日本血吸虫40kDa热休克蛋白（SjHSP40）的免疫学功能研究

发布时间：2018-06-18 10:03

  本文选题：日本血吸虫 + sjHSP40　； 参考：《南方医科大学》2012年硕士论文

【摘要】：目的：构建PGEX-6P-1-SjHSP40(SjHSP40)融合表达载体并在大肠埃希菌(Escherich coli内进行原核表达及重组蛋白纯化,初步探讨SjHSP40的免疫学功能。 方法：首先,对SjHSP40进行生物信息学分析,以PCR方法扩增SjHSP40基因片段,将其克隆入原核表达质粒pGEX-6P-1,经测序验证后转化入E. coli BL-21(DE3)中。随后,重组蛋白经异丙基硫代半乳糖苷(IPTG)诱导表达后以Glutathione-Sepharose4B亲和柱进行纯化,所得产物GST-SjHSP40融合蛋白进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western blot鉴定。最后,将此融合蛋白免疫Balb/c小鼠后,以ELISA法检测血清中SjHSP40特异性抗体水平(IgG及其亚型IgG1和IgG2a),并以流式细胞术通过体内外实验观察SjHSP40对CD4+T淋巴细胞分化(包括Th1、Th2、Th17和Treg细胞)的动态变化。 结果：对SjHSP40进行生物信息学分析发现,SjHSP40的氨基酸序列与曼氏血吸虫的同系物相似性最高,序列一致性达到86%,与人、小鼠HSP40的氨基酸序列一致性分别为37%和39%,SjHSP40含有7个潜在的B细胞表位及多个T细胞表位。测序结果表明,本研究成功构建了pGEX-6P-1-SjHSP40原核表达载体,经IPTG诱导表达出融合蛋白GST-SjHSP40。Western blot结果显示该融合蛋白能被抗GST抗体特异性识别。ELISA检测表明,与GST等对照组相比,此融合蛋白免疫小鼠血清中抗原特异性IgG及其亚型IgGl和IgG2a水平均显著升高。通过体内外实验,运用流式细胞术检测显示,与GST等对照组相比,本实验中未见SjHSP40对CD4+T淋巴细胞分化的具有明显影响。 结论：本实验中SjHSP40可诱导免疫小鼠血清中高水平的特异性IgG,包括其亚型IgG1和IgG2a,但对CD4+T淋巴细胞的分化(包括Thl、Th2、Th17和Treg细胞)却未见明显影响。
[Abstract]:Aim: to construct a fusion expression vector PGEX-6P-1-SjHSP40 (SjHSP40) and express it in E. coli Escherich coli, and to investigate the immunological function of SjHSP40. Methods: firstly, SjHSP40 was analyzed by bioinformatics. The SjHSP40 gene fragment was amplified by PCR and cloned into prokaryotic expression plasmid pGEX-6P-1. The SjHSP40 gene was transformed into E. coli BL-21 (DE3) after sequencing. Then, the recombinant protein was induced by isopropylthione-Sepharose 4B affinity column and purified. The GST-SjHSP40 fusion protein was identified by SDS-PAGEG blot. Finally, after immunizing Balb / c mice with this fusion protein, The specific antibody level of SjHSP40 and its subtypes, IgG1 and IgG2a, were detected by Elisa. The dynamic changes of CD4 T lymphocyte differentiation (including Th1, Th2Th2Th17 and Treg cells) induced by SjHSP40 were observed by flow cytometry in vitro and in vivo. Results: bioinformatics analysis of SjHSP40 showed that the amino acid sequence of SjHSP40 had the highest similarity with the homologue of Schistosoma mansoni. The amino acid sequence consistency of mouse HSP40 was 37% and 39% respectively. SjHSP40 contained 7 potential B cell epitopes and multiple T cell epitopes. The results of sequencing showed that pGEX-6P-1-SjHSP40 prokaryotic expression vector was successfully constructed, and the fusion protein GST-SjHSP40 was induced by IPTG. The results of Western blot showed that the fusion protein could be specifically recognized by anti-GST antibody. Elisa showed that the fusion protein could be compared with GST and other control groups. The levels of antigen-specific IgG and its subtypes IgGl and IgG2a in serum of mice immunized with this fusion protein were significantly increased. In vitro and in vivo experiments, flow cytometry showed that SjHSP40 had no significant effect on CD4 T lymphocyte differentiation compared with GST and other control groups. Conclusion: SjHSP40 can induce high level of specific IgG in serum of immunized mice, including its subtypes IgG1 and IgG2a, but has no significant effect on the differentiation of CD4 T lymphocytes (including ThlnTh2Th17 and Treg cells).
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R383.24

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