本文關(guān)鍵詞：結(jié)核分枝桿菌葉酸代謝相關(guān)基因Rv0992c的性質(zhì)及功能研究　出處：《西南大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 葉酸代謝 5-甲�；�-四氫葉酸-環(huán)連接酶(MTHFS) 結(jié)核分枝桿菌

【摘要】：結(jié)核病嚴(yán)重危害人類(lèi)的生命健康,其致病菌結(jié)核分枝桿菌(Mycobacterium tuberculosis)感染過(guò)全世界近三分之一的人口。隨著人們對(duì)結(jié)核分枝桿菌生物學(xué)特征及生理代謝的了解,相繼研發(fā)了鏈霉素、異煙肼、利福平等治療性藥物,但是這些藥物的不合理利用引起耐藥分枝桿菌的出現(xiàn)。結(jié)核病控制工作中急需新型藥物的出現(xiàn),所以尋找新的結(jié)核藥物作用靶點(diǎn)及開(kāi)發(fā)新型抗結(jié)核藥物己成為當(dāng)前的熱點(diǎn)和難點(diǎn)。這就需要加深對(duì)結(jié)核分枝桿菌生理代謝的了解。葉酸代謝是細(xì)菌不可或缺的生理過(guò)程,其代謝途徑中所涉及的某些酶的編碼基因在結(jié)核分枝桿菌中還不是很清楚。 本研究通過(guò)生物信息學(xué)方法對(duì)M. tuberculosis H37Rv的未知功能基因Rv0992c的蛋白產(chǎn)物進(jìn)行結(jié)構(gòu)和功能的分析及預(yù)測(cè),并對(duì)該基因進(jìn)行克隆、表達(dá)及蛋白產(chǎn)物酶學(xué)活性研究。從之前對(duì)深紅紅螺菌(Rhodospirillum rubrum)的研究得知Rru_A1084基因編碼5-甲�；�-四氫葉酸環(huán)連接酶(5-formyltetrahydrofolate cyclo-ligase,也稱(chēng)為次甲基-四氫葉酸合成酶,MTHFS)。M. tuberculosis中Rv0992c基因是Rru A1084的同源物(二者的氨基酸序列具有32.7%的相似性和21.8%的同源性),所以推測(cè)前者編碼結(jié)核分枝桿菌的5-甲�；�-四氫葉酸環(huán)連接酶。從GenBank數(shù)據(jù)庫(kù)中獲得M. tuberculosis H37Rv的Rv0992c的核苷酸序列,設(shè)計(jì)對(duì)引物,以M. tuberculosis H37Rv全基因組為模板,PCR擴(kuò)增獲得Rv0992c基因。通過(guò)TA克隆,將PCR產(chǎn)物連接到pMD19-T Simple Vector上,之后亞克隆至載體pET28上,經(jīng)菌落PCR,質(zhì)粒酶切以及核苷酸序列測(cè)序證明了成功的構(gòu)建了重組質(zhì)粒pET28-Rv0992c。重組質(zhì)粒轉(zhuǎn)化Escherichia coli BL21 (DE3),以溫度、時(shí)間和IPTG濃度建立三因素三水平的正交實(shí)驗(yàn),優(yōu)化該重組蛋白質(zhì)誘導(dǎo)表達(dá)條件。以最佳表達(dá)條件(0.8mM的IPTG,25℃下誘導(dǎo)4h)進(jìn)行擴(kuò)大培養(yǎng),Ni+凝膠親和層析柱純化獲得高純度的重組Rv0992c蛋白。純化蛋白的酶學(xué)活性通過(guò)反應(yīng)產(chǎn)物5,10-次甲基-四氫葉酸在355nm處吸光度的變化得到證實(shí)。 運(yùn)用Vector NTI 9, Swiss Model等生物信息學(xué)資源對(duì)結(jié)核分枝桿菌Rv0992c編碼蛋白的基本性質(zhì)進(jìn)行了分析,包括對(duì)蛋白質(zhì)等電點(diǎn)和分子量的預(yù)測(cè),二級(jí)結(jié)構(gòu)以及三級(jí)結(jié)構(gòu)的預(yù)測(cè)分析。結(jié)果表明：M. tuberculosis H37Rv Rv0992c基因編碼蛋白分子量為21412.95Da,理論P(yáng)I為9.40；二級(jí)結(jié)構(gòu)是由3個(gè)α螺旋8個(gè)β折疊及無(wú)規(guī)則卷曲構(gòu)成的。在一二級(jí)結(jié)構(gòu)分析的基礎(chǔ)上,利用同源建模的方法完成了其三維結(jié)構(gòu)的建模。分析該蛋白在分枝桿菌屬中的分布情況,結(jié)果顯示M. tuberculosis H37Rv的MTHFS同已報(bào)道的其他分枝桿菌屬來(lái)源的MTHFS有較高的同源性。綜上,本研究揭示了M. tuberculosis H37Rv Rv0992c序列編碼蛋白具有將5-甲酰-四氫葉酸轉(zhuǎn)化為5,10-次甲基-四氫葉酸的生物學(xué)功能。
[Abstract]:Tuberculosis serious harm to human health, the pathogenic bacteria of Mycobacterium tuberculosis (Mycobacterium tuberculosis) infection all over the world nearly 1/3 of the population. With the understanding of the biological characteristics of Mycobacterium tuberculosis bacilli and physiological metabolism, have developed streptomycin, isoniazid, rifampin therapeutic drugs, but not the rational use of these drugs cause the emergence of drug-resistant Mycobacterium tuberculosis. The control work in urgent need for new drugs, so looking for new TB drug targets and the development of new anti tuberculosis drugs have become the hot and difficult. It is necessary to deepen the understanding of the physiological metabolism of Mycobacterium tuberculosis. Folate metabolism is a physiological process of bacterial gene encoding some indispensable. The enzyme involved in the metabolic pathway in Mycobacterium tuberculosis is not very clear.
In this study, analyze and predict the structure and function by bioinformatics analysis of Rv0992c protein of unknown function gene M. of tuberculosis H37Rv, and to clone the gene expression and activity of protein enzyme. From the previous of Rhodospirillum rubrum (Rhodospirillum rubrum) research that Rru_A1084 gene encoding 5- formyl tetrahydrofolate ring ligase (5-formyltetrahydrofolate cyclo-ligase, also known as methylene tetrahydrofolate synthetase, MTHFS) Rv0992c.M. tuberculosis Rru gene is the homolog of A1084 (two amino acid sequence homology with 21.8% similarity and 32.7%), so that the former encoding Mycobacterium tuberculosis 5- formyl tetrahydrofolate ligase ring. Nucleotide sequences obtained M. tuberculosis H37Rv Rv0992c from the GenBank database, a pair of primers were designed with M. tuberculosis H37Rv. The genome as a template, PCR Rv0992c gene was amplified by TA. The PCR products were cloned, connected to pMD19-T Simple Vector, then cloned into the vector pET28. By colony PCR, restriction enzyme analysis and nucleotide sequencing proved the successful construction of the recombinant plasmid pET28-Rv0992c. recombinant plasmid was transformed into Escherichia coli BL21 (DE3), with temperature orthogonal experiment, time and concentration of IPTG to establish three levels of three factors, the optimization of recombinant protein expression conditions. With the optimized expression conditions (0.8mM IPTG, 25 DEG C under the induction of 4h) to expand the training, column chromatography to obtain high purity of recombinant protein Rv0992c affinity gel. Ni+ enzymatic activity of purified protein by reaction products 5,10- - methylene tetrahydrofolate in the change of absorbance at 355nm was confirmed.
The use of Vector NTI 9, Swiss Model and other bioinformatics resources for encoding Rv0992c protein of Mycobacterium tuberculosis and the basic properties are analyzed, including the prediction of protein molecular weight and isoelectric point, analysis of two level structure and prediction of three level structure. The results show that the M. tuberculosis H37Rv Rv0992c gene encoding protein molecular weight is 21412.95Da. The theory of PI is 9.40; the two stage structure is folded by 3 alpha helix 8 beta and random coil structure. Based on the analysis of one or two level structure, using homology modeling method to complete the modeling of the three-dimensional structure. Analyzing the distribution of protein in Mycobacterium species, showed homology there are other high M. of Mycobacterium tuberculosis H37Rv MTHFS reported from MTHFS. In conclusion, this study reveals that the M. tuberculosis H37Rv Rv0992c sequence encoding protein with 5- formyl - The transformation of four hydrofolate is the biological function of 5,10- methylene dihydrofolate.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R378

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 蔡大偉;葉酸代謝酶基因MTHFR、MTR多態(tài)性與膀胱癌遺傳易感性及與RASSF1A基因啟動(dòng)子區(qū)甲基化關(guān)系的研究[D];中國(guó)醫(yī)科大學(xué);2010年

相關(guān)碩士學(xué)位論文 前1條

1 余科;葉酸代謝酶基因多態(tài)性與腫瘤易感性的Meta分析[D];復(fù)旦大學(xué);2009年

，

本文編號(hào)：1384981