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TAT-NDRG2重组蛋白的表达、纯化及其对LNCaP细胞系抗增殖作用的评估

Expression and purification of TAT-NDRG2 recombinant protein and evaluation of its anti-proliferative effect on LNCaP cell line.

作者信息

Farokhinejad Fahimeh, Behbahani Abbas Behzad, Rafiei Dehbidi Gholam Reza, Takhshid Mohammad Ali

机构信息

Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran; Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.

出版信息

Protein Expr Purif. 2017 Oct;138:25-33. doi: 10.1016/j.pep.2017.07.004. Epub 2017 Jul 13.

Abstract

N-myc downstream regulated gene2 (NDRG2) belongs to tumor suppressor protein family of NDRG. Anti-proliferative and anti-metastasis of NDRG2 overexpression has been demonstrated in a number of tumors. The aim of this study was to fuse the gene of Trans Activator of Transcription (TAT) protein transduction domain with NDRG2 gene and express and purify TAT-NDRG2 fusion protein in order to investigate the effects of TAT-NDRG2 protein on proliferation and apoptosis of LNCaP prostate carcinoma cell line. pET28a-TAT-NDRG2 and pET28a-NDRG2 plasmids were constructed and transformed into E. coli-BL21(DE3). TAT-NDRG2 and NDRG2 proteins were expressed in the bacteria, purified using affinity chromatography and verified using western blotting. The effects of TAT-NDRG2 and NDRG2 protein treatment on LNCaP cells proliferation and apoptosis were evaluated using MTT assay and AnnexinV, 7-AAD flow cytometry assay, respectively. Western blot analysis confirmed the expression and purification of TAT-NDRG2 and NDRG2 proteins. Treatment of LNCaP cells with TAT-NDRG2 protein increased cell death and induced apoptosis significantly (P < 0.05) compared to control and NDRG2 protein-treated cells. These results suggest that TAT-NDRG2 protein can be considered as a therapeutic modality for the treatment of tumors.

摘要

N-myc下游调控基因2(NDRG2)属于NDRG肿瘤抑制蛋白家族。NDRG2过表达的抗增殖和抗转移作用已在多种肿瘤中得到证实。本研究的目的是将转录反式激活因子(TAT)蛋白转导结构域基因与NDRG2基因融合,表达并纯化TAT-NDRG2融合蛋白,以研究TAT-NDRG2蛋白对LNCaP前列腺癌细胞系增殖和凋亡的影响。构建了pET28a-TAT-NDRG2和pET28a-NDRG2质粒,并将其转化到大肠杆菌BL21(DE3)中。TAT-NDRG2和NDRG2蛋白在细菌中表达,通过亲和层析纯化,并用蛋白质免疫印迹法进行验证。分别使用MTT法和AnnexinV、7-AAD流式细胞术检测法评估TAT-NDRG2和NDRG2蛋白处理对LNCaP细胞增殖和凋亡的影响。蛋白质免疫印迹分析证实了TAT-NDRG2和NDRG2蛋白的表达和纯化。与对照和NDRG2蛋白处理的细胞相比,用TAT-NDRG2蛋白处理LNCaP细胞可显著增加细胞死亡并诱导凋亡(P<0.05)。这些结果表明,TAT-NDRG2蛋白可被视为一种肿瘤治疗方式。

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