Construction of Recombinant Plasmid pcDNA4.0-ORF5-E2 of PRRSV and CSFV and Its Immunity Test of Mice
|School||Northwest University of Science and Technology|
|Course||Preventive Veterinary Medicine|
|Keywords||porcine reproductive and respiratory syndrome virus(PRRSV) classical swine fever virus(CSFV) eukaryotic expressing plasmid immunity test of mice|
In order to explore new method to control the porcine reproductive and respiratory syndrome virus(PRRSV) and classical swine fever virus(CSFV), the recombinant plasmid was constructed containing the ORF5 of PRRSV and E2 of CSFV envelope protein genes and test the immunoreaction on mice. This study consists of two parts:1. The reverse transcription and polymerase chain reaction(RT-PCR) was used to amplify PRRSV ORF5 gene and CSFV E2 gene. 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-ORF5-E2 was obtained.3. Then, 10 kunming mice were immuned with the purified pcDNA4.0-ORF5-E2 and another with pcDNA4.0 into hind legs tibial muscle(50μg per leg) by intramuscular injection, and were boosted on week 1 and week 2 post-immuned. The levels of PRRSV and CSFVs′ELISA antibodies were detected in peripheral bloods of the boosted mice 10 days after the latest immunization by indirect ELISA. Then ORF5-E2 gene was detected by PCR from heart, blood, liver, lung, spleen, kidney and muscle of mice different time post immunization (PI). The results showed that the expressed envelope protein from the plasmid could induce to produce serum antibodies. Recombinant plasmid pcDNA4.0-ORF5-E2 was detected in the muscle, cardiac muscle, blood, liver, lung, spleen, kidney from 14 d to 56 d PI, and was detected from the muscle till 84 d PI.