细粒棘球绦虫原头节和囊壁抗原筛选与烯醇酶和转酮醇酶生物信息学分析

发布时间：2018-11-23 06:29

【摘要】：目的利用蛋白质组学相关技术筛选出细粒棘球蚴原头节、囊壁蛋白中有望成为免疫学诊断抗原的分子,为早期诊断包虫病提供一定理论基础。方法提取细粒棘球蚴原头节、囊壁组织蛋白,利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离总蛋白,借助蛋白质印迹技术初步掌握细粒棘球绦虫原头节、囊壁蛋白表达谱及免疫原性;利用二维电泳技术分离原头节、囊壁蛋白,选取原头节40个蛋白点、囊壁8个蛋白点切胶,胰蛋白酶酶解,利用基质辅助激光电离解析飞行时间质谱分析技术获得各个蛋白点的肽指纹图谱数据,应用Mascot软件检索NCBInr、SWISS-PROT蛋白数据库进行蛋白质鉴定;通过查阅文献结合生物信息学技术初步分析,选取质谱分析鉴定结果中有望成为候选抗原分子的烯醇酶、转酮醇酶,利用生物信息学软件从理化性质、信号肽、B/T细胞表位、空间结构,亚细胞定位等分析烯醇酶、转酮醇酶成为特异性诊断抗原的可能性。构建烯醇酶、转酮醇酶原核表达载体,获得纯化蛋白为后期实验提供一定基础。结果1.原头节蛋白质在分子量72KD左右有大量浓集,囊壁的蛋白质浓集在72k D、26k D和17k D左右;2.原头节、囊壁中分别有36、6个蛋白点得到鉴定,大致可以分为:(1)细胞骨架蛋白(2)代谢相关酶类(3)细胞信号传导通路蛋白(4)物质转运相关蛋白(5)热休克蛋白(6)蛋白翻译有关蛋白等;3.烯醇酶基因全长1449bp,由3个外显子和2个内含子组成,CDS为1302bp,编码434个氨基酸;相对分子量、等电点、不稳定指数分别为46.56k D、6.48、32.97,半衰期较长,为稳定蛋白;烯醇酶亲水性、柔性区域、抗原性、表面可及性得分较高的氨基酸区域分别有9、12、19、8个;可能的B细胞线性表位有15个;CTL细胞表位和Th细胞表位各有10个;二级结构中主要以α螺旋、无规则卷曲为主;与人类烯醇酶氨基酸序列一致性为66.23%。转酮醇酶基因由7个外显子,6个内含子构成,CDS为1878 bp,编码625个氨基酸;相对分子量为67.76 k D;预测结果显示转酮醇酶可能为跨膜蛋白,位于细胞质,二级结构主要以α螺旋、无规则卷曲为主;有16个潜在的B细胞线性表位,11个CTL细胞表位,13个Th细胞表位;与人类转酮醇酶一致性仅为58.68%;4.成功构建pet28a-Eg EN、pet28-Eg TK原核表达载体,并获得重组蛋白,Eg En、Eg TK与CE病人血清反应阳性率均大于75%,CE与AE有部分交叉反应。结论初步鉴定了细粒棘球蚴原头节、囊壁组织中的部分蛋白点,这些蛋白主要参与代谢、细胞骨架、信号传导、物质转运、蛋白合成等,细粒棘球绦虫青海株原头节、囊壁蛋白表达数据库得以初步建立。初步认为烯醇酶、转酮醇酶可能成为早期诊断特异性抗原,但需进一步的实验证实。
[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, 伪 -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.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R383.3

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