Cloning And Expression The PlpB Gene Of Pasteurella Multocida And Biological Characterization Of The Expressing Proteins

Fowl cholera, which pathogen is pasteurella mutocida, is a kind of acute,contagiousand septicaemia infection disease in domestic poultry and wild birds. Different serotypePM which produced fowl cholera disease has no cross protection to each other. The PlpBprotein which located in the bacterium outer membrane can provide protection to thechallenge of heterologous serotype strains. It is insignificant on the researching of thePlpB protein antigen and developing new-effective fowl cholera vaccine as which canprovide protection to the challenge of heterologous serotype strains. The present studyfocused on the cloning, expression and the expression production of the PlpB gene ofPMC48-1, which were summarized as follows.1. cloning and expressing the PlpB gene of the Pasteurella MultocidaThe oligonucleotide primers for amplifying the PlpB gene of PMC48-1 strain weredesigned according to the sequence of gene of PM70 strain, which published in GenBank.The amplified gene which is 831bp containing a complete open reading frame (ORF) wasinserted into the prokaryote expression vector pGEX-KG, and the resulted recombinantplasmid PKG-PIpB was sequenced. This sequence processes a homology of more than99% compared with the published sequence of PlpB gene of PM70 in GenBank. ThepKG-PlpB was transformed into E.coli BL21 (DE3) competent cells. After induction byIPTG, a 56 kDa recombinant protein was expressed. Western blot analysis revealed thatthe recombinant protein have a good reaction with antisera of fowl cholera.2. the primary research on the expressing protein of pasteurella multocida PlpB geneThe nonimmuned mice was injected with the serum which harvested from theimmuned mice, meanwhile carrying out the active and passive immune protection test.The results indicate that the fusion protein, coded by the recombinant plasmids ofpKG-PlpB, which expressed in the E.coli BL21 can induce the immune reaction in miceand protection to the challenge of the high toxical strain. The immune protection can bepassed passively to the nonimmuned mice, so which can get the power of resistance to thechallenge of the high toxical strain also.In summary, the complete PlpB gene was cloned from the C48-1 strain andexpressed in E.coli, the recombinant of the protein which immune activity has been tested and of which primary animal test has been passed is to be a good groundwork to the nextwork for the cross-protective test and to explorer subunit vaccine of fowl cholera.