【摘要】 为探索猪繁殖与呼吸综合征病毒(PRRSV)核酸疫苗用于免疫预防的可行性,试验用PCR方法扩增出PRRSV HB-3株GP5、M和N基因,通过Linker序列将GP5和M串联为GP5-M,双酶切后和N基因一起插入真核表达载体构建重组质粒pcDNA-GP5-M-N,经酶切鉴定表明GP5、M和N基因和载体连接正确。然后将重组质粒转染至Marc-145细胞,经间接免疫荧光及Western blot分析证实重组蛋白能在Marc-145细胞中表达。然后用重组质粒pcDNA-GP5-M-N免疫Balb/c小鼠,中和抗体检测结果表明,首免后2周即有小鼠产生可检测到的病毒中和抗体(1∶4),随后抗体水平快速升高,第8周抗体效价达到最高(1∶32)。说明本试验构建的重组质粒pcDNA-GP5-M-N能诱发免疫小鼠产生较高水平的中和抗体,为PRRSV核酸疫苗的研究奠定了基础。更多还原

【Abstract】 This experiment was conducted to study the DNA vaccine for preventing PRRSV infection.The GP5,M and N genes of porcine reproductive and respiratory syndrome virus(PRRSV)were amplified by PCR,and then were subcloned into eukaryotic expression vectors pcDNA3.1(+).The recombinant named as pcDNA-GP5-M-N was transfected into Marc-145 cells,and its expression was detected by indirect immune fluorescence detection.The results showed that GP5-MN genes of PRRSV HB-3 strain were expressed in Marc-145 cells.Using pcDNA-GP5-M-N to immunize Balb/c mice,anti-PRRSV neutralizing antibodies were detected at two weeks after primary vaccination and reached a peak of titer(1∶32) at eight weeks.These results showed that recombinant plasmid pcDNA-GP5-M-N has been constructed successfully,and with good immunity.These data indicated that pcDNA-GP5-M-N could be used for further development of DNA vaccines for PRRSV.更多还原