人<em>LYC5</em>溶菌酶基因在毕赤酵母中的重组表达

doi:10.13523/j.cb.20140401

中国生物工程杂志

2014, Vol. 34

Issue (4): 1-8 DOI: 10.13523/j.cb.20140401

研究报告

人LYC5溶菌酶基因在毕赤酵母中的重组表达

卫田田, 于颖, 金晓锋, 陶建军, 余龙

复旦大学遗传工程国家重点实验室遗传学研究所上海 200433

Recombinant Expression of Human LYC5 Lysozyme in Pichia pastoris

WEI Tian-tian, YU Ying, JIN Xiao-feng, TAO Jian-jun, YU Long

State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, China

全文: PDF(1185 KB) HTML

摘要：

LYC5是一种c型人溶菌酶蛋白。根据毕赤酵母密码子的偏爱性，对LYC5的mRNA编码序列进行优化设计，将优化后的基因序列克隆至毕赤酵母分泌型表达载体pPIC9K中，构建重组酵母表达质粒pPIC9K- LYC5 。重组质粒经线性化处理后转化毕赤酵母GS115，应用G418抗性筛选出高拷贝转化子，并对其进行摇瓶诱导表达，产物经SDS-PAGE电泳检测，发现在约15 kDa的位置出现了一条特异蛋白条带，此条带经LTQ Orbitra pelite MS鉴定，证明此蛋白即LYC5溶菌酶蛋白，表达量约为20 mg/L。对表达上清液进行活性分析，发现表达上清对溶壁微球菌具有较好的溶菌活性，活性约为40 000 U/mg，最适酶活反应温度为45℃，最适pH为5.0。采用基因工程方法，首次表达出了有生物学活性的人源LYC5溶菌酶蛋白，为深入探讨人溶菌酶家族成员的抗菌谱及其应用前景的研究奠定了基础。

关键词： 人溶菌酶; LYC5; 毕赤酵母; pPIC9K

Abstract:

LYC5 is a kind of c type human lysozyme protein. To optimized and designed the LYC5 gene based on the coding sequence of mRNA on GenBank and the preference of Pichia yeast codon, then cloned the encoding gene into the expression plasmid pPIC9K,and the resulting recombinant plasmids were named as pPIC9K- LYC5. Then they were linearized and electro-transformed into the Pichia pastorios GS115 express bacterium,the multiple copies of transformants were screened by Minimal Dextrose medium (MD),Minimal Methanol medium (MM),the antibiotic of G418 and PCR identification. The transformants were screened through culturing and induction under methanol in baffled flask,the results of SDS-PAGE indicated that the LYC5 target proteins were secreted into the supernatant, about 15 kDa in sizes. These proteins were identified as LYC5 lysozymes by LTQ orbitrap elite MS analysis,with expression levels being about 20 mg/L. The expression products had obvious antibacterial activity on Micrococcus lysodeikticus,with the activity was about 40 000 U/mg,the optimum temperature and pH for reaction of LYC5 lysozyme were 45℃ and 5.0. The successfully expressed LYC5 lysozyme protein with antibacterial activities,which laid a foundation for deeping discuss the antimicrobial spectrum and application of human lysozyme family.

Key words: Human lysozyme LYC5 Pichia pastorios pPIC9K

收稿日期: 2014-03-03 出版日期: 2014-04-25

ZTFLH:

Q786

基金资助:

国家重大新药创制科技重大专项资助项目（2011ZX09102-007-01）

通讯作者: 余龙 E-mail: longyu@fudan.edu.cn

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引用本文:

卫田田, 于颖, 金晓锋, 陶建军, 余龙. 人LYC5溶菌酶基因在毕赤酵母中的重组表达[J]. 中国生物工程杂志, 2014, 34(4): 1-8.

WEI Tian-tian, YU Ying, JIN Xiao-feng, TAO Jian-jun, YU Long. Recombinant Expression of Human LYC5 Lysozyme in Pichia pastoris. China Biotechnology, 2014, 34(4): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140401 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I4/1

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