粪肠球菌介导的日本血吸虫重组Efs-Sj14-3-3疫苗构建、鉴定及表达  

Construction,identification and expression of Enterococcus faecalis based recombinant Efs-Sj14-3-3 vaccine of Schistosoma japonicum

作　　者：李文桂[1] 欧兴坤 何爱琳 LI Wengui;OU Xingkun;HE Ailin(Institute of Infections and Parasitic Diseases,the First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China)

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,重庆400016

出　　处：《热带医学杂志》2024年第6期818-822,共5页Journal of Tropical Medicine

基　　金：国家自然科学基金项目(30801052,30671835,30500423,30200239)。

摘　　要：目的 构建粪肠球菌(Efs)为载体的日本血吸虫(Sj)重组Efs-Sj14-3-3疫苗,并研究其表达效率。方法 制备重组质粒pGEX-Sj14-3-3和粪肠球菌ATCC47077株感受态。采用电穿孔技术将重组质粒pGEX-Sj14-3-3转化粪肠球菌ATCC47077株,构建重组Efs-Sj14-3-3疫苗。抽提质粒进行PCR扩增鉴定;经异丙基-β-D-硫代半乳糖苷(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)和免疫印迹对表达产物进行分析和鉴定。结果 以从重组Efs-Sj14-3-3抽提的质粒为模板进行PCR,可扩增出约399 bp的Sj14-3-3基因片段,SDSPAGE显示表达产物为40×103 Mr的融合蛋白,表达蛋白约占菌体总蛋白的18%;免疫印迹表明融合蛋白能被日本血吸虫感染的患者血清识别。结论 成功构建了日本血吸虫重组Efs-Sj14-3-3疫苗,表达的融合蛋白具有特异的抗原性。Objective To construct an Enterococcus faecalis(Efs) based recombinant Efs-Sj14-3-3 vaccine of Schistosoma japonicum(Sj) and observe its expression efficiency. Methods The recombinant plasmid p GEX-Sj14-3-3 and competent cells of Enterococcus faecalis ATCC47077 strain were preparated;p GEX-Sj14-3-3 was electroporated into Enterococcus faecalis competent cells to construct recombinant Efs-Sj14-3-3 vaccine;the plasmid was extracted from recombinant Efs-Sj14-3-3 for PCR identification. The recombinant Efs-Sj14-3-3 vaccine was expressed through isopropyl-beta-D-thiogalactopyranoside(IPTG) induction,and the recombinant protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting. Results PCR showed that 399 bp Sj14-3-3 gene was amplified when the extracted plasmid from recombinant Efs-Sj14-3-3 as template;the expressed GST-Sj14-3-3 fusion protein was approximately 40×103 Mr by SDS-PAGE;the amount of the expressed protein was 18% of the total bacterial proteins. Western blotting showed that the expressed protein could be recognized by sera from patients infected with Schistosoma japonicum. Conclusion The recombinant Efs-Sj14-3-3 vaccine of Schistosoma japonicum was successfully constructed, and the expressed fusion protein shows specific antigenicity.

关 键 词：粪肠球菌 日本血吸虫 SJ14-3-3 疫苗 

分 类 号：R383.33[医药卫生—医学寄生虫学]