猪繁殖与呼吸综合征病毒GP3/GP5/M真核表达载体的构建及其体液免疫应答

本试验旨在构建表达猪繁殖与呼吸综合征病毒(PRRSV)GP3、GP5和M蛋白的真核重组质粒。以PRRSV LN株为模板,采用PCR方法扩增出GP3、GP5、M基因片段,将扩增的GP5、M通过Linker序列串联成GP5-M,然后将GP3与GP5-M双酶切后插入pcDNA3.1(+)构建重组质粒pcDNA3.1-GP3-GP5-M,将其转染COS7细胞。PCR鉴定表明重组质粒pcDNA3.1-GP3-GP5-M含有PRRSV GP3、GP5-M基因,间接免疫荧光检测表明GP3、GP5-M蛋白在COS7细胞内获得表达。Western blotting检测证实GP3、GP5、M蛋白获得正确表达,并且所表达的GP3、GP5、M蛋白是融合蛋白。将pcDNA3.1-GP3-GP5-M免疫BALB/c小鼠,首免后2周可检测到特异性PRRSV中和抗体,首免后8周中和抗体效价最高可达1∶32。进一步将pcDNA3.1-GP3-GP5-M免疫断奶仔猪,首免后4周即可产生1∶4～1∶8的中和抗体。本试验成功构建了表达PRRSV GP3、GP5和M融合蛋白的真核重组质粒pcDNA3.1-GP3-GP5-M,中和抗体检测表明pcDNA3.1-GP3-GP5-M具有良好的免疫原性,从而为PRRSV基因工程疫苗的研制奠定基础。

Construction and Humoral Immune Response of Recombinant Plasmid Co-Expressing GP3, GP5 and M Proteins of Porcine Reproductive and Respiratory Syndrome Virus

The aim of this study was to construct a recombinant eukaryotic plasmid co-expressing GP3,GP5 and M proteins of porcine reproductive and respiratory syndrome virus(PRRSV).GP3,GP5 and M protein gene fragments of PRRSV LN strain were amplified by PCR and cloned into vector pcDNA3.1(+) to produce the recombinant plasmid pcDNA3.1-GP3-GP5-M.The plasmid was transfected into COS7 cells,and immunological responses to the plasmid were investigated in BALB/c mice and piglets.The recombinant plasmid pcDNA3.1-GP3-GP5-M was transfected into COS7 cells,PCR identification and indirect immunofluorescence assay proved that GP3,GP5-M gene of PRRSV were expressed in COS7 cells.The result of Western blotting showed that the GP3 and GP5 and M proteins were co-expressed and formed fusion protein.The BALB/c mice were injected with recombinant plasmid pcDNA3.1-GP3-GP5-M to evaluate the induced immunological responses in vivo.The specific detectable anti-PRRSV neutralizing antibodies were produced in the vaccinated mice at 2 weeks and reached a peak 1:32 at 8 weeks after primary vaccination.In addition,it was observed that the 1:4-1:8 neutralizing antibodies of the vaccinated piglets.The results showed that the recombinant plasmid pcDNA3.1-GP3-GP5-M has been constructed successfully,and the recombinant plasmid has well immunity,the recombinant plasmid could be a basis to eukaryotic vector vaccine for PRRSV infection.