【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1 680 bp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。更多还原

【Abstract】 Objective To construct recombinant BCG-EmⅡ/3 vaccine of Echinococcus multilocularis and to analyze expression efficiency of EmⅡ/3 antigen encoding gene in rBCG.Methods The total RNA was extracted from alveolar hydatid cyst by ultrasound-breaking,EmⅡ/3 antigen gene amplified by RT-PCR from the total RNA was cloned into E.coli-Mycobacterium shuttle plasmid pBCG to construct pBCG-EmⅡ/3.The recombinant plasmid was electroporated into BCG to construct rBCG-EmⅡ/3 vaccine,the expression product of this vaccine was identified by Western blotting.Results 1680 bp EmⅡ/3 gene was successfully amplified by RT-PCR and cloned into pBCG by restriction analysis,rBCG-EmⅡ/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-EmⅡ3 vaccine of Echinococcus multilocularis is successfully constructed,which lays the theoretical foundation of exploitation and utilization of this vaccine.更多还原