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Subject: Microbiology × Author: Yadav, Archana × End date: 2009 × Start date: 2007 × Type: Thesis ×
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    ThesisItemOpen Access
    Cloning and characterization of Outer Membrane Protein(s) of Pasteurella multocida serotype B:2 (P 52)
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2008-04) Yadav, Archana; Sharma, Anita
    Pasteurella multocida serotype B:2 is a causative agent of Haemorrhagic septicaemia (HS), a fatal disease of cattle and buffaloes. Formalin inactivated whole cell bacterin is frequently used to prepare vaccines in India against HS. This vaccine has several limitations such as short term immunity, poor syringibility and production problem. To overcome from these problems there is an urgent need of search of a conserved immunogenic protein as vaccine candidate. Outer membrane proteins of P. multocida are reported to be immunogenic and protective. SDS-PAGE analysis of outer membrane proteins of vaccine strain P52 revealed the presence of eight polypeptides with molecular weight ranging from 16 to 87 kDa. Antiserum raised against whole cell antigen showed positive result by agar gel immunodiffusion test. Western blot analysis revealed the presence of seven immunodominant outer membrane proteins. Out of seven, omp87 and omp34 were selected as potent antigens for further study. Primers sets were designed from available sequences in NCBI Gene Bank. Both genes (omp87 and omp34) were amplified using DNA as template by PCR. The amplified fragments were cloned in pGEMT-Easy vector. After sequencing, size of the cloned omp87 gene was assessed and found to be 2121 bp accounting for 707 amino acid residues and size of omp34 was 942 bp accounting for 313 amino acid residues. Both the sequences were analysed using online bioinformatics tools (ExPASY server). On comparison of nucleotide sequence, omp87 showed 94.8% similarity with serotype A:1 while homology at amino acid level was 95.2%. Similarly at nucleotide level 98.3% and at amino acid level 97.5% similarity has been observed in omp34. The sequences were submitted to NCBI Gene Bank and accession number EU570212 and EU162755 were obtained for omp87 and omp34 respectively. On phylogenetic analysis it was found that omp34 of serotype B:2 clustered with serotype 3,4. Probable secondary structures and number of transmembrane helices were also predicted.