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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2015, Vol. 35 Issue (1): 21-26    DOI: 10.13523/j.cb.20150103
研究报告     
拟南芥双油体蛋白融合表达KGF-2及其生物学活性研究
艾君1, 姜潮1,2, 刘敏1,2, 王晓艳1, 田海山1, 李校堃1,2
1. 温州医科大学药学院 浙江省生物技术与制药工程重点实验室 温州 325035;
2. 吉林农业大学生物反应器与药物开发教育部工程研究中心 长春 130118
Two Oleosins Flanking the KGF-2 Improve the Expression Level of KGF-2 in Arabidopsis thaliana and Its Activity Analysis
AI Jun1, JIANG Chao1,2, LIU Min1,2, WANG Xiao-yan1, TIAN Hai-shan1, LI Xiao-kun1,2
1. Key Laboratory of Biotechnology Pharmaceutical Engineering of Zhejiang Province, School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou 325035, China;
2. Engineering Research Center of Bioreactor and Pharmaceutical Development, Ministry of Education, Changchun 130118, China
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摘要:

目的:构建p1301-YO-K2-YO双油体KGF-2融合蛋白表达载体并转化拟南芥,与p1301-YO-K2单油体KGF-2融合蛋白表达载体对比,观察是否能够提高外源蛋白KGF-2的表达量,并对融合蛋白生物学活性进行鉴定。方法:PCR获得拟南芥油体蛋白oleosin和KGF-2核心区基因片段,融合PCR获得 KGF-2-oleosin 融合基因,与包含有一个oleosin的p1301-oleosin载体连接,构建p1301-YO-K2-YO(oleosin-KGF-2-oleosin)表达载体,利用农杆菌介导法将其转入拟南芥中进行表达,除草剂筛选获得转基因植株,通过PCR鉴定、SDS-PAGE、Western blot对KGF-2蛋白表达进行分析,提取油体蛋白涂抹法观察对C57BL/6小鼠的毛囊促生长作用。结果:双油体载体成功转入拟南芥基因组中;Western blot灰度分析可知,与单油体表达载体p1301-YO-K2相比,双油体融合表达策略可使KGF-2蛋白表达量提高2.5倍左右;动物实验结果表明油体融合蛋白具有良好的促毛囊增殖活性。

关键词: 拟南芥角质细胞生长因子-2双油体KGF-2 融合蛋白    
Abstract:

Objective: Construct the expression vector p1301-YO-K2-YO based on the oil body expression technology which has two oleosins flanking the KGF-2,observe whether it can improve the expression level of KGF-2 and identify its biological activity. Methods: KGF-2 core gene and Arabidopsis oleosin gene were amplified by PCR, KGF-2-oleosin fused sequence was obtained by the fusion PCR,ligated with the blank vector also has a oleosin gene(p1301-oleosin),then named it p1301-YO-K2-YO,which can be tanslated into the oleosin-KGF-2-oleosin fusion protein. Agrobacterium-mediated transformation of Arabidopsis thaliana was conducted by floral dip.The total protein of the T2 seeds were extracted for SDS-PAGE,Western blot analysis,the proliferation activity of hair follicles was tested on C57BL /6 mice. Results: The plasmid which has two oleosins flanking the KGF-2 was successfully expressed in Arabidopsis thaliana. Western blot result shows this fusion expression strategy could be twice more higher than the single-oleosin configuration production in KGF-2 protein expression,and has a certain biological activity.

Key words: Arabidopsis thaliana    Keratinocyte growth factor-2    Oleosin-KGF-2-oleosin fusion protein
收稿日期: 2014-10-19 出版日期: 2015-01-25
ZTFLH:  Q78  
基金资助:

浙江省自然科学基金(LY13H300006),2012年省级重点科技创新团队项目(2012R1004210),吉林省科技型中小企业创新基金(13C26212201223)资助项目

通讯作者: 姜潮, 李校堃     E-mail: xiaokunli@163.net;chaojiang10@hotmail.com
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引用本文:

艾君, 姜潮, 刘敏, 王晓艳, 田海山, 李校堃. 拟南芥双油体蛋白融合表达KGF-2及其生物学活性研究[J]. 中国生物工程杂志, 2015, 35(1): 21-26.

AI Jun, JIANG Chao, LIU Min, WANG Xiao-yan, TIAN Hai-shan, LI Xiao-kun. Two Oleosins Flanking the KGF-2 Improve the Expression Level of KGF-2 in Arabidopsis thaliana and Its Activity Analysis. China Biotechnology, 2015, 35(1): 21-26.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150103        https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I1/21


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