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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 the immunogenicity and reduce the molecular weight of murine antibody against human, the gene construction and expression of human activated platelet monoclonal antibody SZ 5 1 scFv was explored, and we hope to find out a stable and efficient expression SZ 5 1ScFv method. Methods Two different SZ 5 1 ScFv expression vectors, pHEN1 5 1ScFv and pET2 0b 5 1ScFv, were constructed and expressed in Escherichia coli HB2 15 1 and BL21 (DE3) plys respectively. Their expression profiles were also compared . Results After pHEN1 5 1 ScFv was induced by IPTG in HB2 15 1, SZ 5 1 ScFv was secreted into the bacterial culture supernatant in a soluble form. After induced by IPTG in BL2 1 (DE3) plys, pET20b 5 1ScFv existed as both soluble and insoluble inclusion bodies, which accounted for 20% of total bacterial proteins. Western blot showed that the expressed products of both systems maintained the ability of the parent antibody to specifically bind to activated platelets. Conclusion The pET20b expression system is a stable and efficient expression system for SZ 5 1ScFv. However, the inclusion complex part of the refolding conditions still need to be further explored.