殊异韦荣菌LDH重组蛋白的表达、纯化及活性分析
发布时间：2019-05-14 21:44
【摘要】：韦荣菌(Veillonella)是一种革兰氏阴性厌氧小球菌,主要寄生在动物与人类的口腔、肠道以及呼吸道中,具有一定的耐酸性,可以在酸性环境下生长。 韦荣菌在菌斑中所扮演的角色非常微妙,可以将酸性较强的乳酸转变成弱酸,而在糖的催化下又可使乳酸大量生成。具有这一功能的是韦荣菌中的乳酸脱氢酶( lactate dehydrogenase, LDH),是细菌的固有酶。它以是否依赖NAD(尼克酰胺腺嘌呤二核苷酸)分为依赖性LDH(nLDH)和非依赖性LDH(iLDH)。而行使这一功能的则是nLDH。其基因的具体功能及作用机制尚不清楚。但可以肯定的是韦荣菌与菌斑的生长以及龋病的发生有着密切的关联,因此有必要继续对韦荣菌深入研究。 前期试验中已克隆了殊异韦荣菌乳酸脱氢酶的基因,构建了pET-28a-LDH,进行了重组蛋白的初步表达。该基因序列现已登陆美国GenBank,序列号为EU518464。 本实验将已经构建好的pET-28a-LDH转入BL21(DE3)和Rosetta(DE3)中,分别进行了高效表达,并且对比其表达量。BandScan5.0扫描分析,pET-28a-LDH质粒转化BL21(DE3)表达量明显高于Rosetta(DE3)。后将目的蛋白进行性质鉴定,SDS-PAGE电泳显示,在上清的约33KD处见明显蛋白表达条带。经HisTRAP FF柱提纯后,用南京建成生物技术公司LDH活性试剂盒测定蛋白有活性。为从生物学角度继续深入探索研究韦荣菌乳酸脱氢酶奠定基础,从而寻找防龋的新途径。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378
本文编号：2477075
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378
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