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

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。

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

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.