Jiang Ming Feng, Hu Ming Jun, Ren Hong Hui, Wang Li
College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan 610041, China.
College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan 610041, China ; Key Laboratory of Animal Genetics & Breeding, Southwest University for Nationalities, State Ethnic Affairs Commission and Ministry of Education, Chengdu, Sichuan 610041, China .
Asian-Australas J Anim Sci. 2015 Dec;28(12):1774-83. doi: 10.5713/ajas.15.0125.
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.
乳溶菌酶是哺乳动物乳汁中普遍存在的酶。在本研究中，从牦牛乳腺组织中克隆了一种新的鸡型（c型）乳溶菌酶基因（YML）的cDNA序列。对一个444 bp的开放阅读框进行了测序，该开放阅读框编码148个氨基酸（16.54 kDa），带有一个18个氨基酸的信号肽。进一步分析表明，牦牛和牛乳溶菌酶之间的核酸和氨基酸序列同一性分别为89.04%和80.41%。重组牦牛乳溶菌酶（rYML）由大肠杆菌BL21和毕赤酵母X33产生。用表达载体pPICZαA在毕赤酵母中表达的异源蛋白rYML5检测到最高的溶菌酶活性（M = 1,864.24 U/mg，SD = 25.75），并且它明显抑制金黄色葡萄球菌的生长。使用定量聚合酶链反应对YML基因表达的结果表明，YML基因在产后30天上调至最高水平，也就是说，在初乳中可以发现相对较高的YML。系统发育树表明，氨基酸序列与牛肾溶菌酶相似，这意味着YML可能从不同的祖先基因如牛乳腺中分化而来。在我们的研究中，我们认为YML是一种在牦牛乳腺中表达的新型c型溶菌酶，它起着宿主免疫的作用。
