摘要
目的 构建多房棘球绦虫（Em）重组卡介苗（BCG—EmⅡ／3）疫苗，分析EmⅡ／3分子在该疫苗中的表达效率。方法 超声粉碎泡球蚴组织提取总RNA，通过RT-PCR扩增EmⅡ／3的抗原编码基因；将该基因定向克隆到大肠埃希菌-分枝杆菌穿梭表达载体pBCG，构建重组质粒pBCG—EmⅡ／3；电穿孔法转化BCG，构建多房棘球绦虫重组BCG-EmⅡ／3疫苗。免疫印迹分析重组BCG-EmⅡ／3疫苗的表达产物。结果 RT-PCR成功扩增出1680bp的EmⅡ／3抗原编码基因；双酶切证实EmⅡ／3抗原编码基因成功插入pBCG中；PCR证实rBCG—EmⅡ／3疫苗构建成功；免疫印迹分析发现重组BCG—EmⅡ／3疫苗的表达产物在相对分子质量（Mr）约为65×10^3处有明显的目的蛋白表达条带，且能被活动性泡球蚴病鼠血清特异识别。结论 成功构建了多房棘球绦虫重组BCG—EmⅡ／3疫苗，为疫苗的开发和利用打下了坚实的理论基础。
Objective To construct recombinant BCG-Em Ⅱ/3 vaccine of Echinococcus multilocularis and to analyze expression efficiency of Em Ⅱ/3 antigen encoding gene in rBCG. Methods The total RNA was extracted from alveolar hydatid cyst by ultrasound-breaking, Em Ⅱ/3 antigen gene amplified by RT-PCR from the total RNA was cloned into E.coli-Mycobacterium shuttle plasmid pBCG to construct pBCG-Em Ⅱ/3. The recombinant plasmid was electroporated into BCG to construct rBCG-Em Ⅱ/3 vaccine, the expression product of this vaccine was identified by Western blotting. Results 1 680 bp Em Ⅱ/3 gene was successfully amplified by RT-PCR and cloned into pBCG by restriction analysis, rBCG-Em Ⅱ/3 vaccine was successfully constructed by PCR, the expression product had an obviously target protein brand with relative molecular mass （Mr） of 65 × 10^3, which could be recognized by sera from mice infected with AE. Conclusions rBCG-Em Ⅱ3 vaccine of Echinococcus multilocularis is successfully constructed, which lays the theoretical foundation of exploitation and utilization of this vaccine.
出处
《中国地方病学杂志》
CAS
CSCD
北大核心
2006年第5期490-493,共4页
Chinese Jouranl of Endemiology
基金
教育部重点项目(205131)
重庆市教委科研基金(KJ050313)
重庆市科委科研基金(03-43-8)
重庆市卫生局科研基金(03-2-099)
关键词
多房棘球绦虫
重组BCG—EmⅡ/3疫苗
构建
表达效率
Echinococcus multilocularis
Recombinant BCG-Em Ⅱ/3 vaccine
Construction
Expression efficiency
