本文選題：鐵氧還蛋白 + 原核表達��； 參考：《蘭州理工大學》2017年碩士論文

【摘要】：嗜酸氧化亞鐵硫桿菌是一種應(yīng)用性很強的工業(yè)微生物菌種,鑒于其較好的重金屬抗性,可用于貴金屬的浸提、環(huán)境污染的治理等領(lǐng)域。鐵氧還蛋白作為一種廣泛存在并參與嗜酸氧化亞鐵硫桿菌新陳代謝過程諸如鐵硫簇組裝等重要環(huán)節(jié)的蛋白質(zhì),研究其結(jié)構(gòu)和功能對了解嗜酸氧化亞鐵硫桿菌的代謝過程、電子傳遞及重金屬抗性具有重要意義。本研究的內(nèi)容和結(jié)果如下:(1)從甘肅省蘭州市、白銀市和隴南市3個典型礦區(qū)采集的樣品中分離出四株嗜酸氧化亞鐵硫桿菌,一株氧化亞鐵鉤端螺旋菌。選取生長周期短、菌體富集量大的菌株BYSW1進行后續(xù)的研究。首先選取溫度、pH值、接種量、能源種類利用單因素實驗對其生長條件進行優(yōu)化。在pH為2,于30℃下接種10%的菌液,BYSW1生長最好,葡萄糖和蛋白胨會明顯抑制其生長。(2)在NCBI數(shù)據(jù)庫中找出鐵氧還蛋白基因序列(fdⅠ、fdⅡ),在BYSW1中擴增出目的片段,連接到pET-32(a),轉(zhuǎn)化大腸桿菌(Escherichia coli,E.coli)DH5α。自BYSW1擴增出的鐵氧還蛋白基因序列(fdⅠ、fdⅡ)與數(shù)據(jù)庫匯總的序列一致性分別為83.92%和85.59%,編碼的蛋白與數(shù)據(jù)庫中的序列一致性分別為89.47%和85.59%。(3)重組的質(zhì)粒轉(zhuǎn)化表達宿主E.coli BL21(DE3)。選取溫度、時間、IPTG濃度進行單因素實驗,優(yōu)化重組蛋白的表達條件,并用蛋白質(zhì)印記法進行驗證。用鎳離子親和層析柱對重組蛋白進行純化。FdⅠ和FdⅡ分別在25℃下加入0.5 mM IPTG誘導6 h、25℃下加入0.3 mM IPTG誘導6 h表達效果最佳。FdⅠ和FdⅡ分別可用100 mM咪唑和50 mM咪唑自親和柱中洗脫下來。(4)挑取五種重金屬(Cd2+、Cu2+、Mn2+、Ni2+、Zn2+),對其重金屬抗性進行研究;對重金屬(Cu2+和Cd2+)馴化前后的嗜酸氧化亞鐵硫桿菌的菌體形態(tài)和基因組變化、重組蛋白表達差異、宿主菌體的生長情況進行了研究。結(jié)果表明:BYSW1具有較強的重金屬抗性,分別在0.04 mol/L Cd2+、0.04 mol/L Cu2+、0.15 mol/L Mn2+、0.08 mol/L Ni2+和0.08 mol/L Zn2+存在時生長良好,但是高濃度(大于0.04 mol/L)的Cu2+和Cd2+會明顯抑制BYSW1的生長。Cu2+和Cd2+脅迫后,BYSW1的菌體細胞明顯變小,且表面粗糙,但基因組仍然能保持完整。不同濃度、不同種類的重金屬脅迫前后,鐵氧還蛋白的表達存在顯著差異,且Cd2+對嗜酸氧化亞鐵硫桿菌生長的影響比Cu2+顯著;不同濃度、不同種類的重金屬對于重組蛋白的表達宿主菌體生長影響不同,0.25 m M的Cu2+和Cd2+能明顯抑制宿主菌體的生長。
[Abstract]:Thiobacillus ferrooxidans acidophilus is a kind of industrial microorganism with strong application. In view of its good resistance to heavy metals, it can be used in the extraction of precious metals and the treatment of environmental pollution. Ferredoxin is a widely existed protein involved in metabolic processes of Thiobacillus ferrooxidans, such as the assembly of iron-sulfur clusters. The structure and function of ferredoxin are studied to understand the metabolic process of Thiobacillus ferrooxidans. Electron transport and heavy metal resistance are important. The contents and results of this study are as follows: (1) four strains of Thiobacillus ferrooxidans and one strain of Heptospira ferrooxidans were isolated from three typical mining areas in Lanzhou, Baiyin and Longnan of Gansu Province. The strain BYSW1 with short growth cycle and high concentration of bacteria was selected for further study. The temperature pH value, inoculation amount and energy type were selected to optimize the growth conditions by single factor experiment. The growth of BYSW1 was best when inoculated at 30 鈩，

本文編號：1863260