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通过对通用型在细菌中可溶性表达scFv-Fc抗体载体的构建,对抗IL-33全人源scFv-Fc抗体进行可溶性表达并鉴定。扩增人IgG1Fc片段及前期筛选的抗IL-33单链抗体(scFv)DNA,分别插入到改建的载体pLZ16中,构建可溶性重组表达pLZ16-scFv-Fc质粒。将重组质粒转入E.coli DH5αF’,菌落经PCR和测序验证表明我们成功构建了pLZ16-scFv-Fc重组质粒。将阳性克隆子分别于32℃IPTG表达5h或25℃无IPTG表达50h,用HRP标记抗人IgG1Fc抗体进行检测,结果显示25℃无IPTG表达50h时,表达蛋白分泌在细菌周质间,可明显检测到scFv-Fc的可溶性表达;western blotting结果显示,25℃无IPTG表达的scFv-Fc为65kD左右,其可溶性表达量较32℃表达量高。本研究成功构建了pLZ16-scFv-Fc重组质粒,并在E.coli DH5αF’中成功可溶性表达抗IL-33全人源scFv-Fc抗体。
The anti-IL-33 full-human scFv-Fc antibody was solublely expressed and identified by construction of a universal universal vector for scFv-Fc antibody expression in bacteria. The human IgG1 Fc fragment and the pre-screened anti-IL-33 single chain antibody (scFv) DNA were amplified and inserted into the modified vector pLZ16 respectively to construct the recombinant plasmid pLZ16-scFv-Fc. The recombinant plasmids were transformed into E. coli DH5αF ’. The colonies were confirmed by PCR and sequencing. We successfully constructed pLZ16-scFv-Fc recombinant plasmids. The positive clones were respectively expressed in IPTG at 32 ℃ for 5h or without IPTG at 25 ℃ for 50h and detected by HRP-labeled anti-human IgG1Fc antibody. The results showed that the expressed protein secreted in bacterial periplasm at 25 ℃ without IPTG for 50h The soluble expression of scFv-Fc was detected. Western blotting showed that the scFv-Fc without IPTG at 25 ℃ was about 65kD, and its expression level was higher than that at 32 ℃. In this study, the pLZ16-scFv-Fc recombinant plasmid was constructed successfully and the soluble anti-IL-33 whole-body scFv-Fc antibody was successfully expressed in E. coli DH5αF ’.