捻转血矛线虫Hc-daf-22基因的原核表达及重组蛋白酶活性测定  

Characteristics of Hc-daf-22 Gene from Haemonchus contortus:Crokaryotic Expression and Its Enzymatic Activity

作　　者：郑秀平[1] 丁豪杰[1] 郭筱璐 杨怡[1] 黄艳[1] 陈学秋[1] 周前进[2] 杜爱芳[1] 

机构地区：[1]浙江大学动物科学学院/浙江省动物预防医学重点实验室,杭州310058 [2]宁波大学生物技术系,浙江宁波315211

出　　处：《中国农业科学》2017年第8期1535-1542,共8页Scientia Agricultura Sinica

基　　金：国家重点基础研究发展计划("973"计划)(2015CB150300);浙江省自然科学基金(LY14C180002)

摘　　要：【目的】捻转血矛线虫(Haemonchus contortus)是反刍动物主要的胃肠道线虫之一,该病在中国呈全国性流行。为研究捻转血矛线虫(Haemonchus contortus)脂肪酸代谢相关蛋白DAF-22的生化特性,对其基因进行了克隆、原核表达,并对重组蛋白进行了体外酶活性测定。以期了解捻转血矛线虫Hc-DAF-22蛋白在过氧化物酶体脂肪酸β氧化中的作用。【方法】根据NCBI公布的H.contortus ZJ株daf-22 cDNA序列(Gen Bank:HQ738470.1)设计特异性引物,克隆Hc-daf-22基因并构建重组质粒pET-22b-Hc-daf-22,经测序鉴定正确后将其转化E.coli BL21,经终浓度为0.1 m mol·L^(-1) IPTG(isopropyl-β-d-thiogalactoside)诱导表达4h,离心菌液,50 mmol·L^(-1)浓度的PBS溶液重悬菌体溶液,冰浴超声破碎后上清沉淀分别进行SDS-PAGE分析。超声破碎后产物以镍柱亲和色谱法分离纯化重组蛋白Hc-DAF-22,并用SDS-PAGE检测蛋白纯化情况及以抗His血清作为一抗Western Blot鉴定。纯化后蛋白用超滤管浓缩除去盐分,并按照蛋白浓度测定试剂盒进行蛋白浓度测定。利用天然状态下的乙酰乙酰CoA(AcAc-CoA)分子会发生酮-烯醇互变形成烯醇化合物特性,酶活试验以乙酰乙酰辅酶a为底物建立标准曲线。硫解酶体外测活体系为(50 mmol·L^(-1) Tris-Cl pH 8.1,20 mmol·L^(-1) MgCl_2,60μmol·L^(-1)CoA,10μmol·L^(-1) AcAc-CoA,加入约0.1μg蛋白),通过记录反应过程中由于底物(AcAc-CoA)的减少而引起303 nm波长下的吸收值的变化,从而计算出硫解反应的初始反应速率,最终确定复性后的Hc-DAF-22的硫解酶活性。在相同条件下,取AcAc-CoA底物浓度为10μmol·L^(-1)的反应体系(50 mmol·L^(-1) Tris-Cl pH 8.1,20 mmol·L^(-1)MgCl_2,60μmol·L^(-1) CoA,10μmol·L^(-1) AcAc-CoA,加入约0.1μg蛋白于室温起始反应),分别调节反应体系的温度及pH梯度,确定Hc-DAF-22最佳酶活反应温度及pH条件。【结果】成功克隆Hc-daf-22基因,测序结果与NCBI已公布的H.【Objective】 Haemonchus contortus is one of the major gastrointestinal nematodes infecting millions of ruminants,the disease（haemonchosis） is nationally epidemic in China.In order to analyze the biochemical properties of Hc-DAF-22 in Haemonchus contortus,the enzyme activity of renatured Hc-DAF-22 was measured in this study.By the results of enzyme activity assay,the role of Hc-DAF-22 protein will be understood in peroxisomal β-oxidation.【Method】 First,the full-length of open reading frame of Hc-daf-22 was amplified by PCR from total c DNA of adult H.contortus ZJ Strain（Gen Bank：HQ738470.1）.A recombinant p ET-22b-Hc-daf-22 was constructed and transformed into E.coli BL21 strain; and its prokaryotic expression was induced for four hours by IPTG（0.1 mmol·L^（-1））.Then the bacterial liquid was centrifuged and the cells were resuspended in PBS solution（50 mmol·L^（-1））.After verification by SDS-PAGE and Western blot,the prokaryotic Hc-DAF-22 was purified by Ni-chelating affinity chromatography and concentrated by ultrafiltration tube.The concentration of protein was measured according to the protein concentration assay kit.Acetoacetylat natural state of CoA can occur keto-enol tautomerase and then enol compound is formed.Using this property,taking acetoacetyl coenzyme A as the substrate,the enzyme activity of renatured Hc-DAF-22 was measured in the reaction system（50 mmol·L^（-1） Tris-Cl pH8.1,20 mmol·L^（-1） MgCl_2,60 μmol·L^（-1） CoA,10 μmol·L^（-1） Ac Ac-CoA,0.1 μg protein）.Different temperatures and pH were set in order to determine the optimum reaction conditions.【Result】 The results showed that Hc-daf-22 gene was successfully cloned and gene similarity was 99.9% compared with H.contortus ZJ strain.Recombinant Hc-daf-22 could be expressed in E.coli BL21 and it could be detected both in the lysate supernate and precipitates,and molecular weight of the fusion protein was about 59 kD.The enzyme assay indicated that it could catalyze the substrate acetoacet

关 键 词：捻转血矛线虫 Hc-daf-22 原核表达 酶活测定 β氧化 

分 类 号：S852.7[农业科学—基础兽医学]