日本血吸虫重组两歧双歧杆菌pGEX-Sj14-3-3疫苗构建及鉴定  

Construction and identification of recombinant vaccine Bifidobacterium bifidum pGEX-Sj14-3-3 of Schistosoma japonicum

作　　者：张宁[1] 李文桂[1] 

机构地区：[1]重庆医科大学附属第一医院传染病寄生虫病研究所寄生虫病研究室,400016

出　　处：《中国地方病学杂志》2011年第4期357-360,共4页Chinese Jouranl of Endemiology

基　　金：重庆市科委地方病重大专项基金（2008AB5055、2008AB5008、2008AB5054）

摘　　要：目的 构建日本血吸虫重组双歧杆菌属两歧双歧杆菌（Bifidobacterium bifidum,Bb）pGEx-Sj14-3-3疫苗,并进行鉴定.方法 从日本血吸虫成虫中提取总RNA,通过RT-PCR扩增Sj14-3-3抗原编码基因.将Sj14-3-3基因定向克隆到大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT中,构建重组质粒pGEX-Sj14-3-3.用重组质粒将大肠埃希菌BL21（DE3）转化为感受态细胞,抽提重组质粒进行双酶切,鉴定载体片段和基因片段长度：采用电穿孔法,用重组质粒pGEX-Sj14-3-3转化Bh,构建Bb（pGEX-Sj14-3-3）疫苗,抽提疫苗的质粒进行PCR鉴定,比较重新构建的pGEX-Sj14-3-3基因片段长度与日本血吸虫成虫中Sjl4-3-3基因片段长度是否相同.结果 RT-PCR扩增出的日本血吸虫成虫Sj14-3-3基因片段长度为399 bp;双酶切鉴定,重组质粒pGEX-Sj14-3-3载体片段长度为4947 bp,Sj14-3-3基因片段长度为399 bp;在重组的Bb（pGEX-Sj14-3-3）疫苗中,得到Sj14-3-3基因片段长度为399 bp,与预期的结果一致.结论 成功构建了日本血吸虫重组Bb（pGEX-Sj14-3-3）疫苗.Objective To construct and identify recombinant vaccine Bifwlobacterium bifidum（Bb）pGEX-Sj14-3-3 of Schistosoma japonicum（Sj）. Methods Total RNA was extracted from adult Sj, antigen encoding gene Sj14-3-3 was amplified by RT-PCR and cloned into Escherichia coli （E. coli）-Bb shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Sj14-3-3. The recombinant plasmid was transformed into E. coli BL21 （DE3）.The plasmid was extracted and identified by using BamH I and EcoR I. Then pGEX-Sjl4-3-3 was electroporated into Bb to construct recombinant Bb （pGEX-Sj14-3-3） vaccine. The extracted plasmid of the recombinant Bb （pGEX-Sj14-3-3） vaccine was identified by PCR, and the size of the products was compared with Sj14-3-3 gene of adult worms.Results Sj14-3-3 of 399 bp in length was amplified by RT-PCR. The products were digested by BamH I and EcoR I , and the fragments length of plasmid pGEX-Sj14-3-3 vector was 4947 bp, and of Sj 14-3-3 gene was 399 bp.The product of 399 bp Sj14-3-3 gene was also amplified by PCR from template of the extracted plasmid of the recombinant Bb（pGEX-Sj14-3-3 ） vaccine. The size of the product obtained was just the same as expected.Conclusion The recombinant Bb（pGEX-Sj14-3-3） vaccine of Sj is successfully constructed.

关 键 词：血吸虫 日本 两歧双歧杆菌 质粒 疫苗 

分 类 号：R686[医药卫生—骨科学]