【摘要】： 目的:建立及優(yōu)化日本血吸蟲成蟲的蛋白質(zhì)組學(xué)分析方法,尋找可溶性蛋白組分中與免疫應(yīng)答相關(guān)的特異性成蟲抗原。方法:純化日本血吸蟲成蟲可溶性蛋白,應(yīng)用雙向電泳(2D-E)結(jié)合免疫印跡技術(shù),獲得相應(yīng)的電泳圖譜和免疫印跡圖譜,應(yīng)用PDQuest雙向電泳圖像分析軟件對圖像進(jìn)行分析比對,分析獲得蛋白的理論分子量(Mr)和等電點(pI)等信息,標(biāo)定特異性的抗原蛋白點;從中選取了30個蛋白質(zhì)點,膠內(nèi)胰蛋白酶酶切成多肽,利用LC-MS/MS質(zhì)譜分析技術(shù)進(jìn)行分析,并用SEQUEST軟件搜索Schistosoma的非冗余蛋白質(zhì)序列數(shù)據(jù)庫。成功獲得了24個蛋白斑點的肽指紋圖譜(PMF)及肽序列檢測數(shù)據(jù),分屬18種蛋白,運用生物信息學(xué)等技術(shù)在線分析這些蛋白的結(jié)構(gòu)和功能。結(jié)果:日本血吸蟲成蟲可溶性蛋白經(jīng)雙向電泳后,考馬斯亮藍(lán)R250染色,電泳圖譜顯示了約152個主要蛋白斑點。其分子量分布約為14 kDa～134 kDa,pI主要集中在4.94～9.50。進(jìn)一步用Western blotting鑒定,結(jié)果顯示實驗組圖像可見的抗原抗體反應(yīng)點數(shù)目約為57個,對照組為0個;對選定的蛋白質(zhì)點進(jìn)行脫色和膠內(nèi)原位消化、酶解。酶解后的肽混合物經(jīng)LC-MS/MS分析,獲得肽質(zhì)量指紋圖,該圖質(zhì)量較高,峰信號較強(qiáng),以m/z=200～2000間的片段峰較多,從獲得的數(shù)據(jù)中解析出多肽序列信息,再通過數(shù)據(jù)庫檢索確定相應(yīng)的蛋白質(zhì);通過Internet在線分析和生物信息學(xué)軟件分析,獲得這些蛋白的結(jié)構(gòu)、功能相關(guān)信息,數(shù)據(jù)庫檢索結(jié)果表明這些蛋白主要與細(xì)胞運動、能量代謝、信號轉(zhuǎn)導(dǎo)、蛋白折疊、修飾、合成等功能相關(guān)。對C4蛋白的編碼基因、蛋白結(jié)構(gòu)和功能做進(jìn)一步的分析,序列分析結(jié)果提示該cDNA序列含有一個816 bp的開放閱讀框序列,編碼271個氨基酸,其編碼蛋白的理論分子量為29.34 kDa ,pI為9.12。蛋白含有磷酸甘油醛脫氫酶樣活性位點、N端NAD(P)結(jié)合區(qū)域、C端糖的運輸和代謝的催化區(qū)域等保守結(jié)構(gòu)功能域,具有潛在的抗原表位區(qū)域。結(jié)論:利用雙向電泳分析技術(shù)、質(zhì)譜鑒定技術(shù)和生物信息學(xué)分析了日本血吸蟲成蟲可溶性抗原,成功鑒定了多個日本血吸蟲成蟲抗原蛋白質(zhì),并展示其結(jié)構(gòu)和功能。給研究日本血吸蟲成蟲抗原機(jī)制提供新的線索和思路,為今后日本血吸蟲的蛋白質(zhì)組學(xué)深入系統(tǒng)研究打下了基礎(chǔ),為開發(fā)抗血吸蟲感染疫苗提供新的候選分子以及篩選新的免疫診斷抗原提供了可能。
[Abstract]:Aim: to establish and optimize the proteomic analysis method of adult Schistosoma japonicum, and to find out the specific adult antigen in soluble protein components related to immune response. Methods: the soluble protein of adult Schistosoma japonicum was purified, and the corresponding electrophoresis and Western blotting patterns were obtained by 2D-E and Western blotting. The image was analyzed and compared by PDQuest two-dimensional electrophoresis image analysis software. The theoretical molecular weight (Mr) and isoelectric point (pI) of the protein were obtained and the specific antigen protein spots were calibrated. Thirty protein spots were selected, which were digested into polypeptides by trypsin. The polypeptides were analyzed by LC-MS/MS mass spectrometry, and the non-redundant protein sequence database of Schistosoma was searched by SEQUEST software. The peptide fingerprint (PMF) and peptide sequence detection data of 24 protein spots belonging to 18 kinds of proteins were obtained successfully. The structure and function of these proteins were analyzed online by bioinformatics and other techniques. Results: the soluble protein of adult Schistosoma japonicum was stained with Coomassie brilliant blue R250 after two dimensional electrophoresis. Its molecular weight distribution of about 14 kDa~134 kDa,pI was mainly concentrated in 4.94 ~ 9.50. The results showed that the number of antigen-antibody reaction points was about 57 in the experimental group and 0 in the control group. The selected protein spots were decolorized and digested in situ and hydrolyzed by enzyme. The peptide mixture was analyzed by LC-MS/MS, and the peptide mass fingerprint was obtained, which was characterized by high quality and strong peak signal. The polypeptide sequence information was analyzed from the obtained data. Then the corresponding protein was identified by database retrieval. The structural and functional information of these proteins were obtained by Internet online analysis and bioinformatics software analysis. The results of database retrieval showed that these proteins were mainly associated with cell movement, energy metabolism, signal transduction, protein folding, modification. Synthesis and other functions related. Further analysis of the encoding gene, protein structure and function of C4 protein showed that the cDNA sequence contained an open reading frame sequence of 816 bp, encoding 271 amino acids, and the theoretical molecular weight of the encoded protein was 29.34 kDa. PI is 9. 12. The protein contains some conserved functional domains, such as glyceraldehyde phosphate dehydrogenase-like sites, N-terminal NAD (P) binding region, C-terminal carbohydrate transport and metabolic catalytic region, and has potential antigen epitopes. Conclusion: the soluble antigens of adult Schistosoma japonicum were analyzed by two-dimensional electrophoresis, mass spectrometry and bioinformatics. Several proteins of adult Schistosoma japonicum were successfully identified, and their structures and functions were demonstrated. It provides new clues and ideas for studying the antigenic mechanism of adult Schistosoma japonicum and lays a foundation for further and systematic study of the proteomics of Schistosoma japonicum in the future. It is possible to develop a new vaccine against Schistosoma japonicum infection and to screen new immunological diagnostic antigens.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 胡梅梅;鐘政榮;羅慶禮;方婷娜;沈繼龍;;基于2-DE和蛋白質(zhì)組技術(shù)篩選的日本血吸蟲鳥氨酸氨基轉(zhuǎn)移酶的表達(dá)及其診斷應(yīng)用[J];中國人獸共患病學(xué)報;2011年06期

相關(guān)碩士學(xué)位論文 前1條

1 胡梅梅;基于蛋白質(zhì)組技術(shù)篩選的日本血吸蟲鳥氨酸氨基轉(zhuǎn)移酶和親免素的克隆表達(dá)及其診斷應(yīng)用[D];安徽醫(yī)科大學(xué);2011年

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