本文選題：細(xì)粒棘球絳蟲 + Eg95��； 參考：《蘭州大學(xué)》2008年碩士論文

【摘要】： 目的:分別構(gòu)建細(xì)粒棘球絳蟲Eg95重組非分泌型和分泌型卡介苗及恥垢分枝桿菌疫苗及免疫學(xué)研究。方法:分別以卡介苗(BCG)基因組DNA和PGEX-4T-Eg95為模板,PCR擴(kuò)增獲120bp的BCG抗原85B信號(hào)肽序列和423bp的Eg95基因序列。先將Eg95基因序列定向克隆至大腸埃希菌-分枝桿菌穿梭質(zhì)粒pMV261,構(gòu)建非分泌型重組質(zhì)粒pMEg95。再將BCG-Ag85B信號(hào)肽序列定向克隆至pMEg95,構(gòu)建分泌型重組質(zhì)粒pSMEg95。電穿孔法將兩型重組質(zhì)粒分別導(dǎo)入BCG菌及恥垢分枝桿菌。分別用兩型重組疫苗免疫小鼠,于第五周和第八周用ELISA法檢測(cè)體液免疫指標(biāo)。結(jié)果:雙酶切、PCR擴(kuò)增及測(cè)序鑒定證實(shí),克隆基因Eg95序列和Ag85B信號(hào)肽序列正確插入載體pMV261,細(xì)粒棘球絳蟲Eg95重組非分泌型和分泌型BCG-Eg95和M.s-Eg95疫苗構(gòu)建成功。SM型酶免分析儀檢測(cè)第五周和第八周的IgG1和IgG2a顯示,分泌型重組疫苗較非分泌型重組疫苗抗體效價(jià)高;重組BCG較重組M.s的抗體效價(jià)高。結(jié)論:構(gòu)建了含有Eg95基因序列和BCG-Ag85B信號(hào)肽序列的細(xì)粒棘球絳蟲Eg95重組非分泌型和分泌型BCG-Eg95和M.s-Eg95分枝桿菌疫苗。比較兩型重組疫苗免疫小鼠后體液免疫水平,重組BCG-Eg95和M.s-Eg95疫苗可在BCG和M.s中表達(dá)Eg95抗原蛋白;分泌型重組疫苗較非分泌型重組疫苗免疫效果強(qiáng);作為疫苗載體,BCG較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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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