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

Abstract

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

CLC Number:

Q782

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

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