Identification of new Corynebacterium pseudotuberculosis antigens by immunoscreening of gene expression library

Cleber Eduardo Galvão,Renata Ribeiro Bastos Pereira,Grácia Maria Soares Rosinha,Stenio Perdigão Fragoso,Carina Elisei De Oliveira,Cleber Oliveira Soares,Odinéia Forner

doi:10.1186/s12866-017-1110-7

Abstract

BackgroundCaseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA.ResultsA wild strain of C. pseudotuberculosis was used for extraction and partial digestion of genomic DNA. Sequences between 1000 and 5000 base pairs (bp) were excised from the gel, purified, and the digested DNA fragments were joined to bacteriophage vector ZAP Express, packaged into phage and transfected into Escherichia coli. For immunoscreening a positive sheep sera pool and a negative sera pool for CLA were used. Four clones were identified that strongly reacted to sera. The clones were confirmed by polymerase chain reaction (PCR) followed by sequencing for genomic comparison of C. pseudotuberculosis in GenBank. The genes identified were dak2, fagA, fagB, NlpC/P60 protein family and LPxTG putative protein family.ConclusionProteins of this type can be antigenic which could aid in the development of subunit or DNA vaccines against CLA as well as in the development of serological tests for diagnosis. Immunoscreening of the gene expression library was shown to be a sensitive and efficient technique to identify probable immunodominant genes.

Highlights

Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans
Construction and validation of gene expression Corynebacterium pseudotuberculosis library The average size of inserts cloned in the library was 3 kb, which, in total, corresponds to 576.000 kb or 5.76 × 108 bp recombinant phage
The amplified product corresponds to 924 bp of the pld gene, confirming the presence of this gene in the gene expression library of C. pseudotuberculosis

Summary

Introduction

Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA. Corynebacterium pseudotuberculosis is a Gram-positive, facultative intracellular bacillus. It is the etiological agent of caseous lymphadenitis disease (CLA), which mainly affects sheep and goats. A factor that drives research for CLA is that it causes economic losses in sheep and goat production. CLA is a chronic and sometimes subclinical infection, which makes its diagnosis and management difficult. CLA pathogenesis is a slow process resulting in a chronic disease [1]. Infections are retained to lymph nodes, by the spread of exotoxin

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