2型猪链球菌β-半乳糖苷酶基因的克隆表达及酶活性测定

doi:10.13523/j.cb.20140207

中国生物工程杂志

2014, Vol. 34

Issue (2): 39-44 DOI: 10.13523/j.cb.20140207

研究报告

2型猪链球菌β-半乳糖苷酶基因的克隆表达及酶活性测定

张凤玉^1,2, 胡丹¹, 龚秀芳¹, 郑峰¹, 潘秀珍¹, 王长军^1,2

1. 南京军区军事医学研究所南京 210002;
2. 南京医科大学基础医学院南京 210029

Cloning, Expression and Identification of Gene Encoding the β-Galactosidase of Streptococcus suis serotype 2

ZHANG Feng-yu^1,2, HU Dan¹, GONG Xiu-fang¹, ZHENG Feng¹, PAN Xiu-zhen¹, WANG Chang-jun^1,2

1. Research Institute for Medicine of Nanjing Command, Nanjing 210002, China;
2. School of Basic Medical Sciences, Nanjing Medical University, Nanjing 210029, China

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摘要： 目的：克隆表达2型猪链球菌β-半乳糖苷酶（BgaC）编码基因，并测定其酶活性。方法：根据05ZYH33基因组序列设计引物，PCR扩增bgaC基因，构建重组表达质粒pET28a-bgaC，转化E.coli BL21，筛选阳性转化子进行IPTG诱导表达，产物通过SDS-PAGE鉴定；最后对表达产物进行亲和层析纯化，获得BgaC纯化蛋白后测定其酶活性。结果：bgaC基因在原核细胞中得到高效表达，重组表达的BgaC分子质量约为69kDa，其酶促反应最适温度为42℃，最佳反应时间为30min，最适反应pH为5.5，最佳底物浓度为10mmol/L。2型猪链球菌BgaC的体外酶活为1615U/ml，酶比活为1076U/mg。结论：2型猪链球菌强毒力株05ZYH33中含有bgaC基因，在原核系统高效表达的BgaC具有良好的酶学活性。

关键词： 2型猪链球菌; β-半乳糖苷酶; 克隆; 酶活性

Abstract: Objective:To clone and prokaryotically express the β-galactosidase of Streptococcus suis serotype 2 and to determine the enzymatic properties of the recombinant protein. Methods: bgaC gene was amplified by PCR using the primers which are on the basis of 05ZYH33 genome sequences and cloned into the expression vector. Thereafter, the gene was cloned into prokaryotic expression plasmid pET28a, and the recombinant plasmid pET28a-bgaC was transformed into E.coli BL21. After the induced expression by IPTG, the isolated BgaC protein was analyzed with SDS-PAGE and purified by chromatography. Thus obtaining the completely purified BgaC protein and its enzymatic activity was measured afterward. Results: bgaC gene could express highly in E.coli. The molecular weight of the recombinant expressed β-galactosidase BgaC was about 69kDa, the enzymatic activity analysis indicated the optimum temperature, action time, pH and the substrate concentration were 42℃,30min, 5.5 and 10mmol/L, respectively. Enzymatic activity of BgaC was about 1615U/ml, specific activity was 1076U/mg. Conclusions: The experimental results showed that the bgaC gene can be highly expressed in prokaryotic system, and the recombinant protein has the best enzymatic activity in optimizal temperature, reactive time and pH.

Key words: Streptococcus suis serotype 2 β-galactosidase Cloning Enzymatic activity

收稿日期: 2013-11-08 出版日期: 2014-02-25

ZTFLH:

Q786

基金资助: 国家自然科学基金（31170124，81171527，81172794，31300119）、科技部传染病专项基金（2013ZX10004103-004，2013ZX10004801-004，2013ZX10004218-008）、江苏省自然科学基金（BK2011097，BK2012080）、军队“十二五”项目（AWS11C001&AWS11L009）资助项目

通讯作者: 王长军 E-mail: science2008@hotmail.com

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引用本文:

张凤玉, 胡丹, 龚秀芳, 郑峰, 潘秀珍, 王长军. 2型猪链球菌β-半乳糖苷酶基因的克隆表达及酶活性测定[J]. 中国生物工程杂志, 2014, 34(2): 39-44.

ZHANG Feng-yu, HU Dan, GONG Xiu-fang, ZHENG Feng, PAN Xiu-zhen, WANG Chang-jun. Cloning, Expression and Identification of Gene Encoding the β-Galactosidase of Streptococcus suis serotype 2. China Biotechnology, 2014, 34(2): 39-44.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140207 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I2/39

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