赖型钩端螺旋体毒力基因mviN的克隆表达及功能研究

发布时间：2018-06-15 01:13

本文选题：钩端螺旋体 + mviN基因　；参考：《四川大学》2007年硕士论文

【摘要】： 钩端螺旋体(Leptospira)是系统发育进化上结构和遗传特征都较特殊的一类微生物，它所导致的钩端螺旋体病是在世界范围内流行的人兽共患自然疫源性疾病。问号钩端螺旋体黄疸出血群赖型56601株全基因组测序于2003年由中国科学家完成，对钩端螺旋体的研究重点将转向功能蛋白组学研究。钩端螺旋体与宿主相互作用是钩端螺旋体致病机制的关键环节。因此，研究与钩端螺旋体粘附侵袭相关的毒力因子的功能，对阐明钩端螺旋体致病机制和免疫机理具有重要意义。通过全基因组生物信息学分析发现，mviN基因是个粘附侵袭毒力基因，存在于许多致病微生物中。钩端螺旋体mviN基因读码框含有1596个核苷酸序列，编码531个氨基酸，表达产物是一跨膜蛋白。本研究以问号钩端螺旋体黄疸出血群赖型017株基因组为实验材料，PCR扩增出含完整读码框的mviN基因，与原核表达质粒pET32a(+)连接构建重组表达质粒pET-mviN，在大肠杆菌BL21(DE3)中诱导表达。以全菌体总蛋白作SDS-PAGE，并以兔抗全钩端螺旋体多价血清为一抗，以HRP标记羊抗兔IgG为二抗作Western-blot分析。结果显示成功表达了分子量约为80kD的MviN融合蛋白。MviN融合蛋白在菌体内主要以可溶性方式表达，经亲和层析纯化，获得了高纯度的MviN融合蛋白。进一步构建了mviN基因与pcDNA3.1(+)的真核重组表达质粒pcDNA3.1-mviN；利用脂质体介导pcDNA3.1-mviN转染COS7细胞，，并以RT-PCR法检测其表达情况。以MviN融合蛋白作用于A549和ECV304细胞，观察该蛋白对细胞增殖的影响作用。结果显示，MviN融合蛋白显著抑制这两种细胞的增殖，且该效应随着MviN融合蛋白浓度升高而越明显。还应用流式细胞术检测方法，观察了MviN融合蛋白对ECV304及A549细胞凋亡的作用，实验结果表明MviN融合蛋白能促进细胞凋亡。本研究初步阐明了mviN基因在钩端螺旋体致病机制和免疫机理方面的作用，为进一步研究奠定了基础。
[Abstract]:Leptospirae (Leptospiraa) is a kind of microorganism with special structural and genetic characteristics in phylogenetic evolution. Leptospirosis caused by Leptospirae is a zoonotic natural epidemic disease in the world. The complete genome sequencing of 56601 Leptospira icterus haemorrhagic Leptospira strains was completed by Chinese scientists in 2003. The focus of the study on Leptospira will be turned to functional proteomics. The interaction between leptospirosis and host is a key link in the pathogenesis of leptospira. Therefore, it is of great significance to study the function of virulence factors related to the adhesion and invasion of leptospirosis in order to elucidate the pathogenesis and immune mechanism of leptospira. Through the whole genome bioinformatics analysis, it was found that the MviN gene was a virulence gene of adhesion and invasion, and existed in many pathogenic microorganisms. The reading frame of leptospira mviN gene contains 1596 nucleotide sequences and encodes 531 amino acids. The expressed product is a transmembrane protein. In this study, MviN gene with complete reading frame was amplified by PCR from the genome of Leptospira interrogans icterohaemorrhagic group 017 strain. The recombinant expression plasmid pET-mviN was constructed by ligating with prokaryotic expression plasmid pET32a(), and was induced to be expressed in E. coli BL21 (DE3). The total bacterial protein was used as SDS-PAGE, the rabbit anti-leptospira polyvalent serum was used as the first antibody, and the HRP-labeled goat anti-rabbit IgG was used as the second antibody for Western-blot analysis. The results showed that the MviN fusion protein with molecular weight of about 80 KD was expressed mainly in soluble way in the bacteria and purified by affinity chromatography to obtain a high purity MviN fusion protein. The recombinant eukaryotic expression plasmid pcDNA3.1-mviN of mviN gene and pcDNA3.1 () was constructed and transfected into COS7 cells by liposome-mediated pcDNA3.1-mviN, and the expression of pcDNA3.1-mviN was detected by RT-PCR. The effect of MviN fusion protein on the proliferation of A549 and ECV304 cells was observed. The results showed that MviN fusion protein significantly inhibited the proliferation of these two cells, and the effect increased with the concentration of MviN fusion protein. The effect of MviN fusion protein on apoptosis of ECV304 and A549 cells was also studied by flow cytometry. The results showed that MviN fusion protein could promote apoptosis of ECV304 and A549 cells. The role of MviN gene in pathogenesis and immune mechanism of Leptospira was elucidated in this study, which laid a foundation for further study.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R377.5

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