人源抗破伤风毒素单链二硫键稳定抗体的重组设计与表达

Construction and expression of recombinant prokaryotic expression vector for disulfide-stabilized anti-TeNT single-chain Fv antibody

目的构建人源抗破伤风毒素重链C端单链二硫键稳定抗体原核表达载体,并进行原核表达和生物学特性鉴定。方法采用PCR定点突变的方法,获得二硫键稳定的抗破伤风毒素重链C端(TeNT-Hc)单链抗体基因(27G-scdsFv)。连接pET22b(+)载体,转化大肠杆菌BL21(DE3)工程菌,IPTG诱导表达,SDS-PAGE、Western blot法鉴定表达产物。ELISA检测27G-scdsFv体外抗原特异结合活性和抗体相对稳定性;非竞争酶免法检测抗体亲和力。采用免疫荧光法检测27G-scdsFv体外中和活性。结果测序结果显示获得正确的27G-scdsFv基因。原核表达scdsFv以包涵体形式存在,表达量约占菌体总蛋白的50%。复性后的27G-scdsFv保持了与TeNT-Hc的特异结合活性,亲和力较其scFv形式略有提升,KD=0.93×10-7mol/L,1 L培养物可获得5 mg scdsFv蛋白。27G-scdsFv的稳定性较scFv形式明显增强。27G-scdsFv在体外可以明显抑制TeNT-Hc与神经元细胞的结合。结论成功构建人源抗破伤风毒素重链C端单链二硫键稳定抗体原核表达载体,并获得有活性的目的蛋白,为27G-scdsFv的进一步生物学功能研究奠定基础。

Objective To construct the prokaryotic expression vector for disulfide-stabilized anti-TeNT single-chain Fv antibody （27G-scdsFv）, express the 27G-scdsFv and characterize its bioactivity. Methods 27G-scdsFv gene was construc- ted by PCR-based point mutagenesis strategy and cloned into plasmid pET22b（＋）. The plasmid was then transformed into E. coil BL21 （ DE3 ） competent cells. The target protein was expressed under the induction of IPTG. SDS-PAGE and Western blotting were used to identify the expression products. The specific binding activity to TeNT-Hc and relative stability in vitro of 27G-scdsFv were assayed by ELISA, and the affinity of the antibody was measured by non-competitive enzyme-linked immu- nosorbentassay. The method of immunofluorescence was employed to determine whether or not the 27G-scdsFv kept the neutralizing activity in vitro. Results Sequencing analysis proved that 27G-scdsFv gene was correctly constructed. The tar- get protein was expressed in the form of inclusion body, accounting for about 50% of total bacterial proteins. After renaturat- ed, the 27G-scdsFv protein still maintained specific binding activity to TeNT-Hc, with a higher affinity constant （ KD ） of O. 93 ×10-7 mol/L as compared with that of 27G-scFv, and the yield of the protein was about 5 mg/L induced culture. The relative stability of 27G-scdsFv was improved obviously as compared with the scFv form. 27G-scdsFv strongly inhibited the binding of TeNT-Hc to PC-12 in vitro. Conclusion The porkaryotic expression vector of 27G-scdsFv has been successfully constructed and the activated target protein has been obtained, which lays a foundation for the further study on the biological function of 27G-scdsFv.