二硫键稳定的抗TNF-α单链抗体的原核表达及生物学特性鉴定

Prokaryotic Expression and Biological Activity of Disulfide-Stabilized TNF-α Single Chain Antibody

目的构建二硫键稳定的抗TNF-α单链抗体原核表达质粒,并进行表达和生物学特性鉴定。方法利用SWISS-PROT软件分析抗TNF-α单链抗体(scFv)的结构,并结合二硫键稳定的单链抗体(ds-scFv)的突变位点设计原则,确定其突变位点,采用PCR定点突变的方法,构建二硫键稳定的抗TNF-α单链抗体的表达质粒pGEX-4T-1-ds-scFv,转化大肠杆菌BL21(DE3),IPTG诱导表达。采用十二烷基肌氨酸钠(Sarkosy)l对表达的目的蛋白进行可溶性纯化,并进行抗原结合活性、相对稳定性及细胞毒中和活性检测。结果重组表达质粒pGEX-4T-1-ds-scFv经菌落PCR及测序鉴定,证明构建正确。抗TNF-α的ds-scFv的表达量占菌体总蛋白的16.6%,主要以包涵体形式表达;纯化的目的蛋白纯度可达94.4%;经ELISA及Western blot验证,ds-scFv与TNF-α抗原的结合活性与scFv相近;相对稳定性优于scFv;在同样的抗体浓度下,scFv和ds-scFv的细胞毒中和活性略低于亲本鼠单抗,ds-scFv略高于scFv。结论已成功构建了二硫键稳定的抗TNF-α单链抗体的原核表达质粒,并获得有活性的目的蛋白,为进一步研究其生物学功能奠定了基础。

Objective To construct a prokaryotic expression vector for disulfide-stabilized single chain antibody （ds-scFv） against TNF-α, express the target protein and determine its biological activity. Methods The structure of ds-scFv against TNF-α was analyzed by using SWISS-PROT software, based on which the mutation site was selected, and recombinant plasmid pGEX-4T-1-dsscFv was constructed by PCR-based point mutagenesis, then transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed protein was purified with sarkosyl and determined for antigen-binding activity, relative stability and neutralizing activity to cytotoxieity. Results PCR and sequencing proved that recombinant plasmid pGEX-4T-1-ds-scFv was constructed correctly. The expressed ds-scFv against TNF-α mainly existed in a form of inclusion body, contained 16.6% of total somatic protein, and reached a purity of 94.4% after purification. ELISA and Western blot proved that the binding activities to TNF-α antigen of ds-scFv were similar to that of scFv, while the relative stability of the former was higher than that of the latter. The neutralizing activities to cytotoxicity were slightly lower in seFv and ds-seFv than in their parental murine McAbs at the same concentrations, However, the neutralizing activity to cytotoxicity of ds-scFv was slightly higther than that of scFv. Conclusion The prokaryotic expression vector for disulfide-stabilized TNF-α single chain antibody was successfully constructed, and the target protein with biological activity was expressed, which laid a foundation of further study on biological function of ds-scFv against TNF-α.