细粒棘球绦虫Eg95重组非分泌型和分泌型分枝杆菌疫苗的构建及免疫学研究

发布时间：2018-05-30 11:35

  本文选题：细粒棘球绦虫 + Eg95　； 参考：《兰州大学》2008年硕士论文

【摘要】： 目的:分别构建细粒棘球绦虫Eg95重组非分泌型和分泌型卡介苗及耻垢分枝杆菌疫苗及免疫学研究。方法:分别以卡介苗(BCG)基因组DNA和PGEX-4T-Eg95为模板,PCR扩增获120bp的BCG抗原85B信号肽序列和423bp的Eg95基因序列。先将Eg95基因序列定向克隆至大肠埃希菌-分枝杆菌穿梭质粒pMV261,构建非分泌型重组质粒pMEg95。再将BCG-Ag85B信号肽序列定向克隆至pMEg95,构建分泌型重组质粒pSMEg95。电穿孔法将两型重组质粒分别导入BCG菌及耻垢分枝杆菌。分别用两型重组疫苗免疫小鼠,于第五周和第八周用ELISA法检测体液免疫指标。结果:双酶切、PCR扩增及测序鉴定证实,克隆基因Eg95序列和Ag85B信号肽序列正确插入载体pMV261,细粒棘球绦虫Eg95重组非分泌型和分泌型BCG-Eg95和M.s-Eg95疫苗构建成功。SM型酶免分析仪检测第五周和第八周的IgG1和IgG2a显示,分泌型重组疫苗较非分泌型重组疫苗抗体效价高;重组BCG较重组M.s的抗体效价高。结论:构建了含有Eg95基因序列和BCG-Ag85B信号肽序列的细粒棘球绦虫Eg95重组非分泌型和分泌型BCG-Eg95和M.s-Eg95分枝杆菌疫苗。比较两型重组疫苗免疫小鼠后体液免疫水平,重组BCG-Eg95和M.s-Eg95疫苗可在BCG和M.s中表达Eg95抗原蛋白;分泌型重组疫苗较非分泌型重组疫苗免疫效果强;作为疫苗载体,BCG较M.s具有更好的免疫效果。
[Abstract]:Aim: to construct recombinant non-secretory and secretory BCG vaccine of Echinococcus granulosus (Echinococcus granulosus) and Mycobacterium pubescens vaccine, and to study the immunology of Echinococcus granulosus. Methods: the BCG antigen 85B signal peptide sequence of 120bp and the Eg95 gene sequence of 423bp were amplified by genomic DNA and PGEX-4T-Eg95 of BCG, respectively. The sequence of Eg95 gene was cloned into Escherichia coli-Mycobacterium coli shuttle plasmid pMV261 to construct non-secretory recombinant plasmid pMEg95. The sequence of BCG-Ag85B signal peptide was cloned into pMEg95, and the secretory recombinant plasmid pSMEg95 was constructed. Two types of recombinant plasmids were introduced into BCG and Mycobacterium smegmatis by electroporation. Mice were immunized with two recombinant vaccines and humoral immune indexes were detected by ELISA method at the fifth week and the eighth week. Results: PCR amplification and sequencing confirmed that, The cloned gene Eg95 sequence and Ag85B signal peptide sequence were correctly inserted into the vector pMV261. The recombinant non-secretory and secretory BCG-Eg95 and M.s-Eg95 vaccines of Echinococcus granulosus were constructed successfully. SM type enzyme immunoassay was used to detect IgG1 and IgG2a at the fifth and eighth weeks. The antibody titer of secreted recombinant vaccine was higher than that of non-secretory recombinant vaccine, and the antibody titer of recombinant BCG was higher than that of recombinant M.S. Conclusion: the recombinant non-secretory and secretory BCG-Eg95 and M.s-Eg95 vaccine of Echinococcus granulosus Eg95 containing Eg95 gene sequence and BCG-Ag85B signal peptide sequence were constructed. Compared the humoral immunity level of mice immunized with recombinant vaccine, recombinant BCG-Eg95 and M.s-Eg95 vaccine could express Eg95 antigen protein in BCG and M.s, secretory recombinant vaccine was more effective than non-secretory recombinant vaccine. As a vaccine vector, BCG has better immune effect than M.S.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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