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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (1): 14-20    DOI: 10.13523/j.cb.20170103
研究报告     
抗病毒蛋白RC28在大肠杆菌和毕赤酵母中的表达及活性比较
胡立强, 郑文, 钟艺, 杜丹, 杨浩, 龚萌
四川大学华西医院 华西-华盛顿线粒体与代谢研究中心 成都 610041
Comparison of Expression and Activity of Antiviral Protein RC28 in Escherichia coli and Pichia pastoris
HU Li-qiang, ZHENG Wen, ZHONG Yi, DU Dan, YANG Hao, GONG Meng
HuaXi-Washington Mitochondria and Metabolism Research Center, West China Hospital, SCU, Chengdu 610041, China
 全文: PDF(635 KB)   HTML
摘要：

RC28蛋白是从野生蕈类皱盖罗鳞伞中分离得到的新型抗病毒蛋白。前期通过3'-RACE方法，从皱盖罗鳞伞总RNA中克隆获得包含RC28编码区的cDNA，并通过TA克隆获得质粒pMD18-T-RC28。以该质粒为模板，设计特异性引物，PCR扩增RC28片段，分别插入大肠杆菌表达载体pET28a（+）和毕赤酵母表达载体pPIC9K中，形成在N端表达组氨酸标签的重组RC28表达粒体。将这两种RC28表达质粒转化各自的宿主菌后，筛选阳性克隆，构建稳定表达体系。诱导表达后，用SDS-PAGE电泳和Western blot分析RC28的表达情况。放大表达体系后用Ni-NTA柱纯化获得RC28蛋白，并用MTT法测试重组表达蛋白质的抗病毒活性。在实验条件下，大肠杆菌和毕赤酵母中均能表达可溶性重组RC28，纯化后蛋白质得率分别是3.5mg/L发酵液及0.2mg/L发酵液。但抗病毒活性实验表明，大肠杆菌体系表达的RC28蛋白活性较差，而毕赤酵母系统表达的RC28蛋白的抗病毒活性与天然蛋白接近。
关键词： 大肠杆菌;  毕赤酵母;  表达纯化;  抗病毒RC28    
Abstract:

RC28 is a novel antiviral protein extracted from the mushroom Rozites caperata. In our previous studies, RC28 cDNA were obtained from Rozites caperata total RNA by 3'-RACE and inserted into pMD18-T plasmid by TA cloning. RC28 cDNA fragments were sub-cloned into E. coli expression vector pET28a(+) and Pichia yeast expression vector pPIC9K separately to form recombinant RC28 expression vectors with N'-terminal histidine tags. The two kinds of RC28 expression vectors were transformed into their respective host strains and positive clones were selected to construct stable expression systems. SDA-PAGE and Western blot were performed to analyze RC28 expression after induction. RC28 protein were purified and collected by using Ni-NTA columns and detection of the antiviral activity of the recombinant protein achieved by MTT assay. In our experiment, soluble recombined RC28 proteins were prepared both in E. coli and Pichica yeast and the yields of purified protein from these two expression systems were about 3.5mg/L and 0.2mg/L, respectively. However, the recombined RC28 protein expressed from E. coli system showed poor antiviral activity while the recombined RC28 protein from Pichica yeast system showed much higher antiviral activity which was close to that of the natural protein.
Key words: Protein expression and purification    RC28 antiviral protein    Escherichia coli expression system    Pichia yeast expression system
收稿日期: 2016-08-22 出版日期: 2017-01-25
ZTFLH:  Q819  
基金资助:

国家自然科学基金青年基金资助项目（30900244）
通讯作者: 龚萌     E-mail: 12346441@qq.com
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引用本文:

胡立强, 郑文, 钟艺, 杜丹, 杨浩, 龚萌. 抗病毒蛋白RC28在大肠杆菌和毕赤酵母中的表达及活性比较[J]. 中国生物工程杂志, 2017, 37(1): 14-20.

HU Li-qiang, ZHENG Wen, ZHONG Yi, DU Dan, YANG Hao, GONG Meng. Comparison of Expression and Activity of Antiviral Protein RC28 in Escherichia coli and Pichia pastoris. China Biotechnology, 2017, 37(1): 14-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170103    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I1/14

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