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 GPS, 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 antibod-ies 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.%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peDNA3.1(+)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.
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