Construction Of OmpH Gene Deletion Mutants Of P.multocida From Yak Of The Tibet Plateau And Evaluation Of Part Of Biological Function

Pasteurella multocida (P. multocida) is the aetiological agent of hemorrhagicsepticaemia in yak, however, the virulence factors that involved in the pathogenesisof P. multocida infection are poorly understood. The outer membrane protein H(OmpH) of P. multocida is the most abundant protein in the outer membrane and alsohas been identified as a multifunctional protein involved in pathogenicity. Previouslyresearches showed that the recombinant OmpH protein from the virulent strain P0910isolated from yak could produce90%of the protection in mice, which suggested thatthe OmpH is an important protective antigen in P. multocida. However, whether theOmpH is involved in the initial adhesion and invasion into host tissues duringcolonization has not been studied yet.In the present study, the upstream and the downstream of ompH gene wereamplifying to construct the recombinant plasmid pEX18AP-ompH, primers designedaccording P. Multocida stain HN06(NC017027), and then introduced the recombinantplasmid into P. multocida strains P0910, C47-8and C45-2by electroporation, andunmarked ompH gene deletion mutants derived from P. multocida strain P0910isolated from a yak of the Tibet plateau and two control strains of P. multocida, C45-2and C47-8, were successful constructed by using gene recombinant and negativeselection technique, setting up a technologic platform for identifying ompH gene’sfunction of P. multocida and carrying out researches on adhesion, invasion, virulenceand immune mechanisms of P. multocida.The pathogenicity of these three mutants was determined in vitro and in vivo bytesting the adherence of parent strains and mutant strains to the Vero cells and thevirulence to the mice. The results showed that the quantity of P0910, C47-8and C45-2strain adhere to each Vero cell were32,20,20, while the quantity of threemutant stains adhere to each Vero cell were approximately2; and the virulence of thethree mutants was also reduced in mice,10parent strains bacteria was lethal to micewhile mice were still alive under107mutant stains bacteria injection, which suggestedthat the ompH gene was associated with the adherence of P. multocida to cells andcontributed to the virulence of P. multocida in vivo. This study provides importanttheoretical basis to interpret pathogenesis and develop gene knockout vaccine, and anew way in preventing, monitoring and cleaning hemorrhagic septicemia of yak inTibetan-plateau.