细粒棘球蚴延伸因子-1和磷酸丙糖异构酶基因克隆表达及免疫诊断研究

发布时间：2018-04-18 22:20

本文选题：细粒棘球绦虫 + 抗原表位　；参考：《青海大学》2017年硕士论文

【摘要】：目的:1.运用生物信息学的方法对细粒棘球蚴延伸因子-1和磷酸丙糖异构酶T、B细胞表位以及各级结构进行预测,获得可取的抗原表位,对两者的免疫诊断性初步分析,了解其诊断价值。2.利用原核表达技术,通过对上述两种基因构建表达载体,获得重组蛋白,为后期免疫保护和诊断价值等研究奠定基础。方法:1.经Expasy系统预测两种基因的理化性质,使用DNAStar软件来分析蛋白亲水性、柔性区域、抗原指数及表面可及性,利用ABCpred与IEDB软件综合预测B细胞线性抗原表位,通过DNAStar软件预测T细胞表位,使用SOPMA软件预测二级结构,SWISS-MODEL网站构建三维结构。使用DNAstar软件对Eg和人类EF-1、TPI的氨基酸序列进行比对。2.通过RT-PCR获得目的基因与克隆载体连接形成重组质粒,重组质粒与p ET-28α(+)经双酶切连接成原核表达重组质粒,表达、纯化目的蛋白。用ELISA方法分别对37例囊型包虫、29例泡型包虫患者、16例正常人的血清检出率测定,了解其诊断价值。结果:1.EgEF-1亲水性得分较高的区域有7个;6个柔性区域;抗原性指数得分较高的区域分布与柔性区域一致;表面可及性得分较高的区域较少;可能的B细胞线性表位区域是:120-130、286-295、306-320、365-378;可能的优势性T细胞表位区域是:34-48、164-177、222-244、280-290、321-330、396-407;Eg EF-1的二级结构中α螺旋占32.81%、β折叠22.54%、β转角10.94%、无规则卷曲33.71%;序列比对,Eg EF-1与人类EF-1的一致性为35.06%,Eg TPI与人类TPI的一致性为8%。Eg TPI亲水性得分较高的区域有8个;8个柔性区域;抗原性指数得分较高的区域分布与柔性区域大部分一致;表面可及性得分较高的区域分布区域相对较少;可能的B细胞线性表位的氨基酸序列为:28-39、50-60、70-80、131-139、150-159、213-231。Eg TPI二级结构中α螺旋占36%、β折叠22.00%、β转角12.00%、无规则卷曲30.00%;可能的优势性T细胞表位区域为:16-30、71-85、98-119、142-154、178-200。2.成功构建重组表达载体,诱导纯化出重组蛋白Eg EF-1的分子量在34KD左右,Eg TPI的分子量在27KD左右。ELISA结果显示,Eg EF-1血清检测阳性率为0,但OD值普遍较高,Eg TPI细粒棘球蚴病患者阳性检出率为86.48%,多房棘球蚴患者阳性检出率为72.41%,与健康人群比较,差异具有统计学意义(P0.05),但CE和AE间差异没有显著性。结论:1.通过对抗原表位的预测,Eg EF-1有4个B细胞表位的区域、6个优势性T细胞表位区域,Eg TPI有6个可能形成B细胞表位的区域、5个优势性T细胞表位区域,这些抗原表位预示可作为免疫诊断和药物治疗靶点2.成功纯化重组蛋白后,Eg EF-1对两种病人血清检测结果无阳性例数;Eg TPI检测结果表明,细粒患者与多房患者之间存在交叉反应,Eg TPI重组蛋白无种属特异性。
[Abstract]:Purpose 1.Bioinformatics method was used to predict the epitopes and structures of Echinococcus granulosus extension factor 1 (Echinococcus granulosus) and phosphoropropyleneisomerase (TG) B cell epitopes and to obtain the desired epitopes. The immunological diagnostics of Echinococcus granulosus were preliminarily analyzed to find out the diagnostic value of the epitopes. 2.By using prokaryotic expression technique, the recombinant protein was obtained by constructing the expression vector of the above two genes, which laid the foundation for the later study of immune protection and diagnostic value.Method 1: 1.The physicochemical properties of the two genes were predicted by Expasy system. The hydrophilicity, flexibility, antigen index and surface accessibility of the two genes were analyzed by DNAStar software. The linear antigen epitopes of B cells were predicted by ABCpred and IEDB software.Using DNAStar software to predict T cell epitopes and SOPMA software to predict secondary structure SWISS-MODEL website to construct 3D structure.The amino acid sequences of EG and human EF-1TPI were compared by DNAstar software.The target gene was ligated with the clone vector to form a recombinant plasmid by RT-PCR. The recombinant plasmid and p ET-28 伪 () were ligated into prokaryotic expression plasmid by double enzyme digestion, and the target protein was expressed and purified.The detection rate of serum in 37 patients with cystic hydatid cyst and 29 patients with vesicular hydatid disease was determined by ELISA method.Results: 1. There were 7 regions with higher score of hydrophilicity in EgEF-1, 6 flexible regions, the distribution of regions with higher antigenicity index was consistent with that of flexible regions, and less areas with higher surface accessibility score.The possible B cell linear epitope region is: 1: 120-130286-295306-320365-378; the possible dominant T cell epitope region is the secondary structure of the cell EF-1 of: 34-48164-177222-244280-29030-330396-4070.The 伪 helix is 32.81, 尾 folding 22.54, 尾 rotation 10.94, irregular curl 33.71; sequence alignment is 35.06g TPI;There are 8 regions with higher hydrophilicity score of 8%.Eg TPI, 8 flexible regions and 8 flexible regions, which are consistent with human TPI.The regional distribution of higher antigenicity index is consistent with that of flexible region, and the area with higher surface accessibility score is relatively small.The amino acid sequence of the possible B cell linear epitope is as follows: 1. The amino acid sequence of the B cell linear epitope is:: 28-39-139150-159213-231Eg TPI secondary structure: a helix is 36x, 尾 folding 22.00m, 尾 rotation 12.00, irregular curl 30.000.The possible dominant T cell epitope region is: B16-3071-85n98-119142-154178-200.2.The recombinant expression vector was successfully constructed.The result of Elisa showed that the positive rate of Eg EF-1 in serum was 0, but the OD value was generally higher. The positive rate of Eg TPI in patients with echinococcosis was 86.48.The positive rate of echinococcosis was 72.41%, which was compared with the healthy population.The difference was statistically significant (P 0.05), but there was no significant difference between CE and AE.Conclusion 1.The antigenic epitopes of Eg EF-1 were predicted to have 4 B cell epitopes, 6 dominant T cell epitope regions and 6 predominance T cell epitope regions to form B cell epitopes and 5 dominant T cell epitopes.These epitopes indicate that they can be used as targets for immunological diagnosis and drug therapy.After the recombinant protein was purified successfully, there were no positive cases of Eg TPI in serum of the two patients. The results showed that there was cross-reaction between granulated patients and multilocular patients, and there was no species-specificity of the recombinant protein Eg TPI.
【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】：R392

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