Immune Protective Efficacy of Recombinant Outer Membrane Protein H of Pasteurella multocida B:2 Associated with Hemorrhagic Septicemia in Pakistan
Immune Protective Efficacy of Recombinant Outer Membrane Protein H of Pasteurella multocida B:2 Associated with Hemorrhagic Septicemia in Pakistan
Abida Mushtaque1, Ali Ahmad Sheikh1, Aamir Ghafoor1*, Wasim Shehzad2 and Nadeem Ahmad3
ABSTRACT
P. multocida B: 2 is a Gram-negative, small, non-flagellated, pleomorphic coccobacillus. It does possess different types of outer membrane proteins (OMP’s) which assist in interaction with host cells. The outer membrane protein H (OmpH) of Pasteurella multocida B:2 is a major transmembrane porin that can be used as a subunit vaccine and development of diagnostic kit against hemorrhagic septicemia (HS) because of its immunogenic nature. Pasteurella multocida has economic importance because of endemic and epizootic diseases in domestic and wild animals. This bacterium is an opportunistic pathogen of upper respiratory tracts of wild and domestic animals and birds as well as in livestock . The ompH gene was amplified with in-house designed primers, the amplified gene (1002 bp) was cloned into pET 40-b (+) vector and transformed into E. coli BL21 (DE3) competent cells using heat shock transformation method to get expression. The product size was confirmed by restriction digestion. The rOmpH protein was purified by immobilized metal affinity chromatography by using His-Ni resins to get desired protein and characterized qualitatively by SDS-PAGE by using His antibodies and western blotting. Once purified the immunogenicity of rOmpH was evaluated in BALB/c mice. The expression of recombinant OmpH protein (rOmpH) resulted in its accumulation in cytosol. The purified rOmpH protein had a molecular weight of 36 kDa and concentration around 0.973 mg/mL. The BALB/c mice group injected with 100 ng of purified rOmpH exhibited significant protection (80%) when challenged with LD50 of P. multocida B:2. From the obtained results it is evident that rOmpH can be used as subunit vaccine against P. multocida B:2 and the purified rOmpH can also be further used to develop diagnostic test i.e., ELISA to screen and assess the immune response of cattle and buffaloes against P. multocida B:2 in developing countries like Pakistan.
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