乐至黑山羊肾溶菌酶基因克隆及原核表达

›› 2013, Vol. 40 ›› Issue (9): 46-51.

乐至黑山羊肾溶菌酶基因克隆及原核表达

胡宇飞^1,2, 江明锋^1,2, 张鹏^1,2, 罗樊^1,2, 朱莲莲^1,2, 王永¹

1. 青藏高原动物遗传资源保护与利用四川省重点实验室, 四川成都 610041;
2. 西南民族大学生命科学与技术学院, 四川成都 610041

收稿日期:2013-02-18 出版日期:2013-09-20 发布日期:2013-09-18
通讯作者: 王永 E-mail:wangyong010101@swun.cn
作者简介:胡宇飞(1991-),女,河南人,学士,研究方向:生物技术。
基金资助:
国家科技支撑计划(2012BAD13B06);四川省科技创新产业链示范工程重大项目(2011NZ0003)。

Cloning and Prokaryotic Expression of Kidney Lysozyme Gene in Lezhi Black Goat

HU Yu-fei^1,2, JIANG Ming-feng^1,2, ZHANG Peng^1,2, LUO Fan^1,2, ZHU Lian-lian^1,2, WANG Yong¹

1. Key Laboratory of Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation, Chengdu 610041, China;
2. College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, China

Received:2013-02-18 Online:2013-09-20 Published:2013-09-18

摘要/Abstract

摘要： 本研究对乐至黑山羊肾溶菌酶(LZLyK)基因进行了克隆、表达,并对其活性进行了测定。采用比较基因组技术,成功克隆了LZLyK基因,其cDNA长444 bp,编码147个氨基酸。同源性分析结果表明,氨基酸同源性与绵羊溶菌酶2(M32498.1)最高,达96.9%。构建原核表达质粒pET- LZLyK,转化大肠杆菌BL21(DE3)并诱导表达。SDS-PAGE分析结果表明,重组蛋白LZLyK得到正确表达,分子质量约为16、30 ku。采用比浊法测定了LZLyK蛋白的活性,其活性为15 U/mL,表明LZLyK蛋白具有一定抗菌活性。运用实时荧光定量PCR发现在乐至黑山羊肝脏、肾脏、气管和皱胃中均有肾溶菌酶基因的表达,在胃中表达量较高。

关键词: 乐至黑山羊; 肾溶菌酶基因; 克隆; 原核表达

Abstract: The kidney lysozyme (LZLyK) gene from Lezhi Black goat was cloned, expressed and its activity was detected in this experiment. With the comparative genomics, the LZLyK gene was cloned successfully obtaining a 444 bp long cDNA which coded 147 amino acids. The phylogenetic analysis showed that the LZLyK gene shared the highest homology up to 96.9% with the sheep's lysozyme NO.2(M32498.1) in amino acids. After being transformed into E.coli BL21 (DE3), the recombinant plasmids pET-LZLyK were expressed in a prokaryotic expression system. The SDS-PAGE analysis revealed that the LZLyK protein had molecular mass of 16 and 30 ku respectively, which were consistent with the prediction. The activity of the protein mentioned above was determined by the way of nephelometry, and consequently it turned out to be 15 U/mL which indicated that the protein of LZLyK possessed a certain antibacterial activity. It was found that the content of lysozyme was more in stomach than other tissues through RF-PCR.

Key words: Lezhi Black goat; lysozyme gene; clone; prokaryotic expression

中图分类号:

胡宇飞, 江明锋, 张鹏, 罗樊, 朱莲莲, 王永. 乐至黑山羊肾溶菌酶基因克隆及原核表达[J]. , 2013, 40(9): 46-51.

HU Yu-fei, JIANG Ming-feng, ZHANG Peng, LUO Fan, ZHU Lian-lian, WANG Yong. Cloning and Prokaryotic Expression of Kidney Lysozyme Gene in Lezhi Black Goat[J]. China Animal Husbandry & Veterinary Medicine, 2013, 40(9): 46-51.

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