Cloning and Sequence Analysis of Hsf, an Outer Membrane Protein Gene of Pasteurella Multocida Serotype B:2

Aim: The present study was undertaken to clone, sequence and analyze the hsf, an outer membrane protein gene of Pasteurella multocida serotype B:2 Materials and Methods: hsf gene was amplified from genomic DNA of P. multocida. Polymerase chain reaction (PCR) product was cloned in pET-32a vector and was characterized. hsf gene was sequenced, analyzed and phylogenetic tree was constructed taking sequences of other strains. Results: Amplicon size was found to be 785 bp. Recombinant got characterized through colony PCR and restriction enzyme analysis. Conclusion: hsf gene of P. multocida serotype B is similar to serotype A, but different from serotype D. Further work is needed to evaluate role of Hsf protein in protection studies and to study the antigenic properties of this recombinant protein as a candidate for vaccine.

HS is endemic in most tropical countries such as India and Africa causing high mortality in livestock [3].A wide range of host such as cattle, buffalo, pig, sheep, goat, deer and camel are susceptible to this infection [4].Though the uses of inactivated and live attenuated vaccines have led to a considerable reduction in overall livestock mortality, still the objective of complete protection has not been achieved [3].
Outer membrane proteins (OMPs) are involved in protective immunity against P. multocida infections.Virulence factors of P. multocida aid in the colonization, invasion, and its pathogenesis are surface located [5].
OMPs have been reported to confer various virulence attributes of the organism like colonization and invasion thus, are potential antigenic candidates [6].Therefore, identification of OMPs is critical to make out the protective antigens and to develop novel diagnostics after proper evaluation of their antigenic potential and the ability to contribute to the host immunogenicity.OMPs of P. multocida based on functional characteristics were categorized and have been shown to stage pivotal role in host pathogen interaction and disease processes [7].The key antigens of P. multocida B:2 that evoke protective immunity to HS in cattle have still not been well defined.Although sequencing of genes encoding various OMPs has been carried out, still for many genes no study has yet been done.
The present study was aimed to characterize important OMP hsf, a high molecular weight auto transporter adhesion protein, is a virulence factor.No systematic study on sequence analysis of hsf gene has been carried out till date, therefore, these proteins can be exploited for development of recombinant antigens based diagnostic test for detection of HS in bovines or as vaccine candidate for providing protection against the ailment.

Ethical approval
The Institutional Animal Ethics Committee (IAEC)/Committee for the Purpose of Control and Supervision of Experiments in Animals (CPCSEA) of Copyright: The authors.This article is an open access article licensed under the terms of the Creative Commons Attributin License (http:// creative commons.org/licenses/by/2.0)which permits unrestricted use, distribution and reproduction in any medium, provided the work is properly cited.
the IVRI, Izatnagar had approved for immunization studies.

Extraction of total genomic DNA of P. multocida serotype B:2
Genomic DNA was extracted from culture of P. multocida serotype B:2 maintained in the Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar by CTAB method and was preserved for further use.
The PCR was carried out in 50 μl reaction volume containing 10 pmol of each primer, 0.1 mM of dNTPs, 1.5 mM of MgCl 2 , 2 U of vent polymerase (NEB, UK) in 1X reaction buffer using 50 ng of genomic DNA as template.The reaction conditions were as follows; initial denaturation 95°C for 5 min.35 cycles denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 1 min followed by one cycle of final extension at 72°C for 10 min.The amplified products were analyzed by electrophoresis in 1.5% agarose containing ethidium bromide (0.5 μg/ml).
The amplified PCR product and pET-32a vector were double digested with restriction endonuclease enzymes BamHI (Fermentas) and SacI (Fermentas) and ligated using T4 DNA ligase (Novagen, USA).Recombinants were screened through colony PCR using gene specific primers as given above and further analyzed by electrophoresis in 1.5% agarose containing ethidium bromide (0.5 μg/ml).
Recombinant plasmid was also characterized through double restriction digestion analysis using BamHI and SacI.The digested products of recombinant plasmid were separated on 1.5% agarose gel.

DNA sequence analysis
The positive recombinant plasmid containing hsf gene fragments of P. multocida was sequenced using both forward and reverse primers of pET-32a vector.Sequence was analyzed and submitted to NCBI GenBank.P. multocida strains 3480 (CP001409.1),Pm70 (AE004439.1)and hsf gene of Haemophilus influenza (AJ277635.1)were retrieved from NCBI.Phylogenetic analysis and percent similarity/divergence were analyzed using Mega Align (DNASTAR, Madison, Wisconsin, USA) software.

Results and Discussion
The ~785 bp amplicon size as visualized in agarose gel electrophoresis indicated the amplification of hsf gene from the P. multocida genomic DNA (Figure -1).PCR product cloned in pET-32a vector was characterized by both colony PCR and restriction enzyme analysis.Colony PCR upon gel separation gave amplicon size ~785 bp that indicated the presence of hsf gene (Figure -2).Recombinant plasmids were also characterized by double restriction enzyme digestion analysis where release of insert of size ~785 bp confirmed the presence of the gene of interest in the recombinant plasmid (Figure -3).Sequencing results were analyzed and further submitted to NCBI GenBank (KM593283).Phylogenetic tree based on hsf nucleotide sequences of three strains of P. multocida (P-52, 3480 and Pm70) and one strain of H. influenza is indicated in Figure -4.The nucleotide alignment results of hsf gene of different strains of P. multocida taking P-52 as reference strain revealed 96.8% homology and 2.3% divergence from Pm70 (P.multocida serotype A:1 isolates from USA) strain whereas 75.6% homology and 29.1% divergence from strain

(P. multocida serotype D isolates from China).
Phylogram suggested that hsf gene of P. multocida serotype B is similar to serotype A, but different from serotype D. As only one isolate was sequenced, so it is not clear whether the substitution from serotype D observed in this study carry signature sequences for capsular type specificity or due to host origin specificity or for geographical location.hsf is an adhesion related gene of P. multocida showed homology with H. influenza surface fibrils.The H. influenza type B Hsf is a vitronectin binding protein which contributes to resistance against antibody [8].Transcriptional activation of hsf gene of P. multocida was also found to be escalated under nutrient deficient conditions [9].These above two findings suggested the role of hsf gene in host resistance.
The P. multocida contains two types of hsf gene namely hsf1 and hsf2.The frequency of hsf gene varies among serotypes with the gene hsf2 being more prevalent.In porcine isolates the hsf2 gene is present in 99.1% isolates whereas hsf1 in 67% strains [10].Among Indian small ruminant isolates hsf2 gene was present in 90.9% isolates whereas hsf1 in 51.1% isolates [11].
The sequence analyses for many other genes encoding OMPs have showed variable degree of homology that was reported by several authors.
Recently, authors have reported that the vacJ gene had greater homogeneity (99.1-100%) among the P. multocida strains [12].Singh et al., 2010 [13] compared plpE gene in different capsular types and observed more than 90% homology whereas Siju et al., 2007 [14] observed 78.4% of similarity between nucleotide sequence of ptfA gene in A:1 and B:2 isolates.Similarly Shivachandra et al., 2005 [15] reported 98.4% homology and 1.5% divergence in tbpA gene of P. multocida B:2 isolate from serotype A:1 isolates.Jain et al., 2013 [16] observed >95% homology among hasR and hgbA gene among different isolates.So this study highlights the need to exploit outer membrane genes and carry out multidimensional study in the field of diagnosis and prophylaxis.

Conclusion
HS is an important disease of cattle and buffaloes.For effective control of the disease, an efficacious and longer duration immunity vaccine is required.Identification and characterization of important immunogens of the bacteria would not only help in designing an improved vaccine but also Identification of the OMPs and exploiting it would serve as a candidate for development of diagnostics.As Hsf protein is one of the important virulence factors of the bacteria therefore further work is needed to evaluate its role in protection studies and to study the antigenic properties of this recombinant protein as a candidate for vaccine.