重组氨基脲敏感型胺氧化酶在293T细胞中的定位及酶活性测定

doi:10.3969/j.issn.1004-616x.2017.04.007

癌变·畸变·突变 ›› 2017, Vol. 29 ›› Issue (4): 277-283.doi: 10.3969/j.issn.1004-616x.2017.04.007

重组氨基脲敏感型胺氧化酶在293T细胞中的定位及酶活性测定

钱九战, 李蕊, 罗红军, 林哲绚, 施金婉, 罗文鸿

汕头大学医学院生物分析实验室, 广东汕头 515041

收稿日期:2017-03-07 修回日期:2017-06-06 出版日期:2017-07-31 发布日期:2017-07-31
通讯作者: 罗文鸿,E-mail:whluo@stu.edu.cn E-mail:whluo@stu.edu.cn
作者简介:钱九战,E-mail:2683000144@qq.com。
基金资助:
国家自然科学基金（31671183）；汕头大学医学院创新强校工程（25012025）

Expression of recombinant semicarbazide-sensitive amine oxidase in 293T cells:localization and activity determination

QIAN Jiuzhan, LI Rui, LUO Hongjun, LIN Zhexuan, SHI Jinwan, LUO Wenhong

Bioanalytical Laboratory, Shantou University Medical College, Shantou 515041, Guangdong, China

Received:2017-03-07 Revised:2017-06-06 Online:2017-07-31 Published:2017-07-31

摘要/Abstract

摘要：

目的：构建氨基脲敏感型胺氧化酶（SSAO）基因真核表达载体，并在293T细胞中进行瞬时表达，确定SSAO体外表达效率、亚细胞定位及酶活性。方法：以pcDNA3-SSAO质粒为模板，PCR扩增获取SSAO编码区全序列，分别构建SSAO与绿色荧光蛋白（GFP）的融合表达质粒pEGFP-N3-SSAO和共表达质粒pIRES2-EGFP-SSAO，并通过酶切和测序验证。再分别将pcDNA3-SSAO、pEGFP-N3-SSAO、pIRES2-EGFP-SSAO和pcFLAG-SSAO重组质粒瞬时转染293T细胞，在荧光显微镜下观察目的蛋白的分布及表达情况。采用高效液相色谱法（HPLC）测定转染上述4种重组质粒及相应空质粒细胞的SSAO活性。结果：测序结果表明重组真核表达质粒构建正确。在瞬时转染pEGFP-N3-SSAO和pIRES2-EGFP-SSAO重组质粒的293T细胞中，转染后3~36 h各组均可观察到绿色荧光，且转染36 h为最佳观察时间点。转染pIRES2-EGFP-SSAO质粒的绿色荧光分布于细胞质中；转染pEGFP-N3-SSAO和pcFLAG-SSAO质粒的绿色荧光及红色荧光均分布于细胞膜上。酶活性检测结果显示转染pcFLAG-SSAO、pIRES2-EGFP-SSAO和pcDNA3-SSAO载体质粒的293T细胞中的SSAO活性分别为111.50±7.07、117.22±9.54、215.74±21.44 nmoL/（mg·h），较各自的空白质粒对照组均显著升高（P均 < 0.01），但转染pEGFP-N3-SSAO质粒的细胞SSAO活性极低，仅为2.09±0.29 nmoL/（mg·h）。结论：293T细胞中，体外重组表达的SSAO蛋白主要分布于细胞膜上，以pcDNA3为载体外源表达的天然状态SSAO活性最高。

关键词: 氨基脲敏感型胺氧化酶, 重组表达, 亚细胞定位, 酶活性测定

Abstract:

OBJECTIVE: To explore the protein expression efficiency,cellular localization and the amine oxidase activity of exogenous recombinant expressed semicarbazide-sensitive amine oxidase (SSAO) in 293T cells. METHODS: The recombinant plasmid pcDNA3-SSAO was used as template to amplify the entire sequence of SSAO coding area. SSAO and green fluorescent protein (GFP) fusion expression and the co-expression plasmids were respectively constructed and identified by restriction enzyme digestion and sequencing. The recombinant plasmids pcDNA3-SSAO,pEGFP-N3-SSAO,pIRES2-EGFP-SSAO and pcFLAG-SSAO were transiently transfected into 293T cells,then the distribution of recombinant protein in 293T cells was observed under fluorescence microscope. The enzymatic activity of SSAO in 293T cells that were transiently transfected with recombinant plasmids and with corresponding empty plasmids were measured using high-performance liquid chromatography. RESULTS: Sequencing results show that the recombinant eukaryotic expression plasmids pEGFP-N3-SSAO and pIRES2-EGFP-SSAO were constructed correctly. The green fluorescence was observed in 293T cells at 3~36 h after pEGFP-N3-SSAO and pIRES2-EGFP-SSAO plasmids' transfection,with optimal observation time of 36 h. The cells transfected with pIRES2-EGFP-SSAO plasmids showed green fluorescence in cytoplasm. The cells transfected with pEGFP-N3-SSAO and pcFLAG-SSAO showed green fluorescence or red fluorescence on the cellular membranes. SSAO activity of 293T cells transfected with pcFLAG-SSAO,pIRES2-EGFP-SSAO and pcDNA3-SSAO recombinant plasmids were 111.50±7.07,117.22±9.54,215.74±21.44 nmoL/(mg·h),respectively,which were significantly higher than that in the control group (P < 0.01). While,the activity of SSAO in cells transfected with pEGFP-N3-SSAO plasmid was only 2.09±0.29 nmoL/(mg·h). CONCLUSION: Recombinant expressed SSAO proteins were localized on the cellular membranes in 293T cells. Cells transfected with pcDNA3 as the carrier plasmids could generate higher SSAO activity.

Key words: expressed semicarbazide-sensitive amine oxidase, enzyme activity, recombinant expression, subcellular localization

中图分类号:

Q782

钱九战, 李蕊, 罗红军, 林哲绚, 施金婉, 罗文鸿. 重组氨基脲敏感型胺氧化酶在293T细胞中的定位及酶活性测定[J]. 癌变·畸变·突变, 2017, 29(4): 277-283.

QIAN Jiuzhan, LI Rui, LUO Hongjun, LIN Zhexuan, SHI Jinwan, LUO Wenhong. Expression of recombinant semicarbazide-sensitive amine oxidase in 293T cells:localization and activity determination[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2017, 29(4): 277-283.

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重组氨基脲敏感型胺氧化酶在293T细胞中的定位及酶活性测定

Expression of recombinant semicarbazide-sensitive amine oxidase in 293T cells:localization and activity determination

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