结核分枝杆菌Rv3717基因的克
发布时间：2018-07-25 15:10
【摘要】：结核分枝杆菌是全球主要的病原体之一,随着多重耐药菌株的增加,其威胁性也随之增大。结核分枝杆菌的细胞壁相对较厚、硬并且具有疏水性,能够有效地保护细菌,因此抗菌药物的开发具有较高的难度。 乙胺丁醇(Ethambutol,EMB),是一种一线抗结核的药物。本课题利用生物信息学的方法,发现一种未知功能的基因Rv3717。当乙胺丁醇作用于结核分枝杆菌后,Rv3717基因的表达上调。该基因被预测具有水解肽聚糖的功能,可能会造成肽聚糖的水解,引起细胞的自溶。 肽聚糖是结核分枝杆菌细胞壁核心结构的重要组成部分,目前关于分枝杆菌细胞壁自溶素(也称为肽聚糖水解酶)的了解并不多。已知枯草芽孢杆菌的cwlB基因是一种已知的细胞壁自溶素,把此基因的序列与结核分枝杆菌的基因组序列进行BLAST比对,找到两种同源基因Rv3915和Rv3717,推测该两种基因表达的蛋白质可能同样具有肽聚糖水解酶的活性。有研究表明,Rv3915基因被成功克隆表达并且经鉴定具有肽聚糖水解酶的活性。因此,Rv3717基因是否也有此活性是本课题所要解决的问题。 对Rv3717基因肽聚糖水解酶活性的确定,有利于揭示EMB抗结核的作用机制,并为发现新的药物靶点进而开发新一代抗结核药物提供理论依据。 目的:通过对Rv3717目的基因的克隆表达,纯化目的蛋白,以对Rv3717基因的功能进行鉴定。 方法:以结核分枝杆菌基因组DNA为模板,利用PCR技术进行目的基因的扩增。使用克隆载体pMD18-T与目的基因以“A-T”连接方式构建克隆质粒pMD18-Rv3717;被扩增的目的片段经测序正确后,载体和目的片段分别经双酶切后进行连接,构建表达质粒。利用不同的载体和不同的宿主菌,通过调节诱导剂的浓度和诱导温度等进行目的蛋白的优化表达。利用SDS-PAGE和Western blotting检测目的基因的表达,利用Ni~(2+)亲和层析柱对目的蛋白进行纯化并用酶谱法对目的蛋白进行功能的检测。 结果:构建了克隆质粒pMD18-Rv3717;扩增的目的基因经测序正确后,构建了无突变碱基的表达质粒pET29b-Rv3717和pET16b-Rv3717。表达质粒pET29b-Rv3717在所使用的各种宿主菌中的蛋白表达量极低。表达质粒pET16b-Rv3717在大肠杆菌BL21(DE3)中的表达量高,但是绝大部分是以包涵体的形式存在,上清液中的可溶性目的蛋白比较少。目的蛋白的上清液经镍柱纯化以后,得到含有少量杂蛋白的目的蛋白的纯化物。纯化的目的蛋白没有检测到肽聚糖水解酶的活性。 结论:利用分子克隆技术克隆了结核分枝杆菌的基因Rv3717;使用表达质粒pET16b在大肠杆菌BL21(DE3)中过表达了目的基因,并且绝大部分目的蛋白是以包涵体的形式存在。本论文论述了目的基因Rv3717与肽聚糖水解酶的关系,未能检测出过表达的目的蛋白的肽聚糖水解酶的活性,但为进一步地探讨两者之间的关系提供了方法学的借鉴和物质基础。
[Abstract]:Mycobacterium tuberculosis is one of the major pathogens in the world. With the increase of multidrug resistant strains, the threat of Mycobacterium tuberculosis is also increasing. The cell wall of Mycobacterium tuberculosis is relatively thick, hard and hydrophobic, and can effectively protect bacteria. Therefore, the development of antibacterial drugs has high difficulty.
Ethambutol (EMB) is a first-line anti tuberculosis drug. This topic uses bioinformatics to discover an unknown function gene Rv3717., when ethambutol acts on Mycobacterium tuberculosis, the expression of Rv3717 gene is up-regulated. This gene is predicted to have the function of hydrolysable peptidoglycan and may cause the hydrolysis of peptidoglycan. It causes autolysis of cells.
Peptidoglycan is an important component of the core structure of the cell wall of Mycobacterium tuberculosis. At present, there is not much knowledge about the cell wall autlysin (also known as peptidoglycan hydrolase) of Mycobacterium tuberculosis. The cwlB gene of Bacillus subtilis is known as a known cell wall autolytic hormone, the sequence of this base and the genome sequence of Mycobacterium tuberculosis. Two homologous genes, Rv3915 and Rv3717 were found, and the protein expressed by the two genes might also have the activity of peptidoglycan hydrolase. Some studies have shown that the Rv3915 gene was successfully cloned and expressed with the activity of peptidoglycan hydrolase. Therefore, whether the Rv3717 gene also has this activity is the subject of this subject. Solve the problem.
The determination of the activity of Rv3717 peptidoglycan hydrolase is helpful to reveal the mechanism of the anti tuberculosis action of EMB, and provide a theoretical basis for the discovery of new drug targets and further development of a new generation of anti tuberculosis drugs.
Objective: To identify the function of Rv3717 gene by cloning, expression and purification of Rv3717 gene.
Methods: the genomic DNA of Mycobacterium tuberculosis was used as the template, and the target gene was amplified by PCR technology. Clone vector pMD18-T was used to construct the clone plasmid pMD18-Rv3717 with the target gene "A-T" connection. After the amplified target fragment was sequenced correctly, the vector and the target fragment were connected by double enzyme, and the expression was constructed. Plasmid. Using different carriers and different host bacteria to optimize the expression of the target protein by regulating the concentration of inducer and induction temperature. Using SDS-PAGE and Western blotting to detect the expression of the target gene, the target protein was purified by Ni~ (2+) affinity chromatography column and the function of the target protein was detected by the enzyme spectrum method.
Results: The Clone plasmid pMD18-Rv3717 was constructed. After sequencing, the expression plasmid pET29b-Rv3717 and pET16b-Rv3717. expression plasmid pET29b-Rv3717 without mutation base were very low. The expression of expression plasmid pET16b-Rv3717 in Escherichia coli BL21 (DE3) was very low. It is high, but most of them exist in the form of inclusion bodies. The soluble protein in the supernatant is less. After purification of the supernatant of the target protein, the purify of the target protein containing a small amount of protein is obtained. The purified target protein does not detect the activity of the peptide polyhydrolysate.
Conclusion: the gene Rv3717 of Mycobacterium tuberculosis was cloned by molecular cloning technology, and the expression plasmid pET16b was used to express the target gene in Escherichia coli BL21 (DE3), and most of the target proteins existed in the form of inclusion body. The relationship between the target gene Rv3717 and peptidoglycan hydrolase was discussed in this paper, and the table was not detected. The peptidoglycan hydrolase activity of the target protein was studied, but it provided methodological reference and material basis for further study of the relationship between the two proteins.
【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：2144205
[Abstract]:Mycobacterium tuberculosis is one of the major pathogens in the world. With the increase of multidrug resistant strains, the threat of Mycobacterium tuberculosis is also increasing. The cell wall of Mycobacterium tuberculosis is relatively thick, hard and hydrophobic, and can effectively protect bacteria. Therefore, the development of antibacterial drugs has high difficulty.
Ethambutol (EMB) is a first-line anti tuberculosis drug. This topic uses bioinformatics to discover an unknown function gene Rv3717., when ethambutol acts on Mycobacterium tuberculosis, the expression of Rv3717 gene is up-regulated. This gene is predicted to have the function of hydrolysable peptidoglycan and may cause the hydrolysis of peptidoglycan. It causes autolysis of cells.
Peptidoglycan is an important component of the core structure of the cell wall of Mycobacterium tuberculosis. At present, there is not much knowledge about the cell wall autlysin (also known as peptidoglycan hydrolase) of Mycobacterium tuberculosis. The cwlB gene of Bacillus subtilis is known as a known cell wall autolytic hormone, the sequence of this base and the genome sequence of Mycobacterium tuberculosis. Two homologous genes, Rv3915 and Rv3717 were found, and the protein expressed by the two genes might also have the activity of peptidoglycan hydrolase. Some studies have shown that the Rv3915 gene was successfully cloned and expressed with the activity of peptidoglycan hydrolase. Therefore, whether the Rv3717 gene also has this activity is the subject of this subject. Solve the problem.
The determination of the activity of Rv3717 peptidoglycan hydrolase is helpful to reveal the mechanism of the anti tuberculosis action of EMB, and provide a theoretical basis for the discovery of new drug targets and further development of a new generation of anti tuberculosis drugs.
Objective: To identify the function of Rv3717 gene by cloning, expression and purification of Rv3717 gene.
Methods: the genomic DNA of Mycobacterium tuberculosis was used as the template, and the target gene was amplified by PCR technology. Clone vector pMD18-T was used to construct the clone plasmid pMD18-Rv3717 with the target gene "A-T" connection. After the amplified target fragment was sequenced correctly, the vector and the target fragment were connected by double enzyme, and the expression was constructed. Plasmid. Using different carriers and different host bacteria to optimize the expression of the target protein by regulating the concentration of inducer and induction temperature. Using SDS-PAGE and Western blotting to detect the expression of the target gene, the target protein was purified by Ni~ (2+) affinity chromatography column and the function of the target protein was detected by the enzyme spectrum method.
Results: The Clone plasmid pMD18-Rv3717 was constructed. After sequencing, the expression plasmid pET29b-Rv3717 and pET16b-Rv3717. expression plasmid pET29b-Rv3717 without mutation base were very low. The expression of expression plasmid pET16b-Rv3717 in Escherichia coli BL21 (DE3) was very low. It is high, but most of them exist in the form of inclusion bodies. The soluble protein in the supernatant is less. After purification of the supernatant of the target protein, the purify of the target protein containing a small amount of protein is obtained. The purified target protein does not detect the activity of the peptide polyhydrolysate.
Conclusion: the gene Rv3717 of Mycobacterium tuberculosis was cloned by molecular cloning technology, and the expression plasmid pET16b was used to express the target gene in Escherichia coli BL21 (DE3), and most of the target proteins existed in the form of inclusion body. The relationship between the target gene Rv3717 and peptidoglycan hydrolase was discussed in this paper, and the table was not detected. The peptidoglycan hydrolase activity of the target protein was studied, but it provided methodological reference and material basis for further study of the relationship between the two proteins.
【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 胡斌，宋彦凤，孙颖，，冯冶兵;细菌肽聚精的研究[J];松辽学刊(自然科学版);1994年01期
2 庄玉辉,何秀云,张小刚,李国利,阙海萍,刘绍军;结核分枝杆菌培养物不同组份蛋白质组研究[J];微生物学报;2002年03期
本文编号：2144205
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