Primary Research On Immune Complex Vaccine Of Fowl Cholera

Fowl cholera, which pathogen is pasteurella mutocida (PM), is a kind of acute, contagious infection disease in domestic poultry and wild birds. Different serotype of PM which caused fowl cholera has no cross protection with each other. It was reported that the lipoprotein E (P1pE) which located in the bacterial outer membrane can provide protection to the challenge of heterologous serotype strains of PM. The present study was focused on the cloning, expression of the P1pE gene from C48-1strain of PM, and immune response of the immune complex with P1pE. The results are as follows:1. Cloning and expression of P1pE gene from Pasteurella MultocidaAccording to the P1pE nucleotide sequence of avian Pasteurella multocida ATCC12948, a pair of primers was designed, the (P1pE) gene was amplified from Pasteurella multocida C48-1 strain by PCR. Results of sequence analysis showed the nucleotide sequence homology was 92% to 99% compared with the reportedsequence, and amino acid homology was 90% to 99%. It was cloned into the prokaryote expression vector pET-28a and recombinant plasmid pET-P1pE was obtained. Recombinant P1pE could be expressed in E.coli BL21 (DE3) via IPTG induction. The expressed product was identified by SDS-PAGE and Western-blotting. The results showed that fusion protein P1pE was about 37.8 kDa and it could react with specific antisera against PM C48-1 strain. Six-week-old mice were immunized with 50μg of purified P1pE, Two weeks later after enhanced immunization, mice were challenged with lOLD50 of C48-1 strain of PM. The survival rate is 100%. These results provided a good groundwork for the further research on the cross-protective test and development of novel subunit vaccine against Fowl cholera.2. The primary research on immune complex vaccine against fowl choleraThree kinds of complex vaccines were prepared from P1pE of Pasteurelta multocida and specific antibodies against P1pE in different ratios, the immune tests were carried out with chicks. Chicks in groups A,B and C were vaccinated with immune complex vaccines, The ratio of antigen and antibody were 2:1,4:1,8:1 respectively. Chicks in group D were vaccinated with protein P1pE, Group E was a control. Sera samples were collected at 1-5 weeks after the first immunization, and antibody levels were tested by ELISA. Chicks in all groups were challenged with C48-1 strain of PM after the last collecting of sera samples.The results showed that the higher levels of antibody and protection ratio in group C. the protection ratio can reached at 90%, the protection ratio in group A, B, D, E was 80%,60%,70%,0 respectively.