細(xì)粒棘球絳蟲原頭節(jié)和囊壁抗原篩選與烯醇酶和轉(zhuǎn)酮醇酶生物信息學(xué)分析
發(fā)布時間：2018-11-23 06:29
【摘要】：目的利用蛋白質(zhì)組學(xué)相關(guān)技術(shù)篩選出細(xì)粒棘球蚴原頭節(jié)、囊壁蛋白中有望成為免疫學(xué)診斷抗原的分子,為早期診斷包蟲病提供一定理論基礎(chǔ)。方法提取細(xì)粒棘球蚴原頭節(jié)、囊壁組織蛋白,利用十二烷基硫酸鈉聚丙烯酰胺凝膠電泳分離總蛋白,借助蛋白質(zhì)印跡技術(shù)初步掌握細(xì)粒棘球絳蟲原頭節(jié)、囊壁蛋白表達(dá)譜及免疫原性;利用二維電泳技術(shù)分離原頭節(jié)、囊壁蛋白,選取原頭節(jié)40個蛋白點、囊壁8個蛋白點切膠,胰蛋白酶酶解,利用基質(zhì)輔助激光電離解析飛行時間質(zhì)譜分析技術(shù)獲得各個蛋白點的肽指紋圖譜數(shù)據(jù),應(yīng)用Mascot軟件檢索NCBInr、SWISS-PROT蛋白數(shù)據(jù)庫進(jìn)行蛋白質(zhì)鑒定;通過查閱文獻(xiàn)結(jié)合生物信息學(xué)技術(shù)初步分析,選取質(zhì)譜分析鑒定結(jié)果中有望成為候選抗原分子的烯醇酶、轉(zhuǎn)酮醇酶,利用生物信息學(xué)軟件從理化性質(zhì)、信號肽、B/T細(xì)胞表位、空間結(jié)構(gòu),亞細(xì)胞定位等分析烯醇酶、轉(zhuǎn)酮醇酶成為特異性診斷抗原的可能性。構(gòu)建烯醇酶、轉(zhuǎn)酮醇酶原核表達(dá)載體,獲得純化蛋白為后期實驗提供一定基礎(chǔ)。結(jié)果1.原頭節(jié)蛋白質(zhì)在分子量72KD左右有大量濃集,囊壁的蛋白質(zhì)濃集在72k D、26k D和17k D左右;2.原頭節(jié)、囊壁中分別有36、6個蛋白點得到鑒定,大致可以分為:(1)細(xì)胞骨架蛋白(2)代謝相關(guān)酶類(3)細(xì)胞信號傳導(dǎo)通路蛋白(4)物質(zhì)轉(zhuǎn)運(yùn)相關(guān)蛋白(5)熱休克蛋白(6)蛋白翻譯有關(guān)蛋白等;3.烯醇酶基因全長1449bp,由3個外顯子和2個內(nèi)含子組成,CDS為1302bp,編碼434個氨基酸;相對分子量、等電點、不穩(wěn)定指數(shù)分別為46.56k D、6.48、32.97,半衰期較長,為穩(wěn)定蛋白;烯醇酶親水性、柔性區(qū)域、抗原性、表面可及性得分較高的氨基酸區(qū)域分別有9、12、19、8個;可能的B細(xì)胞線性表位有15個;CTL細(xì)胞表位和Th細(xì)胞表位各有10個;二級結(jié)構(gòu)中主要以α螺旋、無規(guī)則卷曲為主;與人類烯醇酶氨基酸序列一致性為66.23%。轉(zhuǎn)酮醇酶基因由7個外顯子,6個內(nèi)含子構(gòu)成,CDS為1878 bp,編碼625個氨基酸;相對分子量為67.76 k D;預(yù)測結(jié)果顯示轉(zhuǎn)酮醇酶可能為跨膜蛋白,位于細(xì)胞質(zhì),二級結(jié)構(gòu)主要以α螺旋、無規(guī)則卷曲為主;有16個潛在的B細(xì)胞線性表位,11個CTL細(xì)胞表位,13個Th細(xì)胞表位;與人類轉(zhuǎn)酮醇酶一致性僅為58.68%;4.成功構(gòu)建pet28a-Eg EN、pet28-Eg TK原核表達(dá)載體,并獲得重組蛋白,Eg En、Eg TK與CE病人血清反應(yīng)陽性率均大于75%,CE與AE有部分交叉反應(yīng)。結(jié)論初步鑒定了細(xì)粒棘球蚴原頭節(jié)、囊壁組織中的部分蛋白點,這些蛋白主要參與代謝、細(xì)胞骨架、信號傳導(dǎo)、物質(zhì)轉(zhuǎn)運(yùn)、蛋白合成等,細(xì)粒棘球絳蟲青海株原頭節(jié)、囊壁蛋白表達(dá)數(shù)據(jù)庫得以初步建立。初步認(rèn)為烯醇酶、轉(zhuǎn)酮醇酶可能成為早期診斷特異性抗原,但需進(jìn)一步的實驗證實。
[Abstract]:Objective to screen out the molecules of echinococcus granulosus proganglia and cystic wall proteins which are expected to be immunological diagnostic antigens by using proteomic techniques to provide a theoretical basis for the early diagnosis of hydatid disease. Methods Echinococcus granulosus was extracted from the head ganglion and cyst wall of Echinococcus granulosus. The total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proto-cephalic ganglion of Echinococcus granulosus was preliminarily grasped by Western blotting technique. Cystic wall protein expression profile and immunogenicity; Two dimensional electrophoresis technique was used to isolate the original ganglion and cystic wall protein. Forty protein spots of the original head ganglion were selected, 8 protein spots of the capsule wall were cut glue and trypsin was hydrolyzed by trypsin. Matrix assisted laser ionization time of flight mass spectrometry (TOF-MS) technique was used to obtain the peptide fingerprint data of each protein point, and the NCBInr,SWISS-PROT protein database was searched by Mascot software for protein identification. The enolase, transketol enzyme, which is expected to be a candidate antigen molecule in the result of mass spectrometry analysis and bioinformatics software were used to analyze the physical and chemical properties and signal peptides. B / T cell epitopes, spatial structures, subcellular localization, and so on, analysis of enolase, transketol enzyme become the possibility of specific diagnosis of antigen. A prokaryotic expression vector of enolase and transketonase was constructed and purified protein was obtained to provide a basis for later experiments. Result 1. There is a large concentration of protophore protein in the molecular weight of 72KD, and the protein concentration in the cyst wall is about 72kD 26kD and 17kD, 2. 36 and 6 protein spots were identified in the primary ganglia and cystic wall, respectively. (1) cytoskeletal proteins (2) metabolism-related enzymes (3) cell signal transduction pathway proteins (4) substance transporter associated proteins (5) heat shock proteins (6) proteins and so on; 3. The enolase gene consists of three exons and two introns, the CDS is 1302bp, encoding 434 amino acids, the relative molecular weight, isoelectric point and instability index are 46.56kD 6.48C 32.97, the half-life is longer, and the enolase gene is a stable protein. For enolase hydrophilicity, flexibility, antigenicity and surface accessibility, there were 912g / 19,8 amino acid domains, 15 possible B cell linear epitopes, 10 CTL cell epitopes and 10 Th cell epitopes, respectively. In the secondary structure, 偽 -helix, irregular crimp, and amino acid sequence of human enolase were 66.23 and 66.23 respectively. The transketolase gene was composed of 7 exons and 6 introns. The CDS was 1878 bp, encoding 625 amino acids, and the relative molecular weight was 67.76 KD. The predicted results showed that transketonase might be a transmembrane protein, located in the cytoplasm, with a helix, irregular curl, 16 potential B cell linear epitopes, 11 CTL cell epitopes and 13 Th cell epitopes. The consistency with human transketol enzyme is only 58.68%. The prokaryotic expression vector of pet28a-Eg EN,pet28-Eg TK was successfully constructed and the recombinant protein was obtained. The positive rate of serum reaction between Eg En,Eg TK and CE was higher than that of 75% CE and AE. Conclusion some protein sites in the proto-ganglion and cyst wall of Echinococcus granulosus were identified. These proteins were mainly involved in metabolism, cytoskeleton, signal transduction, substance transport, protein synthesis, and so on, and Echinococcus granulosus was isolated from the primordial head ganglia of the Qinghai strain of Echinococcus granulosus. The expression database of cystic wall protein was established. It is suggested that enolase and transketonase may be specific antigens for early diagnosis, but further experimental results are needed.
【學(xué)位授予單位】：青海大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R383.3
本文編號：2350611
[Abstract]:Objective to screen out the molecules of echinococcus granulosus proganglia and cystic wall proteins which are expected to be immunological diagnostic antigens by using proteomic techniques to provide a theoretical basis for the early diagnosis of hydatid disease. Methods Echinococcus granulosus was extracted from the head ganglion and cyst wall of Echinococcus granulosus. The total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proto-cephalic ganglion of Echinococcus granulosus was preliminarily grasped by Western blotting technique. Cystic wall protein expression profile and immunogenicity; Two dimensional electrophoresis technique was used to isolate the original ganglion and cystic wall protein. Forty protein spots of the original head ganglion were selected, 8 protein spots of the capsule wall were cut glue and trypsin was hydrolyzed by trypsin. Matrix assisted laser ionization time of flight mass spectrometry (TOF-MS) technique was used to obtain the peptide fingerprint data of each protein point, and the NCBInr,SWISS-PROT protein database was searched by Mascot software for protein identification. The enolase, transketol enzyme, which is expected to be a candidate antigen molecule in the result of mass spectrometry analysis and bioinformatics software were used to analyze the physical and chemical properties and signal peptides. B / T cell epitopes, spatial structures, subcellular localization, and so on, analysis of enolase, transketol enzyme become the possibility of specific diagnosis of antigen. A prokaryotic expression vector of enolase and transketonase was constructed and purified protein was obtained to provide a basis for later experiments. Result 1. There is a large concentration of protophore protein in the molecular weight of 72KD, and the protein concentration in the cyst wall is about 72kD 26kD and 17kD, 2. 36 and 6 protein spots were identified in the primary ganglia and cystic wall, respectively. (1) cytoskeletal proteins (2) metabolism-related enzymes (3) cell signal transduction pathway proteins (4) substance transporter associated proteins (5) heat shock proteins (6) proteins and so on; 3. The enolase gene consists of three exons and two introns, the CDS is 1302bp, encoding 434 amino acids, the relative molecular weight, isoelectric point and instability index are 46.56kD 6.48C 32.97, the half-life is longer, and the enolase gene is a stable protein. For enolase hydrophilicity, flexibility, antigenicity and surface accessibility, there were 912g / 19,8 amino acid domains, 15 possible B cell linear epitopes, 10 CTL cell epitopes and 10 Th cell epitopes, respectively. In the secondary structure, 偽 -helix, irregular crimp, and amino acid sequence of human enolase were 66.23 and 66.23 respectively. The transketolase gene was composed of 7 exons and 6 introns. The CDS was 1878 bp, encoding 625 amino acids, and the relative molecular weight was 67.76 KD. The predicted results showed that transketonase might be a transmembrane protein, located in the cytoplasm, with a helix, irregular curl, 16 potential B cell linear epitopes, 11 CTL cell epitopes and 13 Th cell epitopes. The consistency with human transketol enzyme is only 58.68%. The prokaryotic expression vector of pet28a-Eg EN,pet28-Eg TK was successfully constructed and the recombinant protein was obtained. The positive rate of serum reaction between Eg En,Eg TK and CE was higher than that of 75% CE and AE. Conclusion some protein sites in the proto-ganglion and cyst wall of Echinococcus granulosus were identified. These proteins were mainly involved in metabolism, cytoskeleton, signal transduction, substance transport, protein synthesis, and so on, and Echinococcus granulosus was isolated from the primordial head ganglia of the Qinghai strain of Echinococcus granulosus. The expression database of cystic wall protein was established. It is suggested that enolase and transketonase may be specific antigens for early diagnosis, but further experimental results are needed.
【學(xué)位授予單位】：青海大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R383.3
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