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目的制备单链抗体－人肿瘤坏死因子α(hTNF-α)融合蛋白。方法将hTNF-α的成熟肽cDNA以重组PCR技术融合于胶质瘤单链抗体基因的3′端，构建39ScFv-hTNF-α融合基因，克隆入大肠杆菌分泌型表达载体pET-20b(＋) ，并诱导表达。结果融合蛋白的表达量占菌体总蛋白的20％。还原SDS-PAGE和Western blot显示，其相对分子质量(Mr)为44 000,具有识别胶质瘤相关抗原的活性及TNF-α的抑瘤活性。结论完成了39ScFv-hTNF-α融合基因的构建，并于大肠杆菌中表达了双功能融合蛋白，为胶质瘤的临床治疗提供了新的高效免疫导向药物。
Objective To prepare single chain antibody - human tumor necrosis factor alpha (hTNF-α) fusion protein. Methods The mature cDNA of hTNF-α was fused to the 3 ’end of glioma single-chain antibody gene by recombinant PCR. The 39ScFv-hTNF-α fusion gene was constructed and cloned into E.coli secretory expression vector pET-20b (+). , And induced expression. Results The fusion protein expressed 20% of the total bacterial proteins. SDS-PAGE and Western blot showed that the relative molecular mass (Mr) was 44 000, which showed the activity of recognizing glioma-associated antigen and the anti-tumor activity of TNF-α. Conclusion The construction of the 39ScFv-hTNF-α fusion gene was completed and the bifunctional fusion protein was expressed in E. coli, which provided a novel and highly effective immunostimulatory drug for the clinical treatment of glioma.