Construction Of Recombinant Plasmid Of Porcine Reproductive And Respiratory Syndrome Virus And Its Immunoreaction

In order to explore new method to control the porcine reproductive and respiratory syndrome virus(PRRSV), the recombinant plasmid was constructed containing the ORF3,ORF5 and ORF6 of the porcine reproductive and respiratory syndrome virus(PRRSV) envelope protein genes and test the immunoreaction on mice. This study consists of three parts:1. The reverse transcription and polymerase chain reaction(RT-PCR) was used to amplify PRRSV ORF3,ORF5 genes. The RT-PCR products were cloned into pMD18-T in series, and were ligated into the eukaryotic expression vector pcDNA4.0. Then the recombinant plasmid pcDNA4.0-ORF3-ORF5 was obtained.2. The ORF6 were connected in series with ORF3-ORF5, and were cloned into the eukaryotic expression vector pcDNA4.0 to construct recombinant plasmid pcDNA4.0-ORF6 -ORF3-ORF5. Then the recombinant plasmid pcDNA4.0-ORF6- ORF3-ORF5 was obtained.3. Then, 10 kunming mice were inoculated with the purified pcDNA4.0-ORF3-ORF5 and another with pcDNA4.0-ORF6-ORF3-ORF5 into hind legs tibial muscle(50μg per leg) by intramuscular injection, and were boosted on week 2 post-inoculation. The levels of serum antibodies and neutralizing antibodies were detected in peripheral bloods of the inoculated mice or the boosted mice every 7 days post-inoculation or post-booster-inoculation by indirect ELISA. Then ORF6-ORF3-ORF5 gene was detected by PCR from heart, brain, blood, liver, lung, spleen, kidney and muscle of mice and piglets different time post immunization (PI). The results showed that the expressed envelope protein from the plasmid could induce to produce high levels of serum antibodies and neutralizing antibodies. Recombinant plasmid pcDNA4.0-ORF6-ORF3-ORF5 was detected in the brain 7d-42d PI, in the liver, lung, spleen, kidney from 7 d to 70 d PI, in the blood and heart 7d to 84d PI, and was detected from the muscle till 98d PI.