 1 pmol/mL of methyl parathion (MP), which is about a 10,000-fold greater sensitivity than that of the free E2GFP-MPH molecular sensor.
本研究同时还以甲基对硫磷水解酶MPH为识别元件,对pH值敏感的E2GFP为换能元件构建了一个检测有机磷农药的荧光生物分子传感器E2GFP-MPH。为了进一步提高该生物传感器的检测灵敏度,将酵母淀粉样蛋白Sup351-61的基因与E2GFP-MPH融合,得到一种能自组装成蛋白纳米线的生物传感器Sup351-61-E2GFP-MPH。实验表明,此纳米线生物传感器的检测灵敏度在荧光生物分子传感器E2GFP-MPH的基础上提高了约10,000倍,对甲基对硫磷的检测下限为1pmol/mL。
A mature methyl parathion hydrolase gene from E.coli DH5a/pET5a-mph, an existing strain for methyl parathion hydrolase expression, was cloned into the plasmid pET20b (+) and transformed into E.coli BL21 (DE3).Thus an overexpression strain was obtained. The recombinantlipase was purified in a one-step procedure of His-tag affinity chromatography. Through one cycle of error-prone PCR coupled with a sensitive screening method, two mutants of MPH were obtained. The catalytic activity of one mutant, which named A7, was 2-fold higher than that of wild MPH, while the other, which named E6, with 3-fold higher catalytic activity than that of wild MPH.
In this study, combining molecular recognition, MPH with a fluorophore, which acts as a signal transducer, enables a fluorescent molecular biosensor to be constructed based on a pH-sensitive fluorescence probe, E GFP. For a highly sensitive assay, E2GFP-MPH was further attached to a yeast prion protein Sup351-61 through gene fusion. The resultant nanowire fluorescent molecular biosensor could detect as low as 1 pmol/mL of methyl parathion (MP), which is about a 10,000-fold greater sensitivity than that of the free E2GFP-MPH molecular sensor.
引文
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