猪生殖与呼吸综合征病毒多基因重组真核表达质粒的构建及诱导的小鼠体液免疫

Construction of recombinant plasmid pcDNA4.0-ORF6-ORF3-ORF5 of PRRSV and humoral immunity induced by the plasmid in mice

根据猪生殖与呼吸综合征病毒已知序列设计了3对引物,采用RT-PCR方法分别扩增ORF3、ORF5和ORF6基因,并将其依次与真核表达质粒pcDNA4.0定向连接,构建了重组质粒pcDNA4.0-ORF6-ORF3-ORF5,经提纯后,给试验组小鼠后肢胫前肌注射,每只100μg,2周后再注射1次。首免后每隔7 d采血1次,用间接ELISA检测血清抗体水平并测定其中和抗体水平。结果显示,所构建的重组质粒能够诱导小鼠产生很高的ELISA抗体和中和抗体水平。

Based on the published nucleotide sequence of porcine reproductive and respiratory syndrome virus（PRRSV） ,three pairs of primers were designed and used to amplify ORF3,ORF5 and ORF6 genes by RT-PCR,and the amplified products were cloned into eukaryotic expression plasmid pcDNA4.0 to construct recombinant plasmid pcDNA4.0-ORF6-ORF3-ORF5. Then, 10 Kunming mice were inoculated with the purified pcDNA4.0-ORF6-ORF3-ORF5 into hind legs tibial muscle（50 btg per leg） by intramuscular injection,and were boosted on week 2 post-inoculation. The levels of ELISA antibodies and neutralizing antibodies were detected in peripheral bloods of the inoculated mice or the boosted mice every 7 days postinoculation or post-booster-inoculation by indirect ELISA. The results showed that the expressed envelope protein from the plasmid could induce to produce high levels of ELISA antibodies and neutralizing antibodies.