重组TRAIL_((114-281))片段的表达纯化及抗肿瘤活性分析
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摘要
本文主要探讨人TRIAL_((114-281))对肠癌和肺癌细胞增殖的影响.利用大肠杆菌原核分泌表达TRAIL_((114-281))片段,Ni-NTA亲和层析柱对表达蛋白进行纯化,纯化的蛋白经SDS-PAGE和Western blotting鉴定,通过CCK-8实验检测TRAIL片段对SW480、DLD1、A549肿瘤细胞增殖的影响.实验结果显示经SDS-PAGE和Western blotting鉴定纯化的蛋白为目的蛋白His-TRAIL_((114-281)),且纯度在90%以上.CCK-8结果显示,在一定浓度下,TRAIL_((114-281))片段能明显抑制SW480、DLD1、A549肿瘤细胞的增殖,抑制率呈浓度依赖性,且对各细胞的抑制作用效果不同.本研究证实TRIAL_((114-281))片段在抗肠癌及非小细胞肺癌细胞方面具有一定的应用前景.
In order to investigate the anti-tumor activity of TRAIL_((114-281)). Protein was expressed in Escherichia coli and purified by Ni-NTA chromatography column. SDS-PAGE and Western blotting identify the purified His-TRAIL_((114-281)). The changes of cell proliferation were observed by CCK-8 assay. The results show that the target protein was analyzed by SDS-PAGE showing a special band about 20 kDa. The target protein was successfully purified by Ni-NTA chromatography. The purity of target protein was above 90%. Western blotting appeared a good antigenicity of the purified protein. The result of CCK-8 assay indicated that the prolification of SW480,DLD1,A549 cells was inhibited significantly. Moreover,this alteration of prolification was in contentration dependent manner. Recombinant TRAIL_((114-281)) could suppress the prolification of colon and lung cancer,and have some prospect in antitumor effects.
In order to investigate the anti-tumor activity of TRAIL_((114-281)). Protein was expressed in Escherichia coli and purified by Ni-NTA chromatography column. SDS-PAGE and Western blotting identify the purified His-TRAIL_((114-281)). The changes of cell proliferation were observed by CCK-8 assay. The results show that the target protein was analyzed by SDS-PAGE showing a special band about 20 kDa. The target protein was successfully purified by Ni-NTA chromatography. The purity of target protein was above 90%. Western blotting appeared a good antigenicity of the purified protein. The result of CCK-8 assay indicated that the prolification of SW480,DLD1,A549 cells was inhibited significantly. Moreover,this alteration of prolification was in contentration dependent manner. Recombinant TRAIL_((114-281)) could suppress the prolification of colon and lung cancer,and have some prospect in antitumor effects.
引文
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[7]Dai X,Zhang J,Arfuso F,et al.Target TNF-related apoptosis-inducing ligand(TRAIL)receptor by natural products as a potential therapeutic approach for cancer therapy[J].Exp Biol Med,2015,240(6):760~730.
[8]Xiao Xia Xia,Ya Ling Shen,Dong Zhi Wei,et al.Purification and characterization of recombinant s TRAIL expressed in Escherichia coli[J].Acta Biochimica et Biophysica Sinica,2004,36(2):118~122.
[9]Zhihua Lin,Huanzong Lei,Peng Cao.Expression purication and in vitro refolding of soluble tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)[J].Protein Expression and Purication,2007,51:276~282.
[10]杨峰丽,梁雅丽,赵良启,等.人可溶性TRAIL原核分泌表达载体的构建[J].山西大学学报(自然科学版),2011,34(S2):116~119.
[11]Mielczarek Palacz A,Sikora J,Kondera Anasz Z.Assessment of concentration of s TRAIL ligand and its receptor s TRAIL-R1 and s TRAIL-R2-marker monitoring the course of the extrinsic pathway of apoptosis induction:potential application in ovarian cancer diagnostics[J].Arch Med Sci,2017,13(3):624~628.
[12]Chirlei Buneker,Andrea Mohr,Ralf Michael Zwacka.The TRAIL-receptor-1:TRAIL-receptor-3 and-4 ratio is a predictor for TRAIL sensitivity of cancer cells[J].Oncology Reports,2009,21:1289~1295.
[2]Pitti RM,Marsters SA,Ruppert S,et al.Induction of apoptosis by Apo-2 ligand,a new member of the tumor necrosis factor cytokine family[J].The Journal Biological Chemistry,1996,271(22):126,87~90.
[3]Avi Ashkenazi,Roger C Pai,Sharon Fong,et al.Safety and antitumor activity of recombinant soluble Apo2 ligand[J].The Journal of Clinical Investigation,1999,104:155~162.
[4]Peter Holoch,Thomas S Griffith.TNF-related apoptosis-inducing ligand(TRAIL):A new path to anti-cancer therapies[J].Eur J Pharmacol,2009,625(1-3):63~72.
[5]Heath A.Elrod,Shi Yong Sun.Modulation of death receptors by cancer therapeutic agents[J].Cancer Biology﹠Therapy,2008,7(2),163~173.
[6]Vicente Tur,Almer M van der Sloot,Carlos R Reis,et al.DR4-selective tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)variants obtained by structure-based design[J].The Journal Biological Chemistry,2008,283(29):20560~20568.
[7]Dai X,Zhang J,Arfuso F,et al.Target TNF-related apoptosis-inducing ligand(TRAIL)receptor by natural products as a potential therapeutic approach for cancer therapy[J].Exp Biol Med,2015,240(6):760~730.
[8]Xiao Xia Xia,Ya Ling Shen,Dong Zhi Wei,et al.Purification and characterization of recombinant s TRAIL expressed in Escherichia coli[J].Acta Biochimica et Biophysica Sinica,2004,36(2):118~122.
[9]Zhihua Lin,Huanzong Lei,Peng Cao.Expression purication and in vitro refolding of soluble tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)[J].Protein Expression and Purication,2007,51:276~282.
[10]杨峰丽,梁雅丽,赵良启,等.人可溶性TRAIL原核分泌表达载体的构建[J].山西大学学报(自然科学版),2011,34(S2):116~119.
[11]Mielczarek Palacz A,Sikora J,Kondera Anasz Z.Assessment of concentration of s TRAIL ligand and its receptor s TRAIL-R1 and s TRAIL-R2-marker monitoring the course of the extrinsic pathway of apoptosis induction:potential application in ovarian cancer diagnostics[J].Arch Med Sci,2017,13(3):624~628.
[12]Chirlei Buneker,Andrea Mohr,Ralf Michael Zwacka.The TRAIL-receptor-1:TRAIL-receptor-3 and-4 ratio is a predictor for TRAIL sensitivity of cancer cells[J].Oncology Reports,2009,21:1289~1295.