Immune Protective Efficiency Of Recombinant Pm DOT Against The Chellenge Infection Of Pasteurella Multocida In Mice

Pasteurella multocida（Pm）is a global epidemic pathogen.It can cause pasteurellosis such as porcine lung disease,avian cholera,porcine atrophic rhinitis,rabbit pasteurellosis etc.in animals and humans.Pm infection severely inflences health of animals and humans.Nowadays,the vaccines for clinical practice have some weakness of narrow immune spectrum,short protection time and low cross-pretection.So it is necessary to develop new vaccines.1.Cloning and bioinformatics analysis of Pm dot gene In order to screen vaccine candidates with broad-spectrum anti-Pm effect,the virulence gene of dot was cloned from the genome of serum type A Pm by PCR.The results of bioinformatics analysis showed that Pm dot gene consists of 729 bp,encoding a protein of 242 amino acids with the theoretical molecular weight of 27.032 k Da and the isoelectric point（p I）of 7.86.And the first 24amino acids at the N-terminal are signal peptides.It is a Predictive outer membrane protein with a transmembrane region.Pm DOT shared a high homology among different serotypes of Pm.There are six B cell epitopes.From theses,it is concluded that Pm DOT is a potential vaccine candidate with good immunogenicity and cross-immunoprotection.2.The immunoprotective effect of recombinant Pasteurella multocida DOT protein in mice Pm dot was cloned into p ET-28a（+）to construct p ET-Dot,and then pET-Dot transformed into E.coli BL21（DE3）.The recombinant protein rDOT was successfully expressed by IPTG induction.The result of west-blot detection showed that rDOT had good immunogenicity.Forty 20 g female mice were randomly divided into 5 groups and immunized subcutaneously with normal saline（control group）,Frud’s incomplete adjuvant（FIA）（adjuvant control group）,10μg rDOT+FIA（low dosage group）,30μg rDOT+FIA（middle dosage group）and 50μg rDOT+FIA（high dosage group）at 0,2 and 4 weeks.The results of ELISA showed that the level of serum Ig G in the experimental group was significantly higher than that in the control group（adjuvant group and normal saline group）after the third immunization.Two weeks after the third immunization,the mice were challenged with 5×LD₅₀ Pm intraperitoneally.After 3 days,all the mice in the control group died.The survival rates of mice in low dose,middle dose and high dose groups were12.5%,50%and 62.5%,respectively.It concluded that rDOT+FIA can induce partial immune protection against Pm challenge infection.This study accumulated basic data for the further study of broad-spectrum anti-Pm vaccine.