【摘要】：結核分枝桿菌是全球主要的病原體之一,隨著多重耐藥菌株的增加,其威脅性也隨之增大。結核分枝桿菌的細胞壁相對較厚、硬并且具有疏水性,能夠有效地保護細菌,因此抗菌藥物的開發(fā)具有較高的難度。 乙胺丁醇(Ethambutol,EMB),是一種一線抗結核的藥物。本課題利用生物信息學的方法,發(fā)現(xiàn)一種未知功能的基因Rv3717。當乙胺丁醇作用于結核分枝桿菌后,Rv3717基因的表達上調(diào)。該基因被預測具有水解肽聚糖的功能,可能會造成肽聚糖的水解,引起細胞的自溶。 肽聚糖是結核分枝桿菌細胞壁核心結構的重要組成部分,目前關于分枝桿菌細胞壁自溶素(也稱為肽聚糖水解酶)的了解并不多。已知枯草芽孢桿菌的cwlB基因是一種已知的細胞壁自溶素,把此基因的序列與結核分枝桿菌的基因組序列進行BLAST比對,找到兩種同源基因Rv3915和Rv3717,推測該兩種基因表達的蛋白質可能同樣具有肽聚糖水解酶的活性。有研究表明,Rv3915基因被成功克隆表達并且經(jīng)鑒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課題所要解決的問題。 對Rv3717基因肽聚糖水解酶活性的確定,有利于揭示EMB抗結核的作用機制,并為發(fā)現(xiàn)新的藥物靶點進而開發(fā)新一代抗結核藥物提供理論依據(jù)。 目的:通過對Rv3717目的基因的克隆表達,純化目的蛋白,以對Rv3717基因的功能進行鑒定。 方法:以結核分枝桿菌基因組DNA為模板,利用PCR技術進行目的基因的擴增。使用克隆載體pMD18-T與目的基因以“A-T”連接方式構建克隆質粒pMD18-Rv3717;被擴增的目的片段經(jīng)測序正確后,載體和目的片段分別經(jīng)雙酶切后進行連接,構建表達質粒。利用不同的載體和不同的宿主菌,通過調(diào)節(jié)誘導劑的濃度和誘導溫度等進行目的蛋白的優(yōu)化表達。利用SDS-PAGE和Western blotting檢測目的基因的表達,利用Ni~(2+)親和層析柱對目的蛋白進行純化并用酶譜法對目的蛋白進行功能的檢測。 結果:構建了克隆質粒pMD18-Rv3717;擴增的目的基因經(jīng)測序正確后,構建了無突變堿基的表達質粒pET29b-Rv3717和pET16b-Rv3717。表達質粒pET29b-Rv3717在所使用的各種宿主菌中的蛋白表達量極低。表達質粒pET16b-Rv3717在大腸桿菌BL21(DE3)中的表達量高,但是絕大部分是以包涵體的形式存在,上清液中的可溶性目的蛋白比較少。目的蛋白的上清液經(jīng)鎳柱純化以后,得到含有少量雜蛋白的目的蛋白的純化物。純化的目的蛋白沒有檢測到肽聚糖水解酶的活性。 結論:利用分子克隆技術克隆了結核分枝桿菌的基因Rv3717;使用表達質粒pET16b在大腸桿菌BL21(DE3)中過表達了目的基因,并且絕大部分目的蛋白是以包涵體的形式存在。本論文論述了目的基因Rv3717與肽聚糖水解酶的關系,未能檢測出過表達的目的蛋白的肽聚糖水解酶的活性,但為進一步地探討兩者之間的關系提供了方法學的借鑒和物質基礎。
[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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R378

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相關期刊論文 前2條

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