编码基因完整的伪狂犬病毒细菌人工染色体的构建

Construction of a bacterial artificial chromosome containing the complete encoding gene of pseudorabies virus

为了构建包含伪狂犬病毒(PRV)完整编码基因的细菌人工染色体(BAC),首先对PRV基因组进行了分析,确定了单一酶切位点AcclⅠ,用PCR方法扩增出上下游同源臂、EGFP表达盒并同BAC质粒一起克隆到p UC_(18)质粒,获得转移载体;将经限制性内切酶AcclⅠ处理过的病毒全基因组连同转移载体共同转染BHK21细胞,获得重组病毒;利用PCR检测、电镜观察及生长曲线测定对野毒与重组毒的基本特性进行了比较。结果表明:重组病毒可以稳定地表达绿色荧光,BAC在传代过程中能够稳定存在;重组病毒在电镜下的形态与野毒未见差异,其生长曲线也基本同野毒吻合。说明成功构建的编码基因完整的PRV细菌人工染色体可用于该病毒的结构生物学及致病机理研究。

To construct a bacterial artificial chromosome （BAC） containing the complete encoding gene of pseudorabies vires （ PRV）, the PRV genome was firstly analyzed to determine the single restriction site Aecl. The upstream and downstream homologous arms and enhanced green fluorescent protein （EGFP） expression cassettes were amplified by PCR and cloned into pUC18 plasmid with BAC plasmid to obtain the transfer vector. The transfer vector and the full length of genome disposed with the Aecl I were co - transfected into BHK21 cells to generate the recombinant virus. The basic characteristics between the wild - type virus and recombinant virus were compared using PCR detection, electron microscope observation and the detection of growth curve. The results showed that the recombinant virus could stably express green fluorescence, the BAC could stably exist in the process of passage. There is no difference on morphology between the recombinant virus and wild- type vires un- der the electron microscope, and the growth curve of the recombinant virus was basically consistent with that of the wild - type. The results indicate that the successfully constructed bacterial artificial chromosome containing the complete encoding gene of PRV can be used to study the viral structural biology and pathogenesis.