摘要
目的构建细粒棘球绦虫重组BCG-Eg95疫苗,分析Eg95分子在该疫苗中的表达效率。方法超声粉碎细粒棘球蚴组织提取总RNA,通过RT-PCR扩增Eg95的抗原编码基因;将该基因定向克隆到大肠杆菌-分枝杆菌穿梭表达载体pB-CG,构建重组质粒pBCG-Eg95;电穿孔法转化BCG,构建细粒棘球绦虫重组BCG-Eg95疫苗。免疫印迹分析重组BCG-Eg95疫苗的表达产物。结果RT-PCR成功扩增出471bp的Eg95抗原编码基因;双酶切证实Eg95抗原编码基因成功插入pBCG中;PCR证实rBCG-Eg95疫苗构建成功;免疫印迹分析发现重组BCG-Eg95疫苗的表达产物在相对分子质量(Mr)约为16.5×103处有明显的目的蛋白表达条带,且能被感染细粒棘球蚴的鼠血清特异识别。结论成功构建了细粒棘球绦虫重组BCG-Eg95疫苗,为而后的开发利用奠定了理论基础。
Objective To construct recombinant BCG-Eg95 vaccine of Echinococcus and analyze the expression efficiency of the gene encoding Eg95 antigen in rBCG. Methods The total RNA was extracted from hydatid cyst by ultrasound-breaking. Eg95 antigen gene amplified by RT-PCR from the total RNA was cloned into E. coli-Mycobacterium shuttle plasmid pBCG to construct pBCG-Eg95. The recombinant plasmid was electroporated into BCG to construct rBCG-Eg95 vaccine and the expression product of this vaccine was identified by Western blotting. Results Eg95 gene of 471 bp was successfully amplified by RT-PCR and cloned into pBCG by restriction analysis; rBCG- Eg95 vaccine was constructed successfully. The expression product with an approximate Mr of 16.5 ×10^3 could be recognized by sera from mice infected with CE. Conclusion rBCG-Eg95 vaccine of Echinococcus echinococcus is successfully constructed, which lays the theoretical foundation for exploitation and utilization of this vaccine.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第5期494-498,共5页
Immunological Journal
基金
国家自然科学基金资助项目(30671835
30500423和30200239)
关键词
细粒棘球绦虫
重组BCG-EG95疫苗
构建
表达效率
Echinococcus echinococcus
Recombinant BCG-Eg95 vaccine
Construction
Expression efficiency
