鸡传染性支气管炎病毒多表位核酸疫苗的构建和表达

Construction and expression of multi-epitope chimeric gene of IBV in vitro

通过文献报道和计算机软件分析筛选出鸡传染性支气管炎病毒(IBV)结构蛋白基因S1、S2、N基因的T、B细胞的优势抗原表位共7段,采用人工合成及PCR方法将各抗原表位以柔性氨基酸(GP/GA)作为接头串联成一条全新的多表位嵌合基因F,并定向克隆入真核表达质粒pVAX1,采用酶切分析与序列测定方法筛选鉴定阳性重组质粒。阳性质粒pVAX-F通过脂质体转染COS-7细胞进行表达研究。提取转染细胞总RNA,RT-PCR方法检测到目的基因的转录;间接免疫荧光试验(IFA)检测到特异性荧光存在。表明表达产物能与相应抗体特异性结合,证明了具有一定的生物学活性和抗原性,有望成为抗IBV的核酸疫苗。

To design a new gene encoding the multi-epitope chimeric antigen of IBV and express the chimeric gene,the dominant epitopes of S1 ,S2,and N gene of IBV were analyzed and selected by computer software and reported references. A recombinant multi-epitope chimeric gene including IBV seven multi-epitope genes was constructed and cloned into eukaryotic expression vector pVAX1, respectively. Eukaryotic vector pVAX-F was transfected into the COS-7 cells by lipofectamine,and the whole RNA of the COS-7 cells was extracted to detect the mRNA of the chimeric gene by RT-PCR. IFA was also utilized to detect the expression products and its bioactivity. Results showed that the reconstructed plasmid could express product efficiently and the product can specially recognized by the positive anti-IBV/S1 protein/N protein serum, respectively. This proved the reconstructed eukaryotie expression vector pVAX-F might be a candidate vaccine strains for IBV.