Zhang, P., Wang, Y., Jiang, M., Zhu, L., Li, J., Luo, M., Ren, H. and Liu, L. 2014. Molecular cloning, recombinant protein expression, tissue distribution and functional analysis of a new c-type lysozyme from Lezhi black goat rumen. Can. J. Anim. Sci. 94: 27–34. Three major distinct types of lysozymes have been identified in the animal kingdom and most lysozymes cloned from ruminants belong to the chicken-type (c-type). In this study, a new c-type lysozyme gene, named LZRLyz, was cloned and sequenced from the Lezhi black goat rumen. The LZRLyz cDNA has a 444 bp open reading frame (ORF) encoding a 147 amino acid polypeptide. The encoded polypeptide is predicted to have an 18 amino acid signal peptide, and a 129 amino acid mature protein with an isoelectric point (pI) of 6.08. The LZRLyz amino acid sequence shares 70.27% identity with the Capra hircus blood lysozyme and is grouped with other ruminants c-type lysozymes using the phylogenetic tree estimated by Neighbor-Jointing method. The recombinant expressed LZRLyz protein (pET-rLZR) shows a molecular mass of ∼33 kDa, which is consistent with the predicted fusion protein molecular mass and shows antimicrobial activity. Quantitative real-time RT-PCR analyses revealed that LZRLyz transcripts are expressed in all tested tissues with the predominant expression being observed in rumen and the weakest one in spleen. Results of this study suggest that the LZRLyz gene represents a new c-type lysozyme gene that likely functions in Lezhi black goat host immunity and digestive systems.
Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.
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