【摘要】： 傷寒是一種急性全身性感染，包括傷寒沙門氏菌(Salmonella typhi)引起的傷寒和甲、乙、丙型副傷寒沙門氏菌(Salmonella paratyphi A，B and C)引起的副傷寒。甲型副傷寒沙門氏菌是甲型副傷寒的病原菌，通過糞—口途徑傳播，感染劑量為10~3～10~9，人是唯一宿主。人群對(duì)甲型副傷寒沙門氏菌普遍易感，但兒童和青壯年發(fā)病最高�，F(xiàn)在據(jù)估計(jì)每年全球傷寒(包括副傷寒)患者約2200萬，死亡超過20萬人。自1996年以來，我國的甲型副傷寒的發(fā)病率也在逐年升高。更為嚴(yán)重的是，甲型副傷寒沙門氏菌臨床耐藥株以及多重耐藥株(MDR)已經(jīng)相當(dāng)廣泛，使臨床治療變得更加困難，一些患者由于治療不徹底而成為帶菌者。因此，需要研究出安全、高效、簡便、經(jīng)濟(jì)的疫苗來預(yù)防甲型副傷寒。 免疫蛋白質(zhì)組學(xué)是通過雙向電泳分離病原蛋白質(zhì)組，再用不同病人的超免血清鑒定出有強(qiáng)免疫性的蛋白，這一蛋白質(zhì)組的分支，很有希望成為尋找藥物和疫苗靶位的有效途徑(因?yàn)樽C明這些蛋白能引起宿主的免疫應(yīng)答)。甲型副傷寒沙門氏菌是無莢膜的革蘭氏陰性菌，其外膜蛋白在致病和刺激機(jī)體免疫應(yīng)答方面有非常重要的作用。因?yàn)榧?xì)菌的膜蛋白(尤其是外膜蛋白)抗原，往往是一些反應(yīng)性抗原，它們與機(jī)體免疫系統(tǒng)有十分密切的相互作用。因此，在疫苗研究中，病原體的表面蛋白具有很重要的地位。 本實(shí)驗(yàn)提取了甲型副傷寒沙門氏菌的外膜蛋白，建立了甲型副傷寒沙門氏菌的外膜蛋白雙向電泳參考圖譜。在考馬斯亮蘭染色的膠上共取了80個(gè)外膜蛋白點(diǎn)，鑒定出61個(gè)，代表30種蛋白。結(jié)合根據(jù)基因組預(yù)測的外膜蛋白對(duì)鑒定的所有蛋白質(zhì)點(diǎn)的等電點(diǎn)/分子量吻合程度、分布以及功能分類等進(jìn)行了初步的分析。進(jìn)一步采用免疫蛋白質(zhì)組的方法，對(duì)提取細(xì)菌的外膜蛋白進(jìn)行雙向電泳分離后，再轉(zhuǎn)移到PVDF膜上，與甲型副傷寒病人恢復(fù)期的血清進(jìn)行免疫印跡反應(yīng)，記錄具有免疫反應(yīng)的外膜蛋白點(diǎn)。在考馬斯亮蘭染色的膠上取外膜蛋白點(diǎn)以及有免疫反應(yīng)的蛋白點(diǎn)，酶切后進(jìn)行MALDI-TOF質(zhì)譜鑒定。在PVDF膜上共有45個(gè)免疫反應(yīng)點(diǎn)，，鑒定到42個(gè)點(diǎn)，代表23種蛋白質(zhì)抗原，采用psort(www.psort.org)軟件分析，外膜蛋白13種，胞質(zhì)蛋白3種，定位不明確(可能是多處定位)1種，未知定位6種。這些蛋白有希望成為藥靶或疫苗的候選成分。
[Abstract]:Typhoid fever is an acute systemic infection, including typhoid fever caused by (Salmonella typhi) and paratyphoid fever caused by (Salmonella paratyphi A B and C) of Salmonella typhimurium. Salmonella paratyphoid A is the pathogen of paratyphoid A. Paratyphoid A is most susceptible to salmonella A in the population, but it is the highest in children and young adults. It is now estimated that about 22 million people worldwide suffer from typhoid fever (including paratyphoid fever) each year, with more than 200000 deaths. Since 1996, the incidence of paratyphoid A in China has also been increasing year by year. More seriously, the clinical resistant strains and multidrug resistant strains of Salmonella paratyphi A (MDR) have been widely used, which makes clinical treatment more difficult, and some patients become carriers because of incomplete treatment. Therefore, a safe, efficient, simple and economical vaccine is needed to prevent paratyphoid A (paratyphoid A). Immunoproteomics is the separation of pathogenic proteome by two-dimensional electrophoresis, and then the identification of strongly immunized proteins with the superimmune serum of different patients, a branch of the proteome. It is promising to be an effective way to target drugs and vaccines (as these proteins have been shown to elicit host immune responses). Salmonella paratyphoid A is a gram-negative bacterium without capsule, and its outer membrane protein plays an important role in pathogenic and stimulating immune response. Because bacterial membrane proteins (especially outer membrane proteins) antigens are often reactive antigens, they interact very closely with the immune system. Therefore, surface proteins of pathogens play an important role in vaccine research. The outer membrane protein of Salmonella paratyphi A was extracted and the reference map of the outer membrane protein of Salmonella paratyphi A was established. A total of 80 outer membrane protein spots were extracted from Coomassie brilliant blue staining glue and 61 proteins representing 30 proteins were identified. The isoelectric point / molecular weight coincidence degree, distribution and functional classification of all the identified protein spots were preliminarily analyzed in combination with the outer membrane protein predicted by the genome. The outer membrane protein of the extracted bacteria was separated by two dimensional electrophoresis and then transferred to the PVDF membrane. The protein was imprinted with the sera of paratyphoid A patients during the convalescence period. The outer membrane protein spots with immune response were recorded. The outer membrane protein spots and immunoreactive protein spots were extracted from Coomassie brilliant blue stained glue and identified by MALDI-TOF mass spectrometry after enzyme digestion. A total of 45 immunoreactive sites were identified on PVDF membrane, representing 23 protein antigens. Psort (www.psort.org) software was used to analyze 13 kinds of outer membrane proteins, 3 kinds of cytoplasmic proteins, 1 species of unclear localization (possibly multiple loci) and 6 kinds of unknown localization. These proteins are expected to be candidates for drug targets or vaccines.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

【引證文獻(xiàn)】

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2 王斌;李娜;董曉宇;梁昊宇;陳翠萍;陳薇;曾明;;甲型副傷寒沙門菌外膜BtuB蛋白的原核表達(dá)、純化及其免疫保護(hù)性[J];中國生物制品學(xué)雜志;2012年06期

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1 楊艷玲;羊布魯氏菌蛋白質(zhì)組學(xué)分析及免疫候選抗原的篩選與鑒定[D];吉林大學(xué);2011年

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