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  本文關(guān)鍵詞：藤黃微球菌Rpf結(jié)構(gòu)域及其突變體基因的克隆、表達(dá)及生物活性的初步研究　出處：《第四軍醫(yī)大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 藤黃微球菌 恥垢分枝桿菌 Rpf Rpf結(jié)構(gòu)域 突變體 生物學(xué)活性

【摘要】： 結(jié)核病(Tuberculosis,TB)是由結(jié)核分枝桿菌(Mycobacterium tuberculosis,MTB)所致的、以呼吸系統(tǒng)感染為主的傳染病。近年來,由于卡介苗(BCG)保護(hù)性的不完善、耐藥MTB菌株的出現(xiàn)、艾滋病的流行以及人口流動等因素的增加,使得TB的發(fā)病率呈日益上升的趨勢,再次成為危害人類健康的世界性疾病。BCG是目前預(yù)防TB的唯一疫苗,但其不能有效預(yù)防成人TB,因此,積極研究TB的致病及免疫機制,對于開發(fā)更為有效的診斷、預(yù)防和治療TB的新技術(shù)具有重要意義。 藤黃微球菌(M.luteus)中的復(fù)活促進(jìn)因子(Resuscitation-promoting factor,Rpf)是第一個被發(fā)現(xiàn)的細(xì)菌生長因子,它具有促進(jìn)休眠菌的復(fù)蘇以及促進(jìn)細(xì)菌快速生長的作用。序列對比分析發(fā)現(xiàn),在其它的一些革蘭氏陽性細(xì)菌如MTB、麻風(fēng)分枝桿菌、藍(lán)色鏈霉菌、谷氨酸棒桿菌等中也存在30多個與Rpf同源的蛋白。研究發(fā)現(xiàn)MTB基因組可編碼5種Rpf樣蛋白,分別為Rv0867C(Rpf A)、Rv1009(Rpf B)、Rv1884C(Rpf C)、Rv2389C(Rpf D)和Rv2450C(Rpf E),推測這些蛋白也具有促進(jìn)MTB快速生長的作用。此外,這些蛋白均具有Rpf樣結(jié)構(gòu)域,即高度保守的70aa殘基,該保守區(qū)域?qū)pf的復(fù)蘇功能是至關(guān)重要的,單獨的Rpf結(jié)構(gòu)域具有與Rpf蛋白一致的生物學(xué)功能。進(jìn)一步的研究發(fā)現(xiàn),Rpf結(jié)構(gòu)域中都有一個高度保守的谷氨酸殘基(glu54),為Rpf活性所必需,與溶菌酶催化活性相符。 實驗?zāi)康? 利用大腸桿菌表達(dá)系統(tǒng),表達(dá)并純化出M. luteus Rpf結(jié)構(gòu)域及其突變體蛋白,并進(jìn)一步研究其生物學(xué)活性。 實驗方法和結(jié)果: 1. Rpf結(jié)構(gòu)域及其突變體蛋白的表達(dá)及純化 采用聚合酶鏈反應(yīng)(PCR)從M. luteus基因組中擴增出Rpf結(jié)構(gòu)域及其E54A和E54K兩種突變體基因,用限制性內(nèi)切酶消化后克隆入pMD-18T載體中,經(jīng)測序證實,與GenBank公布的序列完全相同,突變位點與設(shè)定一致。將測序正確的基因分別亞克隆到pGEX-4T-1表達(dá)載體中,酶切鑒定陽性重組質(zhì)粒,轉(zhuǎn)化大腸桿菌,并用IPTG進(jìn)行誘導(dǎo)表達(dá)。SDS-PAGE分析表明成功地表達(dá)了Rpf結(jié)構(gòu)域及其突變體融合蛋白,用Rpf結(jié)構(gòu)域單克隆抗體(mAb)進(jìn)行Western-blot分析證實,其大小為32kD,與預(yù)計的融合蛋白大小相符。用GS-4B親和色譜柱純化獲得了目的蛋白。 2. Rpf結(jié)構(gòu)域及其突變體融合蛋白生物學(xué)活性的檢測 將恥垢分枝桿菌(Mycobacterium smegmatis,M.S)休眠菌以1:150的比例稀釋。把各種融合蛋白按照不同的濃度加入到M.S休眠菌中,并分別用相應(yīng)的抗體進(jìn)行干預(yù)。37℃振蕩培養(yǎng),每4 h取少量培養(yǎng)液測A600nm值,根據(jù)測定結(jié)果繪制生長曲線圖。結(jié)果表明:Rpf結(jié)構(gòu)域蛋白對M.S具有促進(jìn)復(fù)蘇和促生長的作用,使M.S的培養(yǎng)周期縮短。而且Rpf結(jié)構(gòu)域蛋白具有和Rpf蛋白相同的生物學(xué)活性。E54K突變后的Rpf蛋白對于M.S的生長有一定的抑制作用,而E54A無明顯的抑制生長作用。 結(jié)論: 獲得了Rpf結(jié)構(gòu)域及其兩種突變體蛋白E54K和E54A,Rpf結(jié)構(gòu)域蛋白與完整的Rpf蛋白一樣對M.S具有一定的復(fù)蘇和促生長作用;而加入Rpf及Rpf結(jié)構(gòu)域抗體后,其作用被抵消,使M.S維持正常生長。E54K突變體對于休眠的M.S的生長有一定的抑制作用。E54A對于休眠的M.S無明顯的抑制作用。這些結(jié)果為研究Rpf結(jié)構(gòu)域突變體在抑制M. luteus的生長,進(jìn)而研究其對MTB生長的影響,以及Rpf結(jié)構(gòu)域和其突變體抗體在TB預(yù)防及臨床快速診斷中的作用奠定基礎(chǔ)。
[Abstract]:Tuberculosis (Tuberculosis, TB) by Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) caused by, on respiratory system infection of infectious diseases. In recent years, due to BCG (BCG) protection is not perfect, the emergence of drug-resistant MTB strains, and to increase the prevalence of HIV / AIDS population movements and other factors, makes the TB the incidence is increasing, harm to human health worldwide disease.BCG is currently the only vaccine against TB become again, but it can not effectively prevent adult TB, therefore, study on the pathogenesis and immunity of TB positive, for the development of more effective diagnosis, prevention and treatment of TB new technology has important significance.
Micrococcus luteus (M.luteus) in the resuscitation promoting factor (Resuscitation-promoting factor, Rpf) is the first discovered bacterial growth factor, it can promote the recovery and promote the role of dormant bacteria, bacteria grow fast. Sequence analysis found that in some other gram positive bacteria such as MTB, Mycobacterium leprae, Streptomyces coelicolor more than 30, homologous with Rpf protein also exists in Corynebacterium glutamicum. Research found that MTB genome encoding 5 Rpf proteins, respectively Rv0867C (Rpf A), Rv1009 (Rpf B), Rv1884C (Rpf C), Rv2389C (Rpf D) and Rv2450C (Rpf E), suggested that these proteins MTB has a role in promoting rapid growth. In addition, these proteins are Rpf like domain that is highly conserved 70AA residues, conserved region of Rpf is crucial to the recovery of the function of the single Rpf domain is consistent with the biology of Rpf protein Further studies revealed that a highly conserved glutamic acid residue (glu54) in the Rpf domain is necessary for Rpf activity, which is consistent with lysozyme catalytic activity.
Objective:
The M. luteus Rpf domain and its mutant protein were expressed and purified by the Escherichia coli expression system, and its biological activity was further studied.
Experimental methods and results:
Expression and purification of 1. Rpf domain and its mutant protein
Polymerase chain reaction (PCR) amplified Rpf domain and its E54A and E54K two kinds of mutant gene from M. luteus genome, with restriction endonuclease digestion and cloned into pMD-18T vector and confirmed by sequencing, identical with the published sequences of GenBank mutation and set. Then the correct sequence of the gene were sub cloned into pGEX-4T-1 expression vector, enzyme digestion of recombinant plasmid was transformed into Escherichia coli, and induced expression of.SDS-PAGE analysis showed that the successful expression of the Rpf domain and its mutants with IPTG fusion protein, Rpf domain monoclonal antibody (mAb) Western-blot analysis confirmed that its size is 32kD, with the expected protein with the size of fusion. Purified the fusion protein by GS-4B affinity.
Detection of biological activity of 2. Rpf domain and its mutant fusion protein
The Mycobacterium smegmatis (Mycobacterium smegmatis M.S) dormant bacteria in 1:150 dilution ratio. The fusion protein with different concentrations of M.S added to the dormant bacteria, respectively and the corresponding antibody intervention.37 C shaking culture, every 4 h to take a small amount of medium A600nm value, according to the determination results of growth curve figure. The results show that the Rpf domain protein can promote the recovery and growth promoting effect on M.S, the M.S and shorten the training cycle. Rpf domain protein has the same biological activity of.E54K and Rpf protein mutation Rpf protein has certain inhibitory effect on the growth of M.S, while E54A had no obvious growth inhibition.
Conclusion:
The Rpf domain and its two mutants E54K and E54A, Rpf domain and complete Rpf protein of M.S has a certain recovery and growth promoting effect; and adding Rpf and Rpf domain antibodies, its role is to offset, M.S to maintain normal growth of.E54K mutant for M.S with dormancy the inhibitory effect of.E54A for dormant M.S had no obvious inhibitory effect. These results for the study of Rpf domain mutants in M. suppressed the growth of luteus, and to study its effects on the growth of MTB, lay the foundation and function of Rpf domain and its mutant antibody in TB prevention and clinical rapid diagnosis.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R378

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 趙明君;海洋弧菌復(fù)蘇促進(jìn)因子家族糖蛋白酶的研究[D];中國海洋大學(xué);2011年

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本文編號：1380762