抗A型肉毒毒素人源单链抗体融合蛋白的重组设计

以获得的抗A型肉毒毒素单链抗体为模板,进行融合改构,将人IgG1的Fc片段连接到ScFv的C端,在大肠杆菌中实现抗体融合蛋白ScFv-Fc的表达,表达量30%以上,蛋白以包含体形式存在,经过体外变复性的抗体融合蛋白ScFv-Fc,进行Protein G Sepharose柱亲和层析纯化,纯度达90%～95%.体外活性检测结果表明,重组抗体融合蛋白ScFv-Fc可以特异结合A型肉毒类毒素抗原,其相对亲和力近似于母本单链抗体,其稳定性高于母本单链抗体.

抗肿瘤单链抗体融合蛋白的研究进展

尽管有手术、放疗和化疗等综合治疗手段,在过去20多年,恶性脑胶质瘤特别是多形胶质母细胞瘤的预后并无明显改善。然而,在成人颅内恶性肿瘤中,多形胶质母细胞瘤每年的发病率占到50%以上[1]。虽然常规手术配合放化疗可以延长患者的生命,但是浸润性生长的特性决定了上述方法都无法有效清除肿瘤细胞,导致肿瘤在短时间内复发,患者的中位生存期一般不超过15个月[2]。

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.

Antitumor effects of an engineered and energized fusion protein consisting of an anti-CD20 scFv fragment and lidamycin

Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases.CD20 antigen,which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma,is an attractive target for the therapy of B-lymphoid malignancies.Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical trials.In this study,we prepared an engineered fusion protein,scFv-LDP,consisting of an anti-CD20 scFv fragment and the apoprotein LDP of LDM using DNA recombination.After purification and refolding,scFv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining assays.The energized fusion protein scFv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and scFv-LDP.MTT assay revealed potent cytotoxicity of scFv-LDP-AE to CD20-positive Raji and Daudi cells,with IC 50 values of 1.21×10-11 and 6.24×10-11 mol L-1,respectively.An in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also utilized.Drugs were given intravenously on day 14 and 21 after tumor transplantation.In terms of maximal tolerated doses,scFv-LDP-AE at 0.3 mg kg-1 suppressed tumor growth by 79.3%,and LDM at 0.05 mg kg-1 by 68.6% (P<0.05).Results suggested scFv-LDP-AE could be a potential candidate for tumor-targeting therapy.