Study On Cloning And Protective Efficacy Of Recombinant Outer Membrane Protein N (rOmpN)-based Vaccine Of Edwarsiella Ictaluri

The ompN complete genes were cloned from the strain Edwarsiella Ictaluri GPY. Based on the nucleotide sequence and the corresponding amino acid sequence of the E. ictaluri ompN genes, we analysed the molecular characteristics of the ompN genes and amino acids of ompN genes by using bioinformatic softwares. Then we cloned and expressed the pompNs genes which signal peptides and transmembrane regions were cut. After that imunogenicity was determined by western blotting. Then rOmpNs were injected into healthy channel catfishes to observe the protective efficacy against E.ictaluri. This experiment covers three main chapters listed below:1. Cloning and sequence analyses of three ompN complete genes of E. ictaluri. Postive plasmid of pMD19-T-omppN1, pMD 19-T-ompN2 and pMD19-T-ompN3 were constructed and analyzed by bioinformatics softwares. The results showed that OmpN1,OmpN2 and OmpN3 belonged to OM_channel family in. Signal peptide and transmembrane areas of all three genes were same, locating at the N-terminus between 1 and 22. OmpN1,OmpN2 and OmpN3 were all hydrophobic proteins. Antigen epitope prediction showed that OmpNl has 15 antigen epitopes, among which 13 distributed on the protein surface; OmpN2 has 14 antigen epitopes,8 on the protein surface; OmpN3 has 15 antigen epitopes,12 on the protein surface. B cell epitope prediction showed that OmpN1, OmpN2 and OmpN3 have 10,9,11 B cell epitopes respectively. Phylogenetic tree was constructed by MEGA. The result showed that OmpN shared closer similarities among other outer membrane of gram-negative bacteria.OmpN2 has a close relatives with E.coli, and OmpNl; OmpN2 and OmpN3 were in different branches.2. Cloning, expression of the ompN immunogenic regions of E. ictaluri and polyclonal antibody preparationBased on the bioinformatics analysis results,we selected the sequences which had removed the signal pepetide to design the specific primers for PCR amplification. The positive recombinant plasmid were identified by bacterial colony PCR and ligated the purified fragment of the pompNl, pompN2 and pompN3 gene into expression plasmid named pET-32a(+)-pompN1, pET-32a(+)-pompN2, pET-32a(+)-p ompN3 and then transformed into E.coli BL21 (DE3) and Rosetta (DE3) competent cell, then induced by IPTG. After that, we used 0.3%(V/V) formalin inactived E. ictaluri by to immune the New Zealand White rabbits for the preparation of anti-serum. Immunogenicity was detected by western-blotting. The results showed that the recombinant protein was expressed as inclusion bodies, which were about 61.8kDa,58.7kDa and 59.1kDa. Agglutination tests showed that the titer of the rabbit anti-serum was 1:32. Rabbit anti-serum could bind specifically to the recombinant rOmpN.3. The immune prective effiacy of rOmpNs to channel catfish against E. ictaluriHealthy channel catfish were immuned with the recombinant protein which were preparaed in Chapter 2, the concentration of 2μg/g fish, and PBS as a control. Channel catfish were divided randomly into five groups rOmpNl, rOmpN2, rOmpN3, mixture and PBS, which were injected intraperitoneally (i.p.) with rOmpN1, rOmpN2, rOmpN3, mixture (mixed rOmpN1, rOmpN2 and rOmpN3 with 1:1:1) and PBS respectively. The serum was collected from vaccinated fish at 7d,14d,21 d,28d,35d, 42d,49d and 56d. ACH50 activity, lysozyme activity, T-SOD, AKP, ACP total protein and specfic antibody titers were performed. Channel catfishes were challenged with E. ictaluri GPY at 29th d post-vaccination, and the mortality was monitored, the percent of the Relative Percent of Survival (RPS) was calculated. The results showed that the recombinant protein OmpNl, OmpN2 and OmpN3 could improve the serum ACH50, lysozyme activity, T-SOD, AKP, ACP, total protein and specfic antibody titers after immuned. And antibody began to increase at 7d post-vaccination and peaked at 28th-35thd, and then decreased at 42thd. The mixture group was the highest (1:128), second was rOmpN3 (1:64), third was rOmpN1 and rOmpN2 (1:32). The mortality rates were 30%,30%,25%,20% and 80% respectively, which correspond to RPS rates of 62.5%,62.5%,68.75% and 75%, respectively. All these results indicated that the three recombinant protein OmpNl,OmpN2 and OmpN3 were effective and had a well defined immunoprotectivity, and the mixture had a best effect, which could be demonstrated as an vaccine against E. ictaluri.