J Vet Sci.  2010 Sep;11(3):227-233. 10.4142/jvs.2010.11.3.227.

Molecular heterogeneity of plpE gene in Indian isolates of Pasteurella multocida and expression of recombinant PlpE in vaccine strain of P. multocida serotype B: 2

• Singh AP
• Singh S
• Ranjan R
• Gupta SK
• Singh VP
• Sharma B

• KMID: 1093497
• DOI: http://doi.org/10.4142/jvs.2010.11.3.227

Abstract

Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.

Keyword

Haemorhhagic septicaemia; Pasteurella multocida; PlpE

MeSH Terms

Animals
Bacterial Outer Membrane Proteins/*genetics/immunology/metabolism
Base Sequence
Blotting, Western
Cattle
Cattle Diseases/*microbiology
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Escherichia coli
*Genetic Variation
Hemorrhagic Septicemia/microbiology/*veterinary
India
Lipoproteins/*genetics/immunology/metabolism
Molecular Sequence Data
Open Reading Frames/genetics
Pasteurella multocida/*genetics/immunology
Sequence Analysis, DNA
Sequence Homology
Serotyping
Species Specificity

Figure

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  Fig. 1 SDS-PAGE of outer membrane preparation. Lane M represents protein molecular weight marker. The most prominent band corresponding to molecular weights of 39 kDa and 32 kDa is indicated.

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  Fig. 2 The plpE gene amplified by PCR using genomic DNA of P. multocida. Lane M represents DNA ladder and Lane 1-3 shows an amplified plpE gene in P. multocida serotypes A: 3, B: 2 and D: 3 respectively.

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  Fig. 3 DNA identity matrix based on pair wise comparison of plpE sequences of different strains of Pasteurella multocida.

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  Fig. 4 Hydrophilicity, antigenicity and surface probability plot of deduced amino acid sequence of PlpE of P. multocida strain P-52 (serotype B: 2). Positive values represent hydrophilic, antigenic and surface exposed regions.

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  Fig. 5 Expression and purification of r-PlpE in E. coli JM109. Lane M represents protein molecular weight marker, Lane 1 contains lysis buffer follow through, Lane 2 contains wash buffer follow through and Lanes 3-7 contains first, second, third, fourth and fifth eluted fractions.

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  Fig. 6 Immunoblot of purified PlpE probed with hyperimmune serum against whole cell lysate of P. multocida serotype B: 2 and rabbit anti-goat peroxidase conjugate. 39 kDa bands corresponding to r-PlpE are indicated. Lane M contains prestained protein molecular weight marker.

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