【摘要】：【目的】捻轉(zhuǎn)血矛線蟲(Haemonchus contortus)是反芻動(dòng)物主要的胃腸道線蟲之一,該病在中國呈全國性流行。為研究捻轉(zhuǎn)血矛線蟲(Haemonchus contortus)脂肪酸代謝相關(guān)蛋白DAF-22的生化特性,對其基因進(jìn)行了克隆、原核表達(dá),并對重組蛋白進(jìn)行了體外酶活性測定。以期了解捻轉(zhuǎn)血矛線蟲Hc-DAF-22蛋白在過氧化物酶體脂肪酸β氧化中的作用�！痉椒ā扛鶕�(jù)NCBI公布的H.contortus ZJ株daf-22 cDNA序列(Gen Bank:HQ738470.1)設(shè)計(jì)特異性引物,克隆Hc-daf-22基因并構(gòu)建重組質(zhì)粒pET-22b-Hc-daf-22,經(jīng)測序鑒定正確后將其轉(zhuǎn)化E.coli BL21,經(jīng)終濃度為0.1 m mol·L~(-1) IPTG(isopropyl-β-d-thiogalactoside)誘導(dǎo)表達(dá)4h,離心菌液,50 mmol·L~(-1)濃度的PBS溶液重懸菌體溶液,冰浴超聲破碎后上清沉淀分別進(jìn)行SDS-PAGE分析。超聲破碎后產(chǎn)物以鎳柱親和色譜法分離純化重組蛋白Hc-DAF-22,并用SDS-PAGE檢測蛋白純化情況及以抗His血清作為一抗Western Blot鑒定。純化后蛋白用超濾管濃縮除去鹽分,并按照蛋白濃度測定試劑盒進(jìn)行蛋白濃度測定。利用天然狀態(tài)下的乙酰乙酰CoA(AcAc-CoA)分子會(huì)發(fā)生酮-烯醇互變形成烯醇化合物特性,酶活試驗(yàn)以乙酰乙酰輔酶a為底物建立標(biāo)準(zhǔn)曲線。硫解酶體外測活體系為(50 mmol·L~(-1) Tris-Cl pH 8.1,20 mmol·L~(-1) MgCl_2,60μmol·L~(-1)CoA,10μmol·L~(-1) AcAc-CoA,加入約0.1μg蛋白),通過記錄反應(yīng)過程中由于底物(AcAc-CoA)的減少而引起303 nm波長下的吸收值的變化,從而計(jì)算出硫解反應(yīng)的初始反應(yīng)速率,最終確定復(fù)性后的Hc-DAF-22的硫解酶活性。在相同條件下,取AcAc-CoA底物濃度為10μmol·L~(-1)的反應(yīng)體系(50 mmol·L~(-1) Tris-Cl pH 8.1,20 mmol·L~(-1)MgCl_2,60μmol·L~(-1) CoA,10μmol·L~(-1) AcAc-CoA,加入約0.1μg蛋白于室溫起始反應(yīng)),分別調(diào)節(jié)反應(yīng)體系的溫度及pH梯度,確定Hc-DAF-22最佳酶活反應(yīng)溫度及pH條件�！窘Y(jié)果】成功克隆Hc-daf-22基因,測序結(jié)果與NCBI已公布的H.contortus ZJ株Hc-daf-22基因序列比對基因相似度為99.9%,并實(shí)現(xiàn)重組子pET-22b-Hc-daf-22在E.coli BL21體內(nèi)進(jìn)行表達(dá),表達(dá)產(chǎn)物經(jīng)SDS-PAGE和Western Blot檢測顯示,pET-22b-Hc-daf-22基因在大腸桿菌中成功表達(dá),呈部分可溶性,融合蛋白的分子量約為59 k D,測得蛋白濃度為1.70μg·μL~(-1);針對該表達(dá)產(chǎn)物的酶活分析結(jié)果表明,原核表達(dá)的Hc-DAF-22具有一定的硫解酶活性,其最適反應(yīng)pH為8.0,最佳反應(yīng)溫度為37℃,酶學(xué)常數(shù)Km值和Vmax值分別為33.765μmol·L~(-1)和1 784 nmol·L~(-1)·min~(-1)�！窘Y(jié)論】捻轉(zhuǎn)血矛線蟲DAF-22是過氧化物酶體脂肪酸β氧化的關(guān)鍵酶之一,本試驗(yàn)通過體外酶活試驗(yàn)成功測定Hc-DAF-22蛋白的酶活性,證明Hc-DAF-22具有一定硫解酶活性,但與秀麗隱桿線蟲(Caenorhabditis elegans)同源蛋白相比硫解酶活性較低。
[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，

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