PRRSV ORF5基因和CSFVE2基因重组真核表达质粒的构建及小鼠免疫试验

Construction of Recombinant Plasmid pcDNA4.0-ORF5-E2 of PRRSV and CSFV and the Immune Test in Mice

根据猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)已知序列设计3对引物,采用RT-PCR方法扩增PRRSV ORF5基因和CSFV E2基因,目的基因依次插入真核表达载体pcDNA4.0,经PCR、酶切及测序分析,重组质粒构建成功,并命名为pcDNA4.0-ORF5-E2。重组质粒回收提纯后,以100μg/只剂量免疫小鼠,免疫3次。三免后10 d采血并制备血清,用间接ELISA检测小鼠血清中PRRSV及CSFV抗体效价。结果显示,所构建的重组质粒能够诱导小鼠产生PRRSV及CSFV抗体。

Based on the published nucleotide sequences of porcine reproductive and respiratory syndrome virus （PRRSV） and classical swine fever virus（CSFV）,three pairs of primers were designed and used to amplify ORF5 and E2 genes by RT-PCR,and the amplified products were directionally cloned into eukaryotic expression plasmid pcDNA4.0 to construct recombinant plasmid. The recombinant plasmid pcDNA4. 0- ORF5-E2 was validated by PCR, enzyme digestion and sequencing. Then, 10 Kunming mice were inoculated with the purified pcDNA4. 0-ORF5-E2 into the tibial muscle of hind legs by intramuscular injection , and were boosted on week 1 and week 2 post-inoculation. The antibody levels of PRRSV CSFV were detected in boosted mice 10 days after the last immunization by indirect ELISA. The results showed that the mice could be induced to produce antibodies against PRRSV and CSFV by the expressed envelope protein from the plasmid.