人活化血小板单抗SZ-51单链抗体的基因构建及高效表达

The expression of single chain Fv fragment of monoclonal antibody against activated platelets in E.coli

目的 为降低鼠源性抗体对人体的免疫原性并降低其分子量 ,进行了人活化血小板单克隆抗体SZ 5 1单链抗体 (ScFv)的基因构建及表达 ,希望摸索出一套稳定、高效表达SZ 5 1ScFv的方法。方法 同时构建了两种不同的SZ 5 1ScFv表达载体pHEN1 5 1ScFv及pET2 0b 5 1ScFv ,并分别导入大肠杆菌HB2 15 1及BL2 1(DE3)plys中进行表达 ,同时对它们的表达特性进行了比较。结果 pHEN1 5 1ScFv在HB2 15 1中经IPTG诱导后 ,SZ 5 1ScFv以可溶性形式分泌至细菌培养上清中。pET2 0b 5 1ScFv在BL2 1(DE3)plys中经IPTG诱导后 ,SZ 5 1ScFv以可溶性与不溶性的包涵体两种形式存在 ,其表达量占菌体总蛋白的 2 0 %。经Westernblot证明两种体系表达产物均维持了亲本抗体与活化血小板特异性结合的能力。结论 pET2 0b表达体系对于SZ 5 1ScFv而言是一种稳定、高效的表达体系。但其包涵体部分的变复性条件仍需进一步探讨。

Objective To reduce immunogenicity and molecular weight of a monoclonal antibody specific for activated human platelet and to express single chain Fv fragment (ScFv) in E.coli . Methods Two expression vectors of SZ-51 ScFv, pHEN1-51 ScFv and pET20b-51 ScFv, were constructed and transformed into E.coli strain HB2151 and BL21(DE3) plys, respectively. The expressed recombinant proteins were analysed. Results Induced by IPTG, HB2151 secreted soluble SZ-51 ScFv to the cultured supernatant. The BL21 (DE3) plys expressed the SZ-51 ScFv the soluble recombinant protein and insoluble recombinant protein in inclusion bodies and constituted 20% of the total cell protein. Western blot analysis showed that the SZ-51 ScFv obtained from two strains of E.coli maintained the binding activity to activated human platelet as their parent ones. Conclusion The results showed that the pET20b expression system is stable and efficient for expressing SZ-51 ScFv. However, the denaturation and protein refolding of the SZ-51 ScFv expressed in inclusion bodies need to be studied further.