目的构建弓形虫Rhomboid基因和猪IL-2基因的重组卡介苗(BCG),并分析其免疫保护效果。方法应用RT-PCR技术分别扩增弓形虫Rhomboid基因和猪IL-2基因,克隆至p MD18-T载体中,双酶切鉴定及测序正确后,将弓形虫Rhomboid基因和猪IL-2基因串联后分别连接至穿梭表达载体pMV261和整合表达载体pMV361中,电转化BCG,制备重组BCG pMV261-IL-2-Rho和pMV361-IL-2-Rho。分别经颈部肌肉注射免疫猪,共3次,每次间隔2周,免疫剂量均为108 CFU/只,采用ELISA法检测免疫前后猪体内抗体(IgG)滴度、Th1型及Th2型细胞因子的变化情况;第3次免疫后2周通过腹腔注射的方式接种弓形虫China I株速殖子1×108个/只进行攻虫试验,观察重组BCG对猪的免疫保护效果。结果成功构建了重组BCG p MV261-IL-2-Rho和p MV361-IL-2-Rho。重组BCG免疫猪后,随着免疫次数的增加,血清中抗体滴度也逐渐升高,且与PBS对照组相比,差异有统计学意义(P<0.01)。3次免疫后,pMV261-IL-2-Rho组血清Th1型细胞因子IL-2...
【英文摘要】
Objective To construct a recombinant BCG(r BCG)with Toxoplasma gondii Rhomboid gene and porcine IL-2(Po IL-2)gene, and observe its immunoprotection. Methods Rhomboid gene of Toxoplasma gondii and Po IL-2 gene were amplified by RT-PCR and cloned into p MD18-T vector. After identification by restriction analysis and sequencing,Rhomboid and Po IL-2 genes were linked and subcloned into pMV261 and pMV361 vectors respectively. The constructed recombinant plasmids were transformed to BCG by electroporation to obta...
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