三个LipidA合成酶基因的克

发布时间：2018-11-13 20:17

【摘要】：布鲁氏菌脂多糖(B.melitensis LPS, B-LPS)是布鲁氏菌细胞壁的主要成分,它主要由O-抗原、核心多糖和类脂A (LipidA)三部分组成。LipidA是B-LPS的疏水基团,可以将亲水性的核心多糖和O-抗原黏附在细菌的外表面构成完整的细胞壁；同时LipidA也是内毒素的活性成分,对B-LPS结构和功能完整性是必不可少的。LipidA的合成是在一系列酶的催化作用下完成的,参与LipidA合成的9种酶及其编码基因在大部分的革兰氏阴性细菌中都存在。Caspase-4(Casp4)属于炎性Caspases家族的一员,在炎症反应的发生上扮演重要角色,它是B-LPS的胞内天然免疫受体,在大多数正常的组织中表达水平很低。本研究第二章对B-LPS LipidA合成有关的三个重要催化酶的基因LpxC、LpxK和KdtA进行了克隆、原核表达、生物信息学分析及序列比对。结果表明：成功对这三个基因进行了克隆和原核表达,通过生物信息学分析,初步了解了这三个催化酶的二级结构和疏水区；对不同brucella种属的这三个基因进行了同源性分析,发现这三个基因序列相对保守,在各种属间同源性较高。通过这部分的研究,能帮助我们更好的了解B-LPS LipidA的合成过程,由于LipidA是B-LPS的活性成分,因此为进一步研究B-LPS的功能奠定了基础。本研究第三章用不同刺激浓度和刺激时间的B-LPS刺激RAW264.7细胞,对Casp4蛋白表达以及对IL-1β、TNF-a的分泌情况进行了分析。结果表明：B-LPS刺激能引起RAW264.7细胞Casp4蛋白表达上调,同时还能引起IL-1β、TNF-α分泌增加。本研究第四章应用小干扰RNA(Small interfering RNA, siRNA)抑制B-LPS刺激条件下Casp4蛋白的表达,应用Western blot方法检测基因沉默效果,通过对转染条件的反复筛选,确定了最适合的转染条件；应用酶联免疫吸附法检测转染后细胞因子分泌水平的变化。结果表明,在B-LPS刺激条件下,siRNA能有效抑制Casp4蛋白的表达；B-LPS刺激条件下,通过siRNA使Casp4knockdown, RAW2647细胞对IL-1β和TNF-α分泌量减少。这些试验结果为研究通过抑制上游的炎症通路从而减弱损伤性炎症反应的实验设想提供了可能,同时为探究布鲁氏菌的致病机理提供了重要依据。
[Abstract]:Brucella lipopolysaccharide (B.melitensis LPS, B-LPS) is the main component of brucella cell wall, which consists of three parts: O- antigen, core polysaccharide and lipopolysaccharide. LipidA is the hydrophobic group of B-LPS. Hydrophilic core polysaccharides and O- antigens can be adhered to the outer surface of the bacteria to form a complete cell wall; At the same time, LipidA is also an active component of endotoxin, which is essential to the structural and functional integrity of B-LPS. The synthesis of LipidA is completed under the catalysis of a series of enzymes. The nine enzymes involved in LipidA synthesis and their coding genes are present in most gram-negative bacteria. Caspase-4 (Casp4) is a member of the inflammatory Caspases family and plays an important role in the pathogenesis of inflammation. It is the intracellular innate immune receptor of B-LPS and its expression level is very low in most normal tissues. In the second chapter, the genes LpxC,LpxK and KdtA of three important catalytic enzymes related to B-LPS LipidA synthesis were cloned, prokaryotic expression, bioinformatics analysis and sequence alignment. The results showed that the three genes were cloned and expressed successfully. The secondary structure and hydrophobic region of the three catalytic enzymes were preliminarily understood by bioinformatics analysis. The homology analysis of these three genes in different brucella species showed that the three genes were relatively conserved and had high homology among different genera. Through this part of the study, can help us to better understand the synthesis process of B-LPS LipidA, because LipidA is the active component of B-LPS, so for the further study of the function of B-LPS laid a foundation. In the third chapter, the expression of Casp4 protein and the secretion of IL-1 尾, TNF-a were analyzed by B-LPS stimulation with different concentration and time. The results showed that B-LPS stimulation could up-regulate the expression of Casp4 protein and increase the secretion of IL-1 尾 and TNF- 伪 in RAW264.7 cells. In the fourth chapter, small interfering RNA (Small interfering RNA, siRNA) was used to inhibit the expression of Casp4 protein under B-LPS stimulation, and Western blot method was used to detect the gene silencing effect. The most suitable transfection conditions were determined by repeated screening of transfection conditions. Enzyme linked immunosorbent assay (Elisa) was used to detect cytokine secretion after transfection. The results showed that siRNA could effectively inhibit the expression of Casp4 protein under the condition of B-LPS stimulation, and under the condition of B-LPS stimulation, the secretion of IL-1 尾 and TNF- 伪 from Casp4knockdown, RAW2647 cells could be reduced by siRNA. These results provide the possibility to study the experimental assumption of attenuating the injurious inflammatory response by inhibiting the upstream inflammatory pathway, and provide an important basis for exploring the pathogenic mechanism of Brucella.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

【相似文献】