結(jié)核分枝桿菌Rv1096的表達及功能鑒定
發(fā)布時間：2018-09-11 21:29
【摘要】：結(jié)核分枝桿菌(Mycobacterium tuberculosis)能夠感染宿主并在宿主體內(nèi)長期存活,其細胞壁起著重要的作用。結(jié)核分枝桿菌細胞壁核心結(jié)構(gòu)由分枝菌酸、聚阿拉伯半乳糖和肽聚糖組成。肽聚糖是由N-乙酰葡糖胺和N-乙酰胞壁酸交替連接形成的多糖鏈與短肽鏈交叉連接形成網(wǎng)狀大分子結(jié)構(gòu),以維持細胞的形狀。結(jié)核分枝桿菌細胞壁中還含有一些蛋白質(zhì)和酶,研究細胞壁蛋白和酶有助于我們深入理解結(jié)核分枝桿菌對宿主細胞的感染機制、細菌的存活及對宿主的免疫調(diào)節(jié)以及細胞壁大分子的合成與降解規(guī)律等。 Rv1096是一種結(jié)核分枝桿菌細胞壁蛋白。生物信息學(xué)分析顯示,Rv1096與肺炎鏈球菌(Streptococcus pneumoniae)的肽聚糖N-乙酰葡糖胺脫乙酰基酶(PgdA)具有31% (76/243)一致性。研究發(fā)現(xiàn),由于肺炎鏈球菌PgdA對肽聚糖N-乙酰葡糖胺的脫乙�；饔�,脫乙酰基的肽聚糖使細菌能夠抵抗溶菌酶的降解作用,使細菌在宿主內(nèi)存活。在本研究中,我們將對結(jié)核分枝桿菌Rv1096進行功能鑒定。 目的:(1)用高保真DNA聚合酶從結(jié)核分枝桿菌H37Rv基因組中擴增出Rv1096基因,并將其克隆到pMD18-T載體中; (2)構(gòu)建pET29b-Rv1096表達載體,在E. coli BL21(DE3)pLysS中表達結(jié)核分枝桿菌Rv1096蛋白;(3)采用親和層析技術(shù)純化Rv1096蛋白,并用SDS-PAGE以及Western blotting鑒定所純化的Rv1096蛋白;(4)對Rv1096的功能進行鑒定。 結(jié)果:1.克隆Rv1096基因 用高保真DNA聚合酶(PrimeStar)從結(jié)核分枝桿菌H37R基因組中擴增出Rv1096基因,將純化Rv1096基因克隆到pMD18-T載體中,構(gòu)建出pMD18-Rv1096。用限制性內(nèi)切酶NdeI鑒定并進行DNA測序。 2.構(gòu)建pET29b-Rv1096表達載體 經(jīng)DNA測序鑒定后,將序列正確的Rv1096基因亞克隆到pET29b質(zhì)粒,構(gòu)建出pET29b-Rv1096重組表達質(zhì)粒。將pET29b-Rv1096轉(zhuǎn)化到E. coliBL21(DE3)pLysS菌株中。 3.誘導(dǎo)Rv1096蛋白在E. coli BL21(DE3)pLysS中的表達 用2.5 mM IPTG,在18℃振蕩培養(yǎng)12小時,誘導(dǎo)E. coli BL21(DE3)pLysS表達重組Rv1096蛋白。用超聲法破碎細菌,對上清組分進行Western blotting分析。結(jié)果表明,在上清中存在表達的Rv1096蛋白,分子量為31.08 kD。 4.采用親和層析技術(shù)純化Rv1096蛋白 pET29b表達載體使重組Rv1096蛋白C端帶有組氨酸標(biāo)簽,因此,用組氨酸-Ni2+親和層析技術(shù)對Rv1096蛋白進行純化。用Western blotting分析洗脫組分1、2,結(jié)果表明,純化所得的蛋白為Rv1096蛋白。用考馬斯亮藍法定量洗脫組分1的濃度為53.78μg/ml,該組分用于酶活性分析。 5.建立脫乙�；富钚缘臏y定方法 將純化的Rv1096蛋白與恥垢分枝桿菌(M. smegmatis)胞壁肽底物在37℃下反應(yīng)3 h,用乙酸檢測試劑盒測定乙�；Ｈ绻鸕v1096具有脫乙�；富钚�,將使肽聚糖脫乙酰基,乙�；M一步轉(zhuǎn)變成乙酸,后者與試劑盒中各組分經(jīng)過一些列的反應(yīng)生成NADH+H+,在340 nm處測定NADH+H+量。結(jié)果表明,Rv1096具有脫乙�；富钚�。 結(jié)論:在本研究中,我們構(gòu)建了pET29b-Tb Rv1096表達載體,在E. coli BL21(DE3)pLysS中表達出可溶性的Rv1096蛋白。并用乙酸試劑盒測定了Rv1096蛋白的肽聚糖脫乙�；富钚�。
[Abstract]:Mycobacterium tuberculosis can infect the host and survive for a long time. Its cell wall plays an important role. The core structure of the cell wall of Mycobacterium tuberculosis is composed of mycobacterial acid, polyarabinogalactose and peptidoglycan. Polysaccharide chains and short peptide chains cross-linked to form a network of macromolecules to maintain the shape of cells. Mycobacterium tuberculosis cell wall also contains some proteins and enzymes. Studying cell wall proteins and enzymes will help us to understand the mechanism of infection of Mycobacterium tuberculosis to host cells, the survival of bacteria and the immune regulation of host cells, and so on. The synthesis and degradation of cell wall macromolecules.
Rv1096 is a cell wall protein of Mycobacterium tuberculosis. Bioinformatics analysis showed that Rv1096 has 31% (76/243) consistency with peptidoglycan N-acetylglucosamine deacetylase (PgdA) of Streptococcus pneumoniae. It was found that PgdA has deacetylation effect on peptidoglycan N-acetylglucosamine. Acetyl peptidoglycans enable bacteria to resist the degradation of lysozyme and enable bacteria to live in host. In this study, we will identify the function of Mycobacterium tuberculosis Rv1096.
Objective: (1) To amplify Rv1096 gene from Mycobacterium tuberculosis H37Rv genome by high fidelity DNA polymerase and clone it into pMD18-T vector; (2) To construct pET29b-Rv1096 expression vector and express Rv1096 protein in E.coli BL21 (DE3) pLysS; (3) To purify Rv1096 protein by affinity chromatography and use SDS-PAGE and Wes-PAGE Tern blotting was used to identify the purified Rv1096 protein; (4) the function of Rv1096 was identified.
Results: 1. cloned Rv1096 gene.
The Rv1096 gene was amplified from Mycobacterium tuberculosis H37R genome by high fidelity DNA polymerase (PrimeStar). The purified Rv1096 gene was cloned into pMD18-T vector and pMD18-Rv1096 was constructed. The Rv1096 gene was identified by restriction endonuclease NdeI and sequenced.
2. construction of pET29b-Rv1096 expression vector.
The recombinant plasmid pET29b-Rv1096 was constructed by subcloning the correct Rv1096 gene into pET29b plasmid. The pET29b-Rv1096 was transformed into E.coli BL21 (DE3) pLysS strain.
3. induce the expression of Rv1096 protein in E. coli BL21 (DE3) pLysS.
The recombinant Rv1096 protein was induced by E.coli BL21 (DE3) pLysS after 12 hours of shaking culture at 18 C for 2.5 mM IPTG. The supernatant of E.coli BL21 (DE3) pLysS was fragmented by ultrasound and analyzed by Western blotting.
4. purification of Rv1096 protein by affinity chromatography
The recombinant Rv1096 protein was purified by histidine-Ni2+ affinity chromatography. The elution fraction 1,2 was analyzed by Western blotting. The result showed that the purified protein was Rv1096. The concentration of elution fraction 1 was 53.78 ug/ml by Coomassie brilliant blue assay. It is used for enzyme activity analysis.
5. establish a method for the determination of deacetylase activity.
The purified Rv1096 protein was reacted with Mycobacterium smegmatis cell wall peptide substrate for 3 hours at 37 C and acetyl group was determined by acetic acid detection kit. DH+H+ was used to determine the NADH+H+ content at 340 nm. The results showed that Rv1096 had the activity of deacetylase.
Conclusion: In this study, we constructed pET29b-Tb Rv1096 expression vector and expressed soluble Rv1096 protein in E.coli BL21 (DE3) pLysS. The activity of peptidoglycan deacetylase of Rv1096 protein was determined by acetic acid kit.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378
本文編號：2237902
[Abstract]:Mycobacterium tuberculosis can infect the host and survive for a long time. Its cell wall plays an important role. The core structure of the cell wall of Mycobacterium tuberculosis is composed of mycobacterial acid, polyarabinogalactose and peptidoglycan. Polysaccharide chains and short peptide chains cross-linked to form a network of macromolecules to maintain the shape of cells. Mycobacterium tuberculosis cell wall also contains some proteins and enzymes. Studying cell wall proteins and enzymes will help us to understand the mechanism of infection of Mycobacterium tuberculosis to host cells, the survival of bacteria and the immune regulation of host cells, and so on. The synthesis and degradation of cell wall macromolecules.
Rv1096 is a cell wall protein of Mycobacterium tuberculosis. Bioinformatics analysis showed that Rv1096 has 31% (76/243) consistency with peptidoglycan N-acetylglucosamine deacetylase (PgdA) of Streptococcus pneumoniae. It was found that PgdA has deacetylation effect on peptidoglycan N-acetylglucosamine. Acetyl peptidoglycans enable bacteria to resist the degradation of lysozyme and enable bacteria to live in host. In this study, we will identify the function of Mycobacterium tuberculosis Rv1096.
Objective: (1) To amplify Rv1096 gene from Mycobacterium tuberculosis H37Rv genome by high fidelity DNA polymerase and clone it into pMD18-T vector; (2) To construct pET29b-Rv1096 expression vector and express Rv1096 protein in E.coli BL21 (DE3) pLysS; (3) To purify Rv1096 protein by affinity chromatography and use SDS-PAGE and Wes-PAGE Tern blotting was used to identify the purified Rv1096 protein; (4) the function of Rv1096 was identified.
Results: 1. cloned Rv1096 gene.
The Rv1096 gene was amplified from Mycobacterium tuberculosis H37R genome by high fidelity DNA polymerase (PrimeStar). The purified Rv1096 gene was cloned into pMD18-T vector and pMD18-Rv1096 was constructed. The Rv1096 gene was identified by restriction endonuclease NdeI and sequenced.
2. construction of pET29b-Rv1096 expression vector.
The recombinant plasmid pET29b-Rv1096 was constructed by subcloning the correct Rv1096 gene into pET29b plasmid. The pET29b-Rv1096 was transformed into E.coli BL21 (DE3) pLysS strain.
3. induce the expression of Rv1096 protein in E. coli BL21 (DE3) pLysS.
The recombinant Rv1096 protein was induced by E.coli BL21 (DE3) pLysS after 12 hours of shaking culture at 18 C for 2.5 mM IPTG. The supernatant of E.coli BL21 (DE3) pLysS was fragmented by ultrasound and analyzed by Western blotting.
4. purification of Rv1096 protein by affinity chromatography
The recombinant Rv1096 protein was purified by histidine-Ni2+ affinity chromatography. The elution fraction 1,2 was analyzed by Western blotting. The result showed that the purified protein was Rv1096. The concentration of elution fraction 1 was 53.78 ug/ml by Coomassie brilliant blue assay. It is used for enzyme activity analysis.
5. establish a method for the determination of deacetylase activity.
The purified Rv1096 protein was reacted with Mycobacterium smegmatis cell wall peptide substrate for 3 hours at 37 C and acetyl group was determined by acetic acid detection kit. DH+H+ was used to determine the NADH+H+ content at 340 nm. The results showed that Rv1096 had the activity of deacetylase.
Conclusion: In this study, we constructed pET29b-Tb Rv1096 expression vector and expressed soluble Rv1096 protein in E.coli BL21 (DE3) pLysS. The activity of peptidoglycan deacetylase of Rv1096 protein was determined by acetic acid kit.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378
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相關(guān)期刊論文 前2條
1 張小霞,嚴(yán)衛(wèi)星,徐海濱;外源蛋白質(zhì)表達系統(tǒng)類型的研究進展[J];國外醫(yī)學(xué)(衛(wèi)生學(xué)分冊);2004年04期
2 李玉華;王鳳山;賀艷麗;;多糖化學(xué)修飾方法研究概況[J];中國生化藥物雜志;2007年01期
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