尿激酶型纤溶酶原激活物生物传感器的构建及鉴定

doi:10.13481/j.1671-587x.20150605

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (06): 1124-1129.doi: 10.13481/j.1671-587x.20150605

尿激酶型纤溶酶原激活物生物传感器的构建及鉴定

崔钢华^1,2, 饶烽^1,2, 王琰^1,2, 曹薇薇², 刘伟², 王维山^1,2, 史晨辉^1,2

1. 石河子大学医学院第一附属医院骨一科, 新疆石河子 832000;
2. 石河子大学新疆地方民族与地方病教育部重点实验室, 新疆石河子 832000

收稿日期:2015-02-02 发布日期:2016-01-11
通讯作者: 王维山,副教授,副主任医师,硕士研究生导师(Tel:0993-2859427,E-mail:wwsmc2002@sina.com);史晨辉,教授,硕士研究生导师(Tel:0993-2859427,E-mail:sch7890@yahoo.com) E-mail:wwsmc2002@sina.com;sch7890@yahoo.com
作者简介:崔钢华(1988-),男,河南省漯河市人,在读医学硕士,主要从事关节外科学方面的研究。
基金资助:
国家自然科学基金资助课题(81160225,81260453,81360451);新疆兵团医药卫生专项基金资助课题(2013BA020);新疆兵团国际交流与合作专项基金资助课题(2012BC002,2011BC004);新疆兵团科技创新团队专项基金资助课题(2014CC002)

Construction and identification of urokinase-type plasminogen activator biosensor plasmid

CUI Ganghua^1,2, RAO Feng^1,2, WANG Yan^1,2, CAO Weiwei², LIU Wei², WANG Weishan^1,2, SHI Chenhui^1,2

1. Department of Orthopedics, First Affiliated Hospital, School of Medical Sciences, Shihezi University, Shihezi 832000, China;
2. Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education, Shihezi University, Shihezi 832000, China

Received:2015-02-02 Published:2016-01-11

摘要/Abstract

摘要：

目的:构建含有增强型青色荧光蛋白-尿激酶型纤溶酶原激活物(uPA)作用底物(substrate)-黄色荧光蛋白变体(YPet)融合蛋白的真核表达载体(ECFP-uPA substrate-linker-YPet),即uPA的生物传感器。方法:以Src-biosensor为模板,Primer Premier 5.0软件设计YPet引物,设计时5'端引入uPA底物序列及Linker,两端连接酶切位点及保护碱基。以pMD^TM-18T为中间载体,通过基因工程方法构建含有ECFP-uPA substrate-linker-YPet的真核表达载体。然后转染293T细胞,24 h后观察转染效率和融合蛋白表达情况,在荧光显微镜下,应用MetaFlour FRET 4.6软件观察并测量uPA生物传感器荧光共振能量转移(FRET)。结果:经过PCR和双酶切鉴定,克隆片段和酶切片段均与uPA substrate分子大小相符。细胞转染后转染效率达40%。免疫荧光检测,uPA生物传感器在293T细胞膜表达,用重组人uPA(rhuPA)刺激转染细胞可以检测到FRET现象。结论:成功构建uPA生物传感器,该生物传感器能够作为活细胞分子探针用于研究uPA的时空变化。

关键词: 尿激酶型纤溶酶原激活物, 生物传感器, 荧光共振能量转移, 骨性关节炎

Abstract:

Objective To construct the eukaryotic expression vector urokinase-type plasminogen activator (uPA) biosensor which was the composition of the fusion protein enhanced cyan fluorescent protein-uPA (substrate)-yellow fluorescent protein variant (ECFP-uPA substrate-linker-YPet). Methods By the template Src-biosensor,the YPet primers were designed by Primer Premier 5.0 software,and the restriction enzyme sites,uPA substrate gene sequence and linker were added in its 5' end.With the intermediate vector pDM^TM-18T,an eukaryotic expression vector which contained a fusion protein of ECFP-uPA substrate-linker-YPet was constructed by genetic engineering.Then the uPA biosensor was transfected into 293T cells.The transfection efficiency and expression of fusion proteins were observed after 24 h.Fluorescence resonance energy transfer (FRET) was observed by the inversion fluorescence microscope and measured by the MetaFlour FRET 4.6 software. Results The uPA biosensor vector was confirmed by the fragment of PCR and double restriction enzyme digestion.The transfection efficiency was nearly 40%.The immunofluorescence detection results displayed that uPA biosensor fusion protein expressed in the 293T cells membrane and the FRET of uPA biosensor in the living 293T cells was observed after incubation with the recombinant human uPA(rhuPA). Conclusion uPA biosensor is successfully constructed and it could be used as a molecular probe to study the temporal and spatial variation of uPA in living cells.

Key words: urokinase-type plasminogen activator, biosensor, fluorescence resonance energy transfer, osteoarthritis

中图分类号:

崔钢华, 饶烽, 王琰, 曹薇薇, 刘伟, 王维山, 史晨辉. 尿激酶型纤溶酶原激活物生物传感器的构建及鉴定[J]. 吉林大学学报(医学版), 2015, 41(06): 1124-1129.

CUI Ganghua, RAO Feng, WANG Yan, CAO Weiwei, LIU Wei, WANG Weishan, SHI Chenhui. Construction and identification of urokinase-type plasminogen activator biosensor plasmid[J]. Journal of Jilin University Medicine Edition, 2015, 41(06): 1124-1129.

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尿激酶型纤溶酶原激活物生物传感器的构建及鉴定

Construction and identification of urokinase-type plasminogen activator biosensor plasmid

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