Immune Protective Efficiency Of Recombinant Pasteurella Multocid Mip Against Chellenge Infection In Mice

Pasteurellosis is caused by Pasteurella multocida（Pm）and distributed in human and a great variety of animals.It is an acute and febrile infectious disease mainly characterized by infectious pneumonia and hemorrhagic inflammation.At present,the clinical practice of Pm vaccine mainly includes inactivated vaccines and attenuated vaccines.Due to the numerous serotypes of Pm and the low cross-protection effect of existing vaccines for the clinical practice,it is necessary to explore new candidate vaccines.1、Cloning and bioinformatics analysis of Pm Mip According to Pm A genome which had been sequenced completely,we found an outer membrane protein called Mip which may be selected as a candidate vaccine antigen to induce effective immune protection.Primer pairs were designed using Primer 5.mip gene fragment was amplificated by PCR from Pm A genome and sequenced.The results of bioinformatics analysis showed that:mip was 717 bp length and its predicat coding protein had 238 amino acids with 2.5768 k D MW.It was an outer membrane protein with one membrane binding region in the range of 8-25 amino acids and a signal peptide region in the position of 24-25 amino acids.It showed seven antigenic determinants by IEDB.Homology analysis showed that it had high homology among different Pm serotypes.So,it was deduced that Pm Mip protein showed a good potential immunogenicity and cross-immunoprotection against the infection of different Pm serotypes.2、Immune protective efficiency of Pm rMip against the chellenge infection in mice The mip gene was inserted into the p ET28a（+）vector to construct recombinant plasmid p ET-Mip.The recombinant plasmid was transfected into BL21 cell and rMip was expressed by IPTG induction.It was showed that rMip was rightly expressed by SDS-PAGE detection.And good immunogenicity by Western blot detection.The immune protective efficiency of rMip was evaluated in SPF ICR mice.The SPF mice were randomly divided into 5 groups and 10 mice in each group.They were immunized subcutaneously with saline group in group A,Freund’s incomplete adjuvant（FIA）in group B,low dose（10μg rMip）+adjuvant in group C,medium dose（30μg rMip）+adjuvant in group D,high dose（50μg rMip）+adjuvant in group E.on day 1,15,30 and 45.The blood samples were collected by cutting the tail vein before the each immunization and challenge infection.The titers of specific Ig G were detected by ELISA.Two weeks after the fourth bolstering immunization,mice were intraperitoneally challenged with LD₅₀Pm A.The lethality were calculated.The results showed that 10μg rMip+Frund’s incompleted adjuvant,30μg rMip+Frund’s incompleted adjuvant and 50μg rMip+Frund’s incompleted adjuvant respectively induced 20%,30%,and 30%survival rate.No mice survived the challenge in adjuvant group and saline group.The rest living mice in experiment groups slowly returned to normal health status.From above,it was concluded that Pm rMip induced a partial immune protection of mice against Pm challenged infection.This study accumulated data for exploring new vaccines of pasteurellosis.