Construction And Characterization Of Carrying The Vectors Of Prrs Virus Orf5 Gene Pleuropneumoniae Actinobacillus Identified

Actinobacillus pleuropneumoniae (APP) is a causative pathogen of porcine pleuropneumonia. Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which caused reproductive failure in sows and respiratory illness and high mortality in piglets. The two pathogens are both considered as the most important respiratory diseases of modern pig industry. Based on the previous research of constructing an attenuated mutant of Actinobacillus pleuropneumoniae by gene deletion, the ORF5 gene segment encoding protective epitopes of PRRSV was inserted into the deleting position of the mutant strain genomic DNA for constructing a bacterial vector containing heterogenous gene. The result will offer a substantial basis on developing a live attenuated APP vaccine against APP and PRRSV infections simultaneously.In order to identify the prokaryotic expression and antigenic epitopes of different domains of PRRSV-ORF5 gene, fragments D, L, N and C obtained by deleting the N-terminal signal peptide sequence and/or the transmembrane domain sequence of ORF5 gene were amplified by PCR. Based on cloning and sequence, these fragments were inserted into the prokaryotic expression vector pET-32a respectively. The recombinant expression plasmid containing fragment L, N or C produced recombinant fusion protein with the expected size of 33 kDa, 25 kDa or 29 kDa respectively, whereas fragment D produced no recombinant fusion protein. The recombinant fusion protein of fragment L showed strong positive reaction with anti-PRRSV serum by Western blotting, fragment C showed weak positive, whereas fragment N showed no positive. The result indicated that the C-terminus of GP5 plays an important role in immunoreactivity.The ORF5-L fragment was inserted into the deletion position of the apxIA gene or the apxIC gene for constructing the mutant. Fragments AU (1.8 kb), ORF5-L (348 bp), Chl~r (1.1 kb) and AD (1.7 kb) were obtained from recombinant plasmids of pTAU, pT-ORF5L, pTChl~r and pTAD by double restricted enzymes digestion respectively. All fragments were linked and inserted into the corresponding site of pUC19 for constructing the transfer plasmid pUIA-L-Chl~r. Fragments CU (2.2kb), ORF5-L (348 bp), Chl~r (1.1 kb) and CL (1.8 kb) were obtained from recombinant plasmids of pTCU, pT-ORF5L, pTChl~r and pTCL by the same digestion. All fragments were linked and inserted into the corresponding site of pUC19 for constructing the transfer plasmid pUIC-L-Chl~r. The two transfer plasmids were introduced into the electrocompetent A. pleuropneumoniae serovar 10 for homologous recombination by electroporation respectively. The mutant strains were obtained and identified by PCR and Southern blotting. Characteristics identification of the mutant strains showed that the ORF5-L fragment were introduced into the genome of the mutant strains successfully, and no influence were observed on growth ability compared with the parent strain. The inserted Chl~r and ORF5-L fragments were stable by continuous passages of the mutant strain. Virulence of the mutant strain decreased at least 100 fold compared with the parent strain. Mice were vaccinated with the mutant strain, and the immunized sera were gathered for detecting the anti-GP5 antibody by ELISA. The antibody titre showed an increasing tendency with more times of vaccination. The result showed that the ORF5-L fragment can express in the mutant strain, and the antibody level in the apxIA mutant was obviously higher than that in the apxIC mutant.