【摘要】：韋榮菌(Veillonella)是一種革蘭氏陰性厭氧小球菌,主要寄生在動物與人類的口腔、腸道以及呼吸道中,具有一定的耐酸性,可以在酸性環(huán)境下生長。 韋榮菌在菌斑中所扮演的角色非常微妙,可以將酸性較強的乳酸轉變成弱酸,而在糖的催化下又可使乳酸大量生成。具有這一功能的是韋榮菌中的乳酸脫氫酶( lactate dehydrogenase, LDH),是細菌的固有酶。它以是否依賴NAD(尼克酰胺腺嘌呤二核苷酸)分為依賴性LDH(nLDH)和非依賴性LDH(iLDH)。而行使這一功能的則是nLDH。其基因的具體功能及作用機制尚不清楚。但可以肯定的是韋榮菌與菌斑的生長以及齲病的發(fā)生有著密切的關聯(lián),因此有必要繼續(xù)對韋榮菌深入研究。 前期試驗中已克隆了殊異韋榮菌乳酸脫氫酶的基因,構建了pET-28a-LDH,進行了重組蛋白的初步表達。該基因序列現(xiàn)已登陸美國GenBank,序列號為EU518464。 本實驗將已經構建好的pET-28a-LDH轉入BL21(DE3)和Rosetta(DE3)中,分別進行了高效表達,并且對比其表達量。BandScan5.0掃描分析,pET-28a-LDH質粒轉化BL21(DE3)表達量明顯高于Rosetta(DE3)。后將目的蛋白進行性質鑒定,SDS-PAGE電泳顯示,在上清的約33KD處見明顯蛋白表達條帶。經HisTRAP FF柱提純后,用南京建成生物技術公司LDH活性試劑盒測定蛋白有活性。為從生物學角度繼續(xù)深入探索研究韋榮菌乳酸脫氫酶奠定基礎,從而尋找防齲的新途徑。
[Abstract]:Violet (Veillonella) is a Gram-negative anaerobes, which is mainly parasitic in the oral, intestinal and respiratory tract of animals and humans. It has certain acid resistance and can grow in acidic environment. The role of Weirong bacteria in plaque is very subtle, which can transform strong acidic lactic acid into weak acid, and can produce a large number of lactic acid under the catalysis of sugar. The lactic dehydrogenase (lactate dehydrogenase, LDH), in Verunculus is the inherent enzyme of bacteria. It is divided into dependent LDH (nLDH) and independent LDH (iLDH). By whether or not it is dependent on NAD (Nick Amine adenine dinucleotides). And it's nLDH. that performs this function. The specific function and mechanism of its gene are not clear. However, it is certain that Verong is closely related to the growth of plaque and the occurrence of caries, so it is necessary to continue the further study of Verunculus. In the previous experiment, the gene of lactic dehydrogenase from Vernon was cloned, and pET-28a-LDH, was constructed to express the recombinant protein. The gene sequence has now been logged into the United States. GenBank, sequence number is EU518464.. In this experiment, the constructed pET-28a-LDH was transferred into BL21 (DE3) and Rosetta (DE3), and the expression level was compared. BandScan 5.0 scanning analysis. The expression of BL21 (DE3) transformed with pET-28a-LDH plasmid was significantly higher than that of Rosetta (DE3). After that, the properties of the target protein were identified. SDS-PAGE electrophoresis showed that the protein expression band was obvious at about 33KD in the upper serum. After purification by HisTRAP FF column, the protein activity was determined by LDH activity kit of Nanjing Biotechnology Company. It lays a foundation for the further study of lactic dehydrogenase from the biological point of view, so as to find a new way to prevent caries.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R378

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