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乙酰胆碱酯酶(Ach E)是有机磷(OP)和氨基甲酸酯类农药的作用靶蛋白,在体外胆碱激酶(choline kinase)可专一性地以三磷酸腺苷(ATP)作为磷酸的供体,催化Ach E的酶促反应产物氯化胆碱发生磷酸化。依据家蚕基因组数据库中的胆碱激酶基因(Bmcok)序列(Gen Bank登录号:AK378994.1)设计合成特异引物,从家蚕5龄第3天幼虫头部克隆了家蚕胆碱激酶基因,在大肠杆菌中进行原核表达并纯化目的蛋白质,获得质量浓度约1.03 mg/m L的家蚕胆碱激酶液。纯化的家蚕胆碱激酶在30℃、p H 9.5时的活性最高,约为115.6 U/mg。通过耦合Ach E和家蚕胆碱激酶的酶催化反应,并结合ATP-荧光素发光反应,建立酶抑制-发光快速检测有机磷和氨基甲酸酯类农药残留的方法。该方法对甲胺磷残留的检测限可达0.05μg/m L,灵敏度高于现有的酶抑制色卡法和酶抑制分光光度计法,初步表明了将家蚕胆碱激酶应用于有机磷和氨基甲酸酯类农药残留快速检测的可行性。
Acetylcholinesterase (AchE) is the target protein of organophosphorus (OP) and carbamate pesticides. In vitro, choline kinase can specifically use adenosine triphosphate (ATP) as donor of phosphoric acid and catalyze Choline chloride, an enzymatic reaction product of Ach E, is phosphorylated. Based on the Bmcok sequence (GenBank accession number: AK378994.1) of Bombyx mori genome database, specific primers were designed and synthesized. The silkworm choline kinase gene was cloned from the 3rd larvae of 5th instar larvae of silkworm (Bombyx mori) In the prokaryotic expression and purification of the target protein to obtain a mass concentration of about 1.03 mg / m L of silkworm choline kinase solution. The purified silkworm choline kinase has the highest activity at p H 9.5 at 30 ° C of about 115.6 U / mg. A method for the rapid determination of organophosphorus and carbamate pesticide residues by enzymatic inhibition-luminescence was established by the enzyme-catalyzed reaction of coupling Ach E and silkworm choline kinase together with ATP-fluorescein luminescence reaction. The limit of detection of methamidophos in this method is up to 0.05μg / m L, and the sensitivity is higher than that of the existing enzyme inhibition colorimetric assay and enzyme inhibition spectrophotometry. It has been initially demonstrated that the application of choline chloride to silkworm, Feasibility of Rapid Detection of Carbamate Pesticide Residues.