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目的:利用原核表达系统诱导表达一种可以用于肿瘤生物治疗ADEPT(单抗导向酶活化前药)的重组蛋白RGD4CβL,并对该蛋白进行纯化以及活性检测。方法:对合成的DNA序列进行测序,将重组载体pColdII-RGD4CβL转化入大肠杆菌E.coli BL(DE3),在大肠杆菌细胞中诱导表达目的蛋白,用His标签纯化树脂Ni-NTA纯化目的蛋白,以流式细胞术(FCM)检测纯化所得蛋白的活性。结果:合成的DNA全长为1125bp,其在E.coli BL(DE3)中经IPTG诱导后表达出相对分子质量(Mr)约为42000的目的蛋白,经纯化后获得了产率和纯度均很高的重组蛋白RGD4CβL,流式细胞检测证明该蛋白具有很好的肿瘤细胞靶向性。结论:成功地对重组蛋白RGD4CβL进行了诱导表达,得到的纯化蛋白具有较好的肿瘤细胞靶向性,该重组蛋白将可能成为一种免疫原性更低的新型双功能蛋白用于ADEPT。
OBJECTIVE: To use a prokaryotic expression system to induce the expression of a recombinant protein RGD4CβL that can be used in tumor biotherapy of ADEPT (monoclonal antibody-directed prodrug-activating prodrug), and to purify this protein and test its activity. Methods: The synthesized DNA sequence was sequenced. The recombinant vector pColdII-RGD4CβL was transformed into E.coli BL (DE3), and the target protein was expressed in E. coli cells. The target protein was purified with His-tag purified Ni-NTA resin, The purified protein was tested for its activity by flow cytometry (FCM). Results: The full-length DNA was 1125bp in length. The recombinant protein was induced by IPTG in E.coli BL (DE3) to express the target protein with a relative molecular mass of about 42000. After purification, the yield and purity of the target protein was very high High recombinant protein RGD4CβL, flow cytometry showed that the protein has good tumor cell targeting. CONCLUSION: The recombinant protein RGD4CβL was induced successfully and the purified protein had good tumor cell targeting. The recombinant protein might become a new type of bifunctional protein with lower immunogenicity for ADEPT.