新型鸡痘病毒穿梭载体的构建及其体外表达

为构建用于筛选的新型鸡痘病毒载体,采用染色体步移技术对鸡痘病毒282E4株TK基因的下游序列进行测序,并结合扩增出的左右同源重组臂TKL、TKR片段、启动子、MCS及终止信号构建出三表达盒穿梭载体pTKE3。采用基因工程技术将绿色荧光蛋白EGFP基因克隆入3个启动子的下游,构建出pTKE3-A、pTKE3-B、pTKE3-C验证表达盒质粒。结果显示,构建的3个验证质粒分别与鸡痘病毒282E4株共转染鸡胚成纤维细胞,均可在转染12h后观察到绿色荧光。结果表明,构建的三表达盒载体均可成功表达外源基因,这为进一步构建多价重组鸡痘病毒活载体疫苗奠定了基础。

Construction and expression in vitrostudies on shuttle vector of fowlpox virus

To construct a new vector for screening recombinant fowlpox virus,thymidine kinase(TK) gene of fowlpox virus(FPV) 282E4 strain was partial sequenced using chromosome walking techniques.Expression cassettes containing promoters,multiple cloning site(MCS) and termination signals were designed and synthesized so as to express three exogenous genes.The expression cassettes were inserted between the two arms of the TKL(TK left) and TKR(TK right) gene,and then the new fowlpox virus shuttle vector pTKE3 was constructed.To verify the features of expression cassette,the enhanced green fluorescent protein gene was cloned into downstream of three promoter to construct three plasmids.The constructed plasmids pTKE3-A,pTKE3-B and pTKE3-C which were transfected by lipofectamine on chicken embryo fibroblasts cell pre-infected with FPV 282E4 respectively,and the expression efficiency was identified by observing fluorescence after transfection at 12 h,which show that the shuttle vector containing three cassettes can express foreign proteins successfully.The present study lays a foundation for further construction of a polyvalent live vector vaccine of recombinant fowlpox virus.