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
发布时间：2018-11-03 13:32
【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.contortus ZJ株Hc-daf-22基因序列比对基因相似度为99.9%,并实现重组子pET-22b-Hc-daf-22在E.coli BL21体内进行表达,表达产物经SDS-PAGE和Western Blot检测显示,pET-22b-Hc-daf-22基因在大肠杆菌中成功表达,呈部分可溶性,融合蛋白的分子量约为59 k D,测得蛋白浓度为1.70μg·μL~(-1);针对该表达产物的酶活分析结果表明,原核表达的Hc-DAF-22具有一定的硫解酶活性,其最适反应pH为8.0,最佳反应温度为37℃,酶学常数Km值和Vmax值分别为33.765μmol·L~(-1)和1 784 nmol·L~(-1)·min~(-1)。【结论】捻转血矛线虫DAF-22是过氧化物酶体脂肪酸β氧化的关键酶之一,本试验通过体外酶活试验成功测定Hc-DAF-22蛋白的酶活性,证明Hc-DAF-22具有一定硫解酶活性,但与秀丽隐杆线虫(Caenorhabditis elegans)同源蛋白相比硫解酶活性较低。
[Abstract]:Objective: (Haemonchus contortus) is one of the main gastrointestinal nematodes in ruminants. In order to study the biochemical characteristics of (Haemonchus contortus) fatty acid metabolism-related protein DAF-22, its gene was cloned and expressed in prokaryotic, and the enzyme activity of recombinant protein was determined in vitro. In order to understand the role of Hc-DAF-22 protein in peroxisome fatty acid 尾 oxidation, specific primers were designed according to the daf-22 cDNA sequence (Gen Bank:HQ738470.1) of H.contortus ZJ strain published by NCBI. Hc-daf-22 gene was cloned and the recombinant plasmid pET-22b-Hc-daf-22, was identified by sequencing. The transformed E.coli BL21, was induced to express at 0.1 m mol L ~ (-1) IPTG (isopropyl- 尾 -d-thiogalactoside for 4 h. Centrifuge solution, PBS solution with 50 mmol L ~ (-1) concentration, and supernatant precipitation after ultrasonic crushing in ice bath were analyzed by SDS-PAGE, respectively. The recombinant protein Hc-DAF-22, was separated and purified by Ni column affinity chromatography after ultrasonic crushing. The purified protein was detected by SDS-PAGE and the anti His serum was used as the first anti Western Blot. The purified protein was concentrated and removed by ultrafiltration tube, and the protein concentration was determined according to the protein concentration determination kit. In this paper, acetyl CoA (AcAc-CoA) molecules in natural state were used to deform keto-enol to form enol compounds, and the standard curve was established by enzyme activity test using acetyl coenzyme a as substrate. The activity system of thiolytic enzyme in vitro was (50 mmol L ~ (-1) Tris-Cl pH 8.1 mmol L ~ (-1) MgCl_2,60 渭 mol L ~ (-1) CoA,10 渭 mol L ~ (-1) AcAc-CoA, added about 0.1 渭 g protein). By recording the changes of absorption value at 303 nm wavelength due to the decrease of substrate (AcAc-CoA), the initial reaction rate was calculated, and the activity of sulpolytic enzyme of Hc-DAF-22 after renaturation was finally determined. Under the same conditions, the reaction system with AcAc-CoA substrate concentration of 10 渭 mol L ~ (-1) (50 mmol L ~ (-1) Tris-Cl pH 8.1 mmol L ~ (-1) MgCl_2,60 渭 mol L ~ (-1) CoA,10 渭 mol L ~ (-1) AcAc-CoA,) was obtained. The reaction temperature and pH gradient of the reaction system were adjusted to determine the optimum reaction temperature and pH conditions of Hc-DAF-22. [results] the Hc-daf-22 gene was cloned successfully. The result of sequencing was 99.9 gene similarity with Hc-daf-22 gene sequence of H.contortus ZJ strain published by NCBI, and the recombinant pET-22b-Hc-daf-22 was expressed in E.coli BL21. SDS-PAGE and Western Blot analysis showed that the pET-22b-Hc-daf-22 gene was partially soluble in E. coli, the molecular weight of the fusion protein was about 59kD, and the protein concentration was 1.70 渭 g 渭 L ~ (-1). The results of enzyme activity analysis showed that the Hc-DAF-22 expressed in prokaryotic cells had a certain activity of thiolytic enzyme. The optimum reaction temperature was 37 鈩，
本文编号：2307934
[Abstract]:Objective: (Haemonchus contortus) is one of the main gastrointestinal nematodes in ruminants. In order to study the biochemical characteristics of (Haemonchus contortus) fatty acid metabolism-related protein DAF-22, its gene was cloned and expressed in prokaryotic, and the enzyme activity of recombinant protein was determined in vitro. In order to understand the role of Hc-DAF-22 protein in peroxisome fatty acid 尾 oxidation, specific primers were designed according to the daf-22 cDNA sequence (Gen Bank:HQ738470.1) of H.contortus ZJ strain published by NCBI. Hc-daf-22 gene was cloned and the recombinant plasmid pET-22b-Hc-daf-22, was identified by sequencing. The transformed E.coli BL21, was induced to express at 0.1 m mol L ~ (-1) IPTG (isopropyl- 尾 -d-thiogalactoside for 4 h. Centrifuge solution, PBS solution with 50 mmol L ~ (-1) concentration, and supernatant precipitation after ultrasonic crushing in ice bath were analyzed by SDS-PAGE, respectively. The recombinant protein Hc-DAF-22, was separated and purified by Ni column affinity chromatography after ultrasonic crushing. The purified protein was detected by SDS-PAGE and the anti His serum was used as the first anti Western Blot. The purified protein was concentrated and removed by ultrafiltration tube, and the protein concentration was determined according to the protein concentration determination kit. In this paper, acetyl CoA (AcAc-CoA) molecules in natural state were used to deform keto-enol to form enol compounds, and the standard curve was established by enzyme activity test using acetyl coenzyme a as substrate. The activity system of thiolytic enzyme in vitro was (50 mmol L ~ (-1) Tris-Cl pH 8.1 mmol L ~ (-1) MgCl_2,60 渭 mol L ~ (-1) CoA,10 渭 mol L ~ (-1) AcAc-CoA, added about 0.1 渭 g protein). By recording the changes of absorption value at 303 nm wavelength due to the decrease of substrate (AcAc-CoA), the initial reaction rate was calculated, and the activity of sulpolytic enzyme of Hc-DAF-22 after renaturation was finally determined. Under the same conditions, the reaction system with AcAc-CoA substrate concentration of 10 渭 mol L ~ (-1) (50 mmol L ~ (-1) Tris-Cl pH 8.1 mmol L ~ (-1) MgCl_2,60 渭 mol L ~ (-1) CoA,10 渭 mol L ~ (-1) AcAc-CoA,) was obtained. The reaction temperature and pH gradient of the reaction system were adjusted to determine the optimum reaction temperature and pH conditions of Hc-DAF-22. [results] the Hc-daf-22 gene was cloned successfully. The result of sequencing was 99.9 gene similarity with Hc-daf-22 gene sequence of H.contortus ZJ strain published by NCBI, and the recombinant pET-22b-Hc-daf-22 was expressed in E.coli BL21. SDS-PAGE and Western Blot analysis showed that the pET-22b-Hc-daf-22 gene was partially soluble in E. coli, the molecular weight of the fusion protein was about 59kD, and the protein concentration was 1.70 渭 g 渭 L ~ (-1). The results of enzyme activity analysis showed that the Hc-DAF-22 expressed in prokaryotic cells had a certain activity of thiolytic enzyme. The optimum reaction temperature was 37 鈩，
本文编号：2307934
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2307934.html
最近更新
教材专著
