重组CD13单链抗体和蝎毒素多肽AGAP融合蛋白真核表达及对NB4 细胞的靶向杀伤作用

中国临床药理学与治疗学 ›› 2008, Vol. 13 ›› Issue (11): 1231-1236.

重组CD₁₃单链抗体和蝎毒素多肽AGAP融合蛋白真核表达及对NB4 细胞的靶向杀伤作用

倪吴花¹, 骆超³, 俞康², 吴建波¹

¹温州医学院附属第一医院医学科学研究所,²血液科,³温州医学院, 温州 325000, 浙江

收稿日期:2008-07-21 修回日期:2008-10-21 出版日期:2008-11-26 发布日期:2020-10-14
通讯作者: 俞康,男, 主任医师, 硕士生导师, 研究方向:血液病诊断与治疗。Tel:13806681379 E-mail: yukang62@126.com
作者简介:倪吴花, 女, 在职硕士研究生, 主管检验技师, 研究方向:预防医学。Tel:13587873920 E-mail: wuhuafeyner@yahoo.com.cn
基金资助:
浙江省自然科学基金资助项目(Y206383)

Establishment of fusion protein eukaryotic expression vector of singlechain antibody fragments reacting with human CD₁₃ and scorpion toxin polypeptide AGAP and the target lethal effect to NB4 cells

NI Wu-hua¹, LUO Chao³, YU Kang², WU Jian-bo¹

¹Institute of Medical Sciences, First Affiliated Hospital of Wenzhou Medical College,²Department of Hematology,³Wenzhou Medical College, Wenzhou 325000, Zhejiang, China

Received:2008-07-21 Revised:2008-10-21 Online:2008-11-26 Published:2020-10-14

摘要/Abstract

摘要： 目的: 本研究应用基因工程技术方法, 构建含重组编码CD₁₃单链抗体和蝎毒素镇痛抗肿瘤缬精甘肽(analgesic antitumoral peptide, AGAP) 融合蛋白基因的表达载体, 并研究CD₁₃-AGAP 融合蛋白对CD₁₃ 阳性白血病细胞NB4 影响。方法: 克隆东亚钳蝎活性肽AGAP 的基因, 插入真核表达载体pSecTag2 /CD₁₃ /RFP, 得到pSecTag2 /CD₁₃ /AGAP重组真核表达载体。酶切及测序鉴定后, 脂质体介导重组质粒转染293T 细胞, 通过RT-PCR 和免疫印迹方法检测融合基因和蛋白的表达。纯化融合蛋白, 作用NB4 细胞, CCK8 检测细胞活性, 流式细胞仪检测细胞周期。结果: 酶切及测序结果证实pSecTag2 /CD₁₃ /AGAP 重组真核表达载体构建成功, 转染293T 细胞后, RT-PCR 和免疫印迹方法结果证实重组质粒得到有效表达, 并纯化真核表达融合蛋白CD₁₃-AGAP 对血液肿瘤CD₁₃阳性白血病细胞NB4 有一定的生长抑制作用, 并且使细胞周期G₁ 期阻滞, S 期减少。结论: 成功构建了融合蛋白真核表达载体pSecTag2 /CD₁₃ /AGAP, 并在293T 细胞中成功表达, 进一步证实融合蛋白对CD₁₃阳性白血病细胞NB4 有杀伤作用。

关键词: 东亚钳蝎, 蝎毒活性肽, 抗肿瘤缬精甘肽, CD₁₃单链抗体, 融合蛋白

Abstract: AIM: To establish the eukaryotic expression vector of single-chain antibody fragments reacting with human CD₁₃ and scorpion toxin polypeptide analgesic antitumoral peptide (AGAP) and the target lethal effect to NB4 cells by genetic engineering method. METHODS: Buthus martensii Karsch active peptide AGAP gene was inserted eukaryotic expression vector pSecTag2 /CD₁₃ /RFP, and the recombinate eukaryotic expression vector pSecTag2 /CD₁₃ /AGAP was obtained. After double enzyme digestion and DNA sequencing identification, the vector was transfected into 293T cell line, the AGAP recombinant gene and protein expression were detected by RT-PCR and western blot methods. The recombinant protein was purified by ProBond(tm) Nickel-Chelating Resin kit and used to treat NB4 cells. NB4 cell cytoactive was measured by CCK-8 and the cell cycle was analyzed by FCM. RESULTS: The eukaryotic expression vector pSecTag2/ CD₁₃ /AGAP was successfully established by double enzyme digestion and DNA sequencing identification. AGAP gene and protein expression were detected by RT-PCR and Western Blot after the vectors were transfected into 293T cells. After treatment with the purified recombinant protein CD₁₃-AGAP, NB4 cells viability were decreased and CD₁₃-AGAP recombinant protein arrested NB4 cell cycle in G₁ phase and decreased in S phase. CONCLUSION: The recombinant plasmid pSecTag2 /CD₁₃ /AGAP was successfully established and expressed in 293T cells, the recombinant protein CD₁₃- AGAP had lethal effect to NB4 cells.

Key words: Buthus martensii Karsch, scorpion venom active peptides, analgesic antitumoral peptide, CD₁₃ single chain fragments, fusion protein

中图分类号:

R730
Q786

倪吴花, 骆超, 俞康, 吴建波. 重组CD₁₃单链抗体和蝎毒素多肽AGAP融合蛋白真核表达及对NB4 细胞的靶向杀伤作用[J]. 中国临床药理学与治疗学, 2008, 13(11): 1231-1236.

NI Wu-hua, LUO Chao, YU Kang, WU Jian-bo. Establishment of fusion protein eukaryotic expression vector of singlechain antibody fragments reacting with human CD₁₃ and scorpion toxin polypeptide AGAP and the target lethal effect to NB4 cells[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2008, 13(11): 1231-1236.

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重组CD₁₃单链抗体和蝎毒素多肽AGAP融合蛋白真核表达及对NB4 细胞的靶向杀伤作用

Establishment of fusion protein eukaryotic expression vector of singlechain antibody fragments reacting with human CD₁₃ and scorpion toxin polypeptide AGAP and the target lethal effect to NB4 cells

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