日本血吸虫重组质粒pGEX-Sj14-3-3-Sj32的构建及其在大肠埃希菌BL21（DE3）中的表达  

Construction and expression of a recombinant plasmid pGEX-Sj14-3-3-Sj32 of Schistosoma japonicum inEscherichia coli BL21 （DE3）

作　　者：覃婷[1] 李文桂[1] 谭建蓉[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,400016

出　　处：《中华地方病学杂志》2015年第10期723-728,共6页Chinese Journal 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质粒pGEX—Sjl4—3—3-Sj32，探讨重组质粒在大肠埃希菌BL21（DE3）中的表达。方法以重庆医科大学附属第-医院传染病寄生虫病研究所保存的质粒pGEX—sj14—3-3和pET28ct。Si32为模板，通过PCR扩增Sil4—3-3和Si32抗原编码基因，然后采用基因拼接法（geneSOEing）剪接Sil4-3-3和Sj32基因，得到Sjl4-3-3-Sj32融合基因。定向克隆人穿梭载体pGEX-1kT，构建重组质粒pGEX-Sjl4-33Sj32，并采用双酶切法进行验证。将重组质粒转化大肠埃希菌BL21（DE3），经异丙基硫代-B-D-半乳糖苷（IPTG）诱导表达后，用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹（Westernblot）法对表达产物进行分析鉴定。结果基因拼接法得到约1750bp的Sjl4—3—3-Sj32融合基因；双酶切证实sj14-3-3-Sj32融合基因成功插入pGEX-1kT载体中；SDS—PAGE分析显示，表达产物为相对分子质量约为73×100的重组蛋白；Westernblot显示，重组蛋白可被日本血吸虫感染的兔血清识别。结论成功构建日本血吸虫重组质粒pGEX—sj14-33Sj32，该重组质粒在大肠埃希菌BL21（DE3）中得到了高效融合表达，且表达的融合蛋白具有特异的抗原性。Objective To construct and express a recombinant plasmid pGEX-Sj14-3-3-Sj32 of Schistosoma japonicum in Escherichia coli （E. Coli） BL21 （DE3）. Methods Sj14-3-3 and Sj32 antigen genes were amplified by PCR from template of plasmids pGEX-Sjl4-3-3 and pET28a-Sj32 which were extracted from recombinant bacteria BL21 （pET28ct-Sj32） and BL21 （pGEX-Sjl4-3-3） stored in Institute of Infectious and Parasitic Disease of the First Affiliated Hospital of Cbongqing Medical University. Sj14-3-3-Sj32 fusion gene obtained with gene SOEing was cloned into the vector pGEX-lkT to construct pGEX-Sj14-3-3-Sj32 which was identified by double digestion. The recombinant plasmid pGEX-Sj14-3-3-Sj32 was transformed into E. Coli BL21 （DE3）L The recombinant strains were induced by isopropyl-13-d-thiogalactoside （IPTG）, and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and Western blotting. Results The 1 750 bp Sj14-3-3-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into the vector pGEX-lkT verified by restriction analysis, the recombinant plasmid pGEX-Sj 14-3-3-Sj32 was successfully constructed. The molecular mass of the expressed recombinant protein was proximately 73 x 10^3 as detected by SDS-PAGE. Western blotting confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosomajaponicum. Conclusion The recombinant plasmid pGEX-Sj 14-3-3-Sj32 is successfully constructed and could be highly expressed in E. coli and the expressed recombinant protein has specific antigenicity.

关 键 词：血吸虫 日本 质粒 大肠埃希菌 基因表达 

分 类 号：R383.24[医药卫生—医学寄生虫学]