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目的构建并表达抗酸性同功铁蛋白(acidicisoferritin,AIF)的免疫细胞因子,并研究其抗肿瘤特性。方法在克隆小鼠趋化因子CXCL10全长基因的基础上,构建抗AIF单链抗体与CXCL10组成的重组免疫细胞因子,并在小鼠骨髓瘤细胞NSO中进行表达;使用流式细胞技术(FCM)和体外细胞趋化试验等方法来研究该重组蛋白的抗肿瘤特性。结果重组免疫细胞因子(IP10scFv)真核表达产物的相对分子质量(Mr)约为41.1×103,纯化后的蛋白浓度为68mgL,抗体亲和常数(KDIP10scFv)为2.28×10-8molL。IP10scFv能够特异识别分泌AIF的SMMC7721细胞,并能与激活后的小鼠T淋巴细胞表面CXCR3受体特异性结合,对小鼠激活的T淋巴细胞有较强的趋化作用。结论成功地制备了抗AIF单链抗体与趋化因子CXCL10构成的重组免疫细胞因子,该免疫细胞因子是肿瘤治疗的潜在制剂。
Objective To construct and express anti-acidic isoferritin (AIF) immunocytokines and to study their anti-tumor properties. Methods Based on the cloning of the full-length CXCL10 gene of murine chemokine CXCL10, a recombinant immunocytokine consisting of anti-AIF single chain antibody and CXCL10 was constructed and expressed in mouse myeloma NSO cells. Flow cytometry (FCM) ) And in vitro cell chemotaxis assay to study the anti-tumor properties of the recombinant protein. Results The relative molecular mass (Mr) of recombinant eukaryotic expression product (IP10 scFv) was about 41.1 × 103. The purified protein concentration was 68 mgL and the antibody affinity constant (KDIP10 scFv) was 2.28 × 10-8 molL. IP10scFv can specifically recognize AIF-secreting SMMC7721 cells and bind specifically with the activated CXCR3 receptor on the surface of mouse T lymphocytes. IP10scFv has a strong chemotactic effect on mouse activated T lymphocytes. Conclusion Recombinant immune cytokines composed of anti-AIF single-chain antibody and chemokine CXCL10 were successfully prepared, and the immunocytokines are potential agents for tumor therapy.