结核分枝杆菌Rv1096的表达及功能鉴定
发布时间：2018-09-11 21:29
【摘要】：结核分枝杆菌(Mycobacterium tuberculosis)能够感染宿主并在宿主体内长期存活,其细胞壁起着重要的作用。结核分枝杆菌细胞壁核心结构由分枝菌酸、聚阿拉伯半乳糖和肽聚糖组成。肽聚糖是由N-乙酰葡糖胺和N-乙酰胞壁酸交替连接形成的多糖链与短肽链交叉连接形成网状大分子结构,以维持细胞的形状。结核分枝杆菌细胞壁中还含有一些蛋白质和酶,研究细胞壁蛋白和酶有助于我们深入理解结核分枝杆菌对宿主细胞的感染机制、细菌的存活及对宿主的免疫调节以及细胞壁大分子的合成与降解规律等。 Rv1096是一种结核分枝杆菌细胞壁蛋白。生物信息学分析显示,Rv1096与肺炎链球菌(Streptococcus pneumoniae)的肽聚糖N-乙酰葡糖胺脱乙酰基酶(PgdA)具有31% (76/243)一致性。研究发现,由于肺炎链球菌PgdA对肽聚糖N-乙酰葡糖胺的脱乙酰基作用,脱乙酰基的肽聚糖使细菌能够抵抗溶菌酶的降解作用,使细菌在宿主内存活。在本研究中,我们将对结核分枝杆菌Rv1096进行功能鉴定。 目的:(1)用高保真DNA聚合酶从结核分枝杆菌H37Rv基因组中扩增出Rv1096基因,并将其克隆到pMD18-T载体中; (2)构建pET29b-Rv1096表达载体,在E. coli BL21(DE3)pLysS中表达结核分枝杆菌Rv1096蛋白;(3)采用亲和层析技术纯化Rv1096蛋白,并用SDS-PAGE以及Western blotting鉴定所纯化的Rv1096蛋白;(4)对Rv1096的功能进行鉴定。 结果:1.克隆Rv1096基因 用高保真DNA聚合酶(PrimeStar)从结核分枝杆菌H37R基因组中扩增出Rv1096基因,将纯化Rv1096基因克隆到pMD18-T载体中,构建出pMD18-Rv1096。用限制性内切酶NdeI鉴定并进行DNA测序。 2.构建pET29b-Rv1096表达载体 经DNA测序鉴定后,将序列正确的Rv1096基因亚克隆到pET29b质粒,构建出pET29b-Rv1096重组表达质粒。将pET29b-Rv1096转化到E. coliBL21(DE3)pLysS菌株中。 3.诱导Rv1096蛋白在E. coli BL21(DE3)pLysS中的表达 用2.5 mM IPTG,在18℃振荡培养12小时,诱导E. coli BL21(DE3)pLysS表达重组Rv1096蛋白。用超声法破碎细菌,对上清组分进行Western blotting分析。结果表明,在上清中存在表达的Rv1096蛋白,分子量为31.08 kD。 4.采用亲和层析技术纯化Rv1096蛋白 pET29b表达载体使重组Rv1096蛋白C端带有组氨酸标签,因此,用组氨酸-Ni2+亲和层析技术对Rv1096蛋白进行纯化。用Western blotting分析洗脱组分1、2,结果表明,纯化所得的蛋白为Rv1096蛋白。用考马斯亮蓝法定量洗脱组分1的浓度为53.78μg/ml,该组分用于酶活性分析。 5.建立脱乙酰基酶活性的测定方法 将纯化的Rv1096蛋白与耻垢分枝杆菌(M. smegmatis)胞壁肽底物在37℃下反应3 h,用乙酸检测试剂盒测定乙酰基。如果Rv1096具有脱乙酰基酶活性,将使肽聚糖脱乙酰基,乙酰基进一步转变成乙酸,后者与试剂盒中各组分经过一些列的反应生成NADH+H+,在340 nm处测定NADH+H+量。结果表明,Rv1096具有脱乙酰基酶活性。 结论:在本研究中,我们构建了pET29b-Tb Rv1096表达载体,在E. coli BL21(DE3)pLysS中表达出可溶性的Rv1096蛋白。并用乙酸试剂盒测定了Rv1096蛋白的肽聚糖脱乙酰基酶活性。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378
本文编号：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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378
【参考文献】
相关期刊论文 前2条
1 张小霞,严卫星,徐海滨;外源蛋白质表达系统类型的研究进展[J];国外医学(卫生学分册);2004年04期
2 李玉华;王凤山;贺艳丽;;多糖化学修饰方法研究概况[J];中国生化药物杂志;2007年01期
，本文编号：2237902
本文链接：https://www.wllwen.com/xiyixuelunwen/2237902.html
最近更新
教材专著
