日本血吸虫18kDa天然分子抗原及其候选基因疫苗的研究

发布时间：2018-01-04 17:08

本文关键词：日本血吸虫18kDa天然分子抗原及其候选基因疫苗的研究　出处：《中南大学》2006年硕士论文　论文类型：学位论文

【摘要】： 目的：筛选和鉴定新的有价值的日本血吸虫疫苗候选分子，为天然分子多价复合疫苗的制备提供实验依据。本论文对日本血吸虫不同靶分子量大小的天然抗原分子进行了分离纯化与鉴定，针对SjAWA18kDa分子抗原进行免疫生化特性研究，并比较了SjAWA18kDa天然分子抗原及其相关编码候选基因SJCWL01DNA免疫小鼠产生的抗血吸虫病免疫保护效果。方法：1采用柱层析方法和SDS-PAGE结合凝胶洗脱分离纯化日本血吸虫成虫(SjAWA)和未成熟虫卵可溶性抗原(SIEA)，SDS-PAGE结合银染色、考马斯亮兰染色进行分析鉴定。2采用多途径免疫法制备SjAWA18kDa纯化抗原的单特异免疫血清，以此为探针进行免疫学分析。3将筛选尾蚴cDNA文库获得的新基因(编码18kDa蛋白，命名为SJCWL01，GENBANK登录号为AY855919)克隆入真核表达载体pcDNA3，经PCR、限制性酶切筛选和鉴定阳性克隆。将pcDNA3／SJCWL01质粒转化大肠杆菌DH5α、大量制备DNA疫苗；将该天然分子抗原编码候选基因与SjAWA18kDa免疫小鼠，然后经腹部皮肤感染40±1条日本血吸虫尾蚴，45天后剖杀，计算减虫率和减卵率。采用免疫组化法检测重组DNA疫苗SJCWL01在局部肌肉组织中的表达。结果：1通过柱层析法或SDS-PAGE结合凝胶洗脱得到了五种不同大小血吸虫分子抗原SjAWA18kDa，SjAWA26／28kDa，SIEA42kDa，SIEA130kDa，SjAWA14kDa。2成功制备了抗日本血吸虫18kDa单特异兔血清，SjAWA18kDa与新基因编码的重组融合蛋白有共同的抗原表位。免疫组化显示SjAWA18kDa主要定位于成虫的肠上皮、表皮和实质细胞。3将SJCWL01基因亚克隆至pcDNA3，然后，转化大肠杆菌，大量制备纯化的重组质粒DNA(pcDNA3／SJCWL01)；以此重组DNA和SjAWA18kDa天然分子免疫小鼠，分别获得了27.6％，30.7％减虫率，，39.5％和50.7％减卵率。免疫组化结果显示SJCWL01在小鼠骨胳肌的细胞浆中得到表达。结论：1实验证明柱层析法或SDS-PAGE电泳切胶、结合凝胶洗脱是一种快速分离纯化血吸虫单一组分抗原简单而有效的方法。2成功制备了抗SjAWA18kDa血清，SjAWA18kDa主要定位于成虫的肠腔和实质细胞。3成功构建了天然分子抗原编码候选基因(18kDa)DNA疫苗，该疫苗与SjAWA18kDa可诱导小鼠产生部分抗血吸虫感染的保护力。
[Abstract]:Objective: screening and identification of new valuable Schistosoma japonicum vaccine candidate molecules, provide an experimental basis for the natural molecular multivalent vaccine preparation. The natural antigens of Schistosoma japonicum in different target molecular size of isolation and identification of immune, biochemical characteristics for SjAWA18kDa molecular antigen, and compared the anti effect of schistosomiasis immune protection in mice immunized with SjAWA18kDa molecular antigen and related candidate gene SJCWL01DNA encoding.
Methods: 1 by column chromatography and SDS-PAGE method combined with gel elution purification of Schistosoma japonicum (SjAWA) and soluble immature egg antigen (SIEA), SDS-PAGE combined with silver staining, Kaumas bright blue were identified by.2 analysis of immune preparation single specific immune serum SjAWA18kDa purified antigen by multi-channel staining to identify novel genes for cercariae cDNA Library as a probe for immunological analysis of.3 (encoding 18kDa protein, named SJCWL01, GENBANK accession No. AY855919) was cloned into eukaryotic expression vector pcDNA3 by PCR, restriction enzyme screening and identification of positive clones. The pcDNA3 / SJCWL01 plasmid was transformed into Escherichia coli DH5 alpha, a preparation of the DNA vaccine; molecular antigen encoding candidate genes in mice immunized with SjAWA18kDa, then the abdominal skin infection 40 + 1 cercariae, 45 days after the killing, calculating the worm reduction rate and egg reduction Rate. Immunohistochemical method was used to detect the expression of recombinant DNA vaccine SJCWL01 in local muscle tissue.
Results: 1 by column chromatography or gel elution with SDS-PAGE got five different size of Schistosoma japonicum antigen SjAWA18kDa, SjAWA26 / 28kDa, SIEA42kDa, SIEA130kDa, anti Schistosoma japonicum 18kDa single specific rabbit serum was prepared successfully with SjAWA18kDa SjAWA14kDa.2, the new gene encoding the recombinant fusion proteins have a common epitope. Immunohistochemistry showed that SjAWA18kDa was mainly localized in adult intestinal epithelial, epidermal and parenchymal cells of.3 SJCWL01 gene was subcloned into pcDNA3, then transformed into Escherichia coli, the recombinant plasmid DNA was purified for the (pcDNA3 / SJCWL01); the recombinant DNA and SjAWA18kDa molecular immunized mice were obtained respectively 27.6%, 39.5% and 30.7% of worm reduction rate 50.7% egg reduction rate. Immunohistochemistry showed that the expression of SJCWL01 in cytoplasm of mouse skeletal muscle.
Conclusion: the experiment proved that the 1 column chromatography or SDS-PAGE electrophoresis, gel elution is a rapid purification method of schistosome antigen of simple and effective single group.2 were successfully prepared anti SjAWA18kDa serum, SjAWA18kDa was mainly located in the adult intestinal cavity and parenchymal cells.3 successfully constructed molecular antigen encoding gene (18kDa) DNA vaccine, the vaccine and SjAWA18kDa can protect part against the infection of Schistosoma japonicum in mice.

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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