Department of Pathology, Nanjing Drum Tower Hospital affiliated Nanjing University Medical School, Nanjing, People's Republic of China.
BMC Biotechnol. 2012 Nov 28;12:91. doi: 10.1186/1472-6750-12-91.
The development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Transferrin receptor (TfR) is an endocytic receptor and identified as tumor relative specific due to its overexpression on most tumor cells or tissues, and TfR binds and intakes of transferrin-iron complex. We have previously generated an anti-TfR single-chain variable fragments of immunoglobulin (scFv) which were cloned from hybridoma cell line producing antibody against TfR linked with a 20 aa-long linker sequence (G4S)4. In the present study, the anti-TfR single-chain antibody (TfRscFv) was fused to DNA-binding domain of the yeast transcription factor GAL4. The recombinant fusion protein, designated as TfRscFv-GAL4, is expected to mediate the entry of DNA-protein complex into targeted tumor cells.
Fusion protein TfRscFv-GAL4 was expressed in an E. coli bacterial expression system and was recovered from inclusion bodies with subsequent purification by metal-chelate chromatography. The resulting proteins were predominantly monomeric and, upon refolding, became a soluble biologically active bifunctional protein. In biological assays, the antigen-binding activity of the re-natured protein, TfRscFv-GAL4, was confirmed by specific binding to different cancer cells and tumor tissues. The cell binding rates, as indicated by flow cytometry (FCM) analysis, ranged from 54.11% to 8.23% in seven different human carcinoma cell lines. It showed similar affinity and binding potency as those of parent full-length mouse anti-TfR antibody. The positive binding rates to tumor tissues by tissue microarrays (TMA) assays were 75.32% and 63.25%, but it showed weakly binding with hepatic tissue in 5 cases, and normal tissues such as heart, spleen, adrenal cortex blood vessel and stomach. In addition, the re-natured fusion protein TfRscFv-GAL4 was used in an ELISA with rabbit anti-GAL4 antibody. The GAL4-DNA functional assay through the GAL4 complementary conjugation with the GAL4rec-GFP-pGes plasmid to verify the GLA4 activity and GAL4rec-recognized specificity functions. It also shows the complex, TfRscFv-GAL4-GAL4rec-GFP-pGes, could be taken into endochylema to express the green fluorescent protein (GFP) with 8 to 10-fold transfection efficiency.
Results of our study demonstrated that the biofunctianality of genetically engineered fusion protein, TfRscFv-GAL4, was retained, as the fusion protein could both carry the plasmid of GAL4rec-pGes and bind TfR on tumour cells. This product was able to transfect target cells effectively in an immuno-specific manner, resulting in transient gene expression. This protein that can be applied as an effective therapeutic and diagnostic delivery to the tumor using endogenous membrane transport system with potential widespread utility.
细胞特异性基因传递载体的发展是基因治疗策略的主要目标。转铁蛋白受体（TfR）是一种内吞受体，由于其在大多数肿瘤细胞或组织上过度表达而被鉴定为肿瘤相对特异性，并且 TfR 结合并摄取转铁蛋白-铁复合物。我们之前从产生针对 TfR 的抗体的杂交瘤细胞系中克隆了抗 TfR 单链可变片段的免疫球蛋白（scFv），该 scFv 与 20 个氨基酸长的接头序列（G4S）4 相连。在本研究中，抗 TfR 单链抗体（TfRscFv）与酵母转录因子 GAL4 的 DNA 结合域融合。重组融合蛋白，命名为 TfRscFv-GAL4，预计将介导 DNA-蛋白复合物进入靶向肿瘤细胞。
融合蛋白 TfRscFv-GAL4 在大肠杆菌细菌表达系统中表达，并通过金属螯合层析从包涵体中回收。所得蛋白质主要是单体，并且在重折叠后成为可溶的生物活性双功能蛋白。在生物测定中，通过与不同的癌细胞和肿瘤组织的特异性结合来证实重折叠蛋白 TfRscFv-GAL4 的抗原结合活性。通过流式细胞术（FCM）分析，七种不同的人癌细胞系中的细胞结合率在 54.11%至 8.23%之间。它表现出与亲本全长鼠抗 TfR 抗体相似的亲和力和结合效力。组织微阵列（TMA）分析的肿瘤组织阳性结合率为 75.32%和 63.25%，但在 5 例中与肝组织结合较弱，与正常组织如心脏、脾脏、肾上腺皮质血管和胃结合较弱。此外，重折叠融合蛋白 TfRscFv-GAL4 用于 ELISA 中，用兔抗 GAL4 抗体。通过 GAL4 互补共轭与 GAL4rec-GFP-pGes 质粒进行 GAL4-DNA 功能测定，以验证 GAL4 活性和 GAL4rec 识别特异性功能。它还表明，复合物 TfRscFv-GAL4-GAL4rec-GFP-pGes 可以进入内体，以 8 到 10 倍的转染效率表达绿色荧光蛋白（GFP）。
我们的研究结果表明，遗传工程融合蛋白 TfRscFv-GAL4 的生物功能得到保留，因为融合蛋白既能携带 GAL4rec-pGes 质粒，又能与肿瘤细胞上的 TfR 结合。该产物能够以免疫特异性方式有效转染靶细胞，从而导致瞬时基因表达。该蛋白可通过内源性膜转运系统有效递送至肿瘤，具有潜在的广泛应用。
