以组织因子为靶点的肿瘤免疫治疗结合物的合成及其对结直肠癌细胞的影响

目的:构建含hFⅦ-LC+hIgG1-Fc cDNA的重组真核表达载体,并转染中国仓鼠卵巢细胞亚株CHO-K1获得以组织因子（TF）为靶点的免疫治疗结合物用于肿瘤的靶向治疗。方法:用分子生物学方法从汉族人肝组织和淋巴细胞中分别获得hFⅦ-LC和hIgG1-Fc DNA片段并用连接酶正向克隆入真核表达质粒pcDNA3.1（+）中。以脂质体法转染阳性质粒入CHO-K1细胞,G418加压筛选获得阳性单克隆细胞;Excel 301无血清培养液扩大培养,培养液Ni-NTA亲和层析纯化和制备hFⅧ-LC+hIgG1-Fc融合蛋白,ELISA法检测该融合蛋白与TF的靶向作用,免疫激活物与结肠癌细胞HT-29及NK细胞混合培养检测结合物激活NK细胞的能力。结果:以汉族人肝组织和淋巴细胞为模板扩增的hFⅦ-LC和hIgG1-Fc DNA片段经测序证实与基因库报道的系列完全一致;经酶切分析、测序证实构建的hFⅦ-LC+hIgG1-Fc融合基因片段完整地落在真核表达质粒pcDNA3.1（+）的阅读框架中;Lipofectamine^TM2000转染试剂能获得含pcDNA3.1（+）-hFⅦ-LC+hIgG1-Fc阳性的CHO-K1细胞,经1g/L G418加压筛选可以获得分泌hFⅦ-LC+hIgG1-Fc融合蛋白的单克隆细胞株;ILExcel301无血清培养液用Ni-NTA亲和层析纯化可以制备1.3 mg hFⅦ-LC+hIgG1-Fc融合蛋白,经ELISA法鉴定与TF具有高亲和力,免疫激活物与结肠癌细胞HT-29及NK细胞混合培养表现出明显的激活NK细胞的能力。结论:成功构建了hFⅦ-LC+hIgG1-Fc融合蛋白的真核表达载体并获得了分泌hFⅦ-LC+hIgG1-Fc融合蛋白的CHO-K1单克隆细胞株,为制备以TF为靶点的肿瘤免疫治疗结合物奠定了基础。

Construction of an immunoconjugate targeting tissue factor and its effect on colorectal cancer cells

AIM:To construct a recombinant eukaryotic expression vector pcDNA3.1(+ )-hFVII-LC + hlgG1-Fc,and to produce and purify the immunoconjugate hFVII-LC + hlgG1-Fc protein.METHODS:The target sequences were amplified by RT-PCR from hepatic tissue and lymphocyte RNA,and cloned into eukaryotic expression vector pcDNA3.1(+ ).After confirmed by restriction endonuclease digestion and DNA sequencing,the recombinant plasmid was transfected into CHO-K1 cells by lipofectamine 2000.The transfectant clones were selected by G418 screening. The positive monoclonals were grown in CHO-K1 serum-free medium Excel 301 and the culture medium was collected.The hFVII-LC + hlgG1-Fc protein was purified by affinity Ni-NTA resin.The immunoconjugate was identified by ELISA with tissue factor(TF) affinity and specificity.Induction of NK cell -mediated antibody -dependent cell cytotoxicity(ADCC) was examined in HT-29 colorectal cancer cell line.RESULTS:Human liver tissue and lymphocytes from Han population were used as template for amplification of hFVII-LC and hlgG1-Fc DNA fragments,which were confirmed by sequencing and were exactly the same as those GenBank reported.The eukaryotic expression vector pcDNA3.1(+ )-hFVII-LC + hlgGl-Fc was successfully constructed,and 1.3 mg of hFVII-LC + hlgG1-Fc protein could be prepared from 1 liter of Excel 301 serum-free culture medium through Ni-NTA affinity chromatography.The immunoconjugate was specially bound to TF and induced a significant ADCC response in HT-29 cells.CONCLUSION:The human hFVII-LC + hlgGl-Fc recombinant plasmid and the hFVII-LC + hlgGl-Fc immunoconjugate are obtained,which provide the basis for further study of cancer-targeted therapy.