Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue.

Ming Feng Jiang,Hong Hui Ren,Ming Jun Hu,Li Wang

doi:10.5713/ajas.15.0125

Abstract

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

Highlights

Lysozyme is a kind of N-acetylmuramideglycanohydrolase which is ubiquitous in plants, animals, and microorganisms
Cloning and sequence analysis of the yak milk lysozyme (YML) gene The cDNA encoding the YML gene was amplified by qPCR
The subsequent sequencing and analysis demonstrated that the cloned gene was the YML gene and the sequence was submitted to the GenBank database (GenBank No.JX946731)

Summary

INTRODUCTION

Lysozyme is a kind of N-acetylmuramideglycanohydrolase which is ubiquitous in plants, animals, and microorganisms. One solution to increase lysozyme in cow milk is to clone the genes to express the human lysozyme in cow mammary glands. There 5×PrimeScript buffer, 1 μL PrimeScript RT Enzyme MixI, has been no accomplished comprehensive study about 1 μL RT Primer Mix, and 4 μL RNase free water were characterization of the milk lysozyme gene from yak added to the previous mixture. The cloning of YML, the phylogenetic analysis, and its expression levels during the lactation cycle of yak Cloning of the cDNA sequence of YML mammary gland tissue and the lysozyme activities of the. The DNA analyzer (ABI, USA) to confirm the insertion of the polymerase chain reaction (PCR) reaction composition was coding sequence of yak milk lysozyme. China) to form recombinant vectors, namely, pET32-YML1 recombinant plasmid pPICZαA-YML5 (5 to 10 μg) was pET32-YML2, pET32-YML3, and pET32-YML4 (Table 1). linearized with SacI and transformed into P. pastoris X33

F: Native N-terminal of mature peptide of YML pPICZαA-YML5

RESULTS

DISCUSSION