本文選題：鉤端螺旋體 + mviN基因　； 參考：《四川大學(xué)》2007年碩士論文

【摘要】： 鉤端螺旋體(Leptospira)是系統(tǒng)發(fā)育進(jìn)化上結(jié)構(gòu)和遺傳特征都較特殊的一類微生物，它所導(dǎo)致的鉤端螺旋體病是在世界范圍內(nèi)流行的人獸共患自然疫源性疾病。問(wèn)號(hào)鉤端螺旋體黃疸出血群賴型56601株全基因組測(cè)序于2003年由中國(guó)科學(xué)家完成，對(duì)鉤端螺旋體的研究重點(diǎn)將轉(zhuǎn)向功能蛋白組學(xué)研究。鉤端螺旋體與宿主相互作用是鉤端螺旋體致病機(jī)制的關(guān)鍵環(huán)節(jié)。因此，研究與鉤端螺旋體粘附侵襲相關(guān)的毒力因子的功能，對(duì)闡明鉤端螺旋體致病機(jī)制和免疫機(jī)理具有重要意義。 通過(guò)全基因組生物信息學(xué)分析發(fā)現(xiàn)，mviN基因是個(gè)粘附侵襲毒力基因，存在于許多致病微生物中。鉤端螺旋體mviN基因讀碼框含有1596個(gè)核苷酸序列，編碼531個(gè)氨基酸，表達(dá)產(chǎn)物是一跨膜蛋白。 本研究以問(wèn)號(hào)鉤端螺旋體黃疸出血群賴型017株基因組為實(shí)驗(yàn)材料，PCR擴(kuò)增出含完整讀碼框的mviN基因，與原核表達(dá)質(zhì)粒pET32a(+)連接構(gòu)建重組表達(dá)質(zhì)粒pET-mviN，在大腸桿菌BL21(DE3)中誘導(dǎo)表達(dá)。以全菌體總蛋白作SDS-PAGE，并以兔抗全鉤端螺旋體多價(jià)血清為一抗，以HRP標(biāo)記羊抗兔IgG為二抗作Western-blot分析。結(jié)果顯示成功表達(dá)了分子量約為80kD的MviN融合蛋白。MviN融合蛋白在菌體內(nèi)主要以可溶性方式表達(dá)，經(jīng)親和層析純化，獲得了高純度的MviN融合蛋白。 進(jìn)一步構(gòu)建了mviN基因與pcDNA3.1(+)的真核重組表達(dá)質(zhì)粒pcDNA3.1-mviN；利用脂質(zhì)體介導(dǎo)pcDNA3.1-mviN轉(zhuǎn)染COS7細(xì)胞，，并以RT-PCR法檢測(cè)其表達(dá)情況。 以MviN融合蛋白作用于A549和ECV304細(xì)胞，觀察該蛋白對(duì)細(xì)胞增殖的影響作用。結(jié)果顯示，MviN融合蛋白顯著抑制這兩種細(xì)胞的增殖，且該效應(yīng)隨著MviN融合蛋白濃度升高而越明顯。還應(yīng)用流式細(xì)胞術(shù)檢測(cè)方法，觀察了MviN融合蛋白對(duì)ECV304及A549細(xì)胞凋亡的作用，實(shí)驗(yàn)結(jié)果表明MviN融合蛋白能促進(jìn)細(xì)胞凋亡。 本研究初步闡明了mviN基因在鉤端螺旋體致病機(jī)制和免疫機(jī)理方面的作用，為進(jìn)一步研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R377.5

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