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
李文桂 1欧兴坤 1何爱琳1
作者信息
- 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
- 折叠
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,SDS-PAGE显示表达产物为40×103Mr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.
Abstract
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 pGEX-Sj14-3-3 and competent cells of Enterococcus faecalis ATCC47077 strain were preparated;pGEX-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Key words
Enterococcus faecalis/Schistosoma japonicum/Sj14-3-3/Vaccine引用本文复制引用
基金项目
国家自然科学基金项目(30801052)
国家自然科学基金项目(30671835)
国家自然科学基金项目(30500423)
国家自然科学基金项目(30200239)
出版年
2024
NETL
NSTL
万方数据