猪带绦虫重组双歧杆菌疫苗人工传代后的稳定性研究  

Stability of the recombinant Bifidobacteriumvaccines against Taenia soliumafter passaging

作　　者：周必英[1] 刘美辰[1] 杨凤娇[1] 

机构地区：[1]贵州遵义医学院寄生虫学教研室,贵州遵义563003

出　　处：《中国病原生物学杂志》2014年第8期710-712,I0003,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81160206);贵州省教育厅资助项目(No.2010040);遵义医学院博士启动基金项目(No.ZMKD2013-016)

摘　　要：目的研究猪带绦虫TSO45W-4B基因、TSOL18基因和TSO45W-4B-TSOL18融合基因重组双歧杆菌(Bb)疫苗人工传代后的稳定性。方法分别将猪带绦虫重组质粒pGEX-TSO45W-4B、pGEX-TSOL18和pGEX-TSO45W-4B-TSOL18电转入长双歧杆菌(Bifidobacteria longum),获得阳性菌株pGEX-TSO45W-4B/Bl、pGEX-TSOL18/Bl和pGEX-TSO45W-4B-TSOL18/Bl,分别经人工传代10次后抽提质粒,进行PCR鉴定和稳定性分析。结果经PCR鉴定,从第7代开始,pGEX-TSOL18/Bl菌株出现质粒丢失的现象,随着培养时间的延长,质粒丢失现象越来越严重,第8代,第9代均出现丢失现象。而pGEX-TSO45W-4B/Bl菌株从第8代开始出现质粒丢失现象,随后的第9代,第10代也陆续出现丢失现象。pGEX-TSO45W-4B-TSOL18/Bl菌株相对稳定,但从第10代开始,开始出现了质粒丢失现象。结论重组质粒转化菌pGEX-TSO45W-4B/Bl、pGEX-TSOL18/Bl和pGEX-TSO45W-4B-TSOL18/Bl人工培养时分别从人工传代培养第8代、第7代和第10代开始出现质粒丢失现象,可稳定遗传7代、6代和9代,显示具有不同程度的遗传稳定性,这为猪带绦虫重组Bb疫苗的进一步研究奠定了基础。Objectives To develop recombinant Bifidobacteriumvaccines with the TSO45W-4Bgene or TSOL18 gene of Taenia soliumor with the TSO45W-4B-TSOL18 fusion gene and to study their stability after passaging. MethodsThe recombinant plasmids pGEX-TSO45W-4B,pGEX-TSOL18,and pGEX-TSO45W-4B-TSOL18 featuring genes from T.solium were transformed into Bifidobacterialongum in order to obtain the positive strains pGEX-TSO45W-4B/Bl,pGEX-TSOL18/Bl,and pGEX-TSO45W-4B-TSOL18/Bl.After 10 passages,the stability of the positive strains was determined with PCR amplification,using the extracted plasmid as a template. Results PCR identification suggested that the plasmid pGEX-TSOL18 disappeared in B.longumafter the 7thof 10 passages.The phenomenon of plasmid loss worsened with a longer incubation time and was evident in the 8thand 9thpassages.The plasmid pGEX-TSO45W-4Bdisappeared in B.longumafter the 8thpassage and was not evident in the subsequent 9thand 10 th passages.The plasmid pGEX-TSO45W-4B-TSOL18 was relatively stable,but the phenomenon of plasmid loss began to occur in B.longumafter the 10 thpassage. Conclusion The recombinant plasmid pGEX-TSO45W-4Bdisappeared in B.longumafter the 8thpassage,pGEX-TSOL18 disappeared after the 7thpassage,and pGEX-TSO45W-4B-TSOL18 disappeared after the 10 thpassage.pGEX-TSO45W-4Bexisted stably in B.longum for 7passages,pGEX-TSOL18 existed for 6,and pGEXTSO45W-4B-TSOL18 existed for 9passages.Different degrees of genetic stability were exhibited in vitro.These results lay the foundation for the further study of recombinant Bifidobacteriumvaccines against T.solium.

关 键 词：猪带绦虫 重组双歧杆菌疫苗 传代 稳定性 

分 类 号：R383.3[医药卫生—医学寄生虫学]