日本血吸虫信号蛋白14-3-3的免疫诊断及虫卵cDNA文库的构建

发布时间：2018-04-27 23:01

本文选题：血吸虫病 + 信号蛋白14-3-3　；参考：《安徽医科大学》2007年硕士论文

【摘要】： 目的:诱导表达本室保种的含有日本血吸虫信号蛋白14-3-3(Sj14-3-3)编码基因的重组表达载体pET28a(+)-Sj14-3-3,制备并纯化出重组蛋白Sj14-3-3(rSj 14-3-3),建立重组抗原间接ELISA方法(rSj-ELISA)诊断日本血吸虫病,并与日本血吸虫可溶性虫卵抗原(SjSEA)的间接ELISA、间接血凝试验(IHA)和环卵沉淀试验(COPT)平行检测,比较几种方法的敏感性和特异性之间的差异,以探讨重组抗原用于诊断血吸虫病的实用性和可靠性。方法:诱导含Sj14-3-3编码基因的重组质粒pET28a(+)-Sj14-3-3表达出rSj14-3-3。SDS-PAGE和Western blotting验证表达量和免疫活性,使用亲和层析法纯化重组蛋白,并用Bradford法检测纯化蛋白的浓度。将rSj14-3-3和SEA包被反应板,棋盘滴定分别确定最适抗原包被量和血清稀释度,建立rSj-ELISA和SEA-ELISA诊断日本血吸虫病方法。用rSj-ELISA,SEA-ELISA,SEA-IHA和COPT四种方法平行检测急性日本血吸虫病患者64例,慢性日本血吸虫病患者56例,华支睾吸虫患者28例,肠道线虫患者以及正常人血清各32例,比较不同方法之间敏感性,特异性以及交叉反应性。结果:诱导表达重组质粒pET28a(+)-Sj14-3-3,Ni2+亲和层析法纯化获得rSj14-3-3,SDS-PAGE检测其分子量与理论值一致;Western blotting显示rSj14-3-3能被其特异性单克隆抗体识别。以rSj14-3-3作为诊断抗原分子,经过条件优化建立了rSj-ELISA诊断日本血吸虫病的方法。用rSj-ELISA,SEA-ELISA,SEA-IHA和COPT四种方法平行检测急、慢性血吸虫病患者,华支睾吸虫患者,肠道线虫患者以及正常人血清,结果示rSj14-3-3用于日本血吸虫病免疫诊断与SEA-ELISA以及SEA-IHA具有相似的敏感性和特异性; rSj-ELISA的敏感性显著优于COPT,但特异性(假阳性以及与其他寄生虫病的交叉反应性)逊于COPT。结论:本研究成功表达并纯化了rSj14-3-3抗原,以此抗原单独包被建立rSj-ELISA方法诊断日本血吸虫病。重组抗原制备简便,成本低廉,制备周期短,方法易于标准化,更适合商品化生产和流行区血吸虫病的血清学辅助诊断。目的:构建日本血吸虫虫卵cDNA文库并用日本血吸虫急性感染患者血清对其进行免疫学筛选,以寻找有效的早期诊断分子和疫苗候选分子。方法:收集日本血吸虫感染6周虫卵,提取总RNA,逆转录生成cDNA,PCR合成cDNA双链。经过纯化、sfi I酶切、过柱除去小片段后,与λTriplEx2噬菌体载体两臂连接,体外包装后形成日本血吸虫虫卵cDNA文库。用大肠杆菌预吸收的急性血吸虫病患者血清对文库进行免疫学筛选,对复筛得到的14个阳性克隆进行插入片段的核苷酸序列测定,结果呈送GenBank进行同源性分析,根据其理论氨基酸组成,初步确定该cDNA插入片段所编码蛋白的特性并利用生物信息学软件分析和预测其结构和用途。结果:所构建的日本血吸虫虫卵cDNA初始文库的容量为2.7x106个重组子,经感染XL1-Blue菌后扩增文库的滴度达1010pfu/ml。任意挑取克隆经自身环化后提取质粒,酶切后片段大小范围为400- 1500bp,重组率85%以上。用急性日本血吸虫患者血清初筛得到28个阳性克隆,复筛得到14个持续阳性的克隆,测序后送GenBank进行BLAST比对,其中9号为空载序列,2号无同源基因匹配为可能的新基因,其余序列均为匹配的日本血吸虫相关基因。经生物信息学分析示2号克隆基因具有一个432bp的完整开放阅读框架,Antheprot软件和多种基于网页的在线软件分析其结构和功能并预测可能为一跨膜蛋白。结论:本试验成功地构建了日本血吸虫虫卵cDNA文库,筛选所得到的阳性克隆有望成为早期诊断抗原或保护性疫苗分子,值得进一步研究。
[Abstract]:Objective: to induce the recombinant expression vector pET28a (+) -Sj14-3-3 containing the encoding gene of Schistosoma japonicum signal protein 14-3-3 (Sj14-3-3), to prepare and purify the recombinant protein Sj14-3-3 (rSj 14-3-3), to establish a recombinant antigen indirect ELISA method (rSj-ELISA) for the diagnosis of schistosomiasis japonicum and to the soluble egg antigen of Schistosoma japonicum. (SjSEA) indirect ELISA, indirect hemagglutination test (IHA) and cyclic egg precipitation test (COPT) parallel detection, compare the differences between the sensitivity and specificity of several methods, in order to explore the practicability and reliability of recombinant antigen used in the diagnosis of schistosomiasis. Method: the recombinant plasmid pET28a (+) -Sj14-3-3 containing Sj14-3-3 encoding gene expressed rSj14-3- 3.SDS-PAGE and Western blotting were used to verify the expression and immune activity. The recombinant protein was purified by affinity chromatography, and the concentration of purified protein was detected by Bradford. RSj14-3-3 and SEA were coated with the reaction plate, and the optimum antigen envelope and serum dilution were determined by chessboard titration, and rSj-ELISA and SEA-ELISA were built to diagnose the Japanese schistosomiasis side. Methods: four methods of rSj-ELISA, SEA-ELISA, SEA-IHA and COPT were used to detect 64 cases of acute schistosomiasis, 56 cases of chronic schistosomiasis, 28 cases of Clonorchis sinensis, intestinal nematode and 32 normal human serum. The sensitivity, specificity and cross reactivity between different methods were compared. The plasmid pET28a (+) -Sj14-3-3, Ni2+ affinity chromatography was purified to obtain rSj14-3-3, and the molecular weight of SDS-PAGE was consistent with the theoretical value; Western blotting showed that rSj14-3-3 could be identified by its specific monoclonal antibody. RSj14-3-3 was used as a diagnostic antigen, and the method was optimized to establish rSj-ELISA for the diagnosis of schistosomiasis japonica. Four methods of ELISA, SEA-ELISA, SEA-IHA and COPT were used to detect acute, chronic schistosomiasis, Clonorchis sinensis, intestinal nematode and normal human serum. The results showed that rSj14-3-3 was similar in sensitivity and specificity to SEA-ELISA and SEA-IHA in the immunodiagnosis of schistosomiasis japonica, and the sensitivity of rSj-ELISA was significantly better than that of COPT. But the specificity (false positive and cross reactivity with other parasitic diseases) is inferior to the COPT. conclusion: This study successfully expressed and purified the rSj14-3-3 antigen, and the antigen was successfully established by the rSj-ELISA method for the diagnosis of schistosomiasis japonicum. The preparation of recombinant antigen is simple, low cost, short preparation period, easy to standardize and more suitable for commodity. Serological diagnosis of schistosomiasis in chemical production and endemic areas.
Objective: to construct the cDNA Library of Schistosoma japonicum eggs and to screen it with sera from acute infected patients with Schistosoma japonicum to find effective early diagnostic molecules and vaccine candidates. Methods: to collect the eggs of 6 weeks infected by Schistosoma japonicum, extract total RNA, reverse transcriptase cDNA and PCR to synthesize cDNA double strands. After purification, SFI I enzyme digestion, After the small fragments were removed, the cDNA Library of Schistosoma japonicum eggs was formed after packing with the two arms of lambda TriplEx2 phage carrier. The sera of patients with acute schistosomiasis preabsorbed by Escherichia coli were immunologically screened, and the nucleotide sequence of the 14 positive clones was detected by the insertion fragment. The result was Ge NBank homology analysis, based on its theoretical amino acid composition, preliminarily determined the characteristics of the cDNA inserted fragment and analyzed and predicted its structure and use by bioinformatics software. Results: the capacity of the initial library of the egg cDNA of Schistosoma japonicum was 2.7x106 recombinant, and after infection of XL1-Blue bacteria, Kuo Zengwen The titer of the library was 1010pfu/ml. randomly selected to extract the plasmid after its own cyclization. The size of the fragment was 400- 1500bp and the recombination rate was more than 85%. 28 positive clones were screened from the sera of acute Schistosoma japonicum patients and 14 continuous positive clones were screened and then sent to GenBank for BLAST comparison, of which 9 was empty load. Sequence, No. 2 homologous genes are matched as possible new genes, and the rest of the sequence are matched genes of Schistosoma japonicum. By bioinformatics analysis, the 2 clone gene has a complete open reading frame of 432bp, Antheprot software and a variety of web based online software to analyze its structure and function and predict that it may be a transmembrane Conclusion: this experiment successfully constructed the cDNA Library of Schistosoma japonicum eggs. The screening of the positive clones is expected to be an early diagnostic or protective vaccine molecule. It is worth further study.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383

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