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目的基于N-糖基化部分改造毕赤酵母构建表达分泌型人全长抗体库。方法基于酵母分泌型表达载体pPICZαA构建双启动子串联表达重链和轻链恒定区基因的载体pPICZαA-C H-C L,经基因序列分析和Western blot分析验证。设计44对简并引物经逆转录多聚酶链式反应(RT-PCR)从人外周血单个核细胞(PBMC)中扩增获得轻链可变区V L和重链可变区V H基因库。通过PCR将其插入上述恒定区表达载体构建全长抗体表达库pPICZαA-V H-C H-V L-C L。将该库电转化N-糖基化部分改造后宿主菌株GS115Y。随机挑取20个平板克隆进行菌液PCR鉴定、基因分析,并提交IgBLAST-imgt数据库鉴定抗体库正确性与多样性。结果获得表达重链和轻链恒定区的表达载体pPICZαA-C H-C L,并在此基础上初步获得全长抗体表达库pPICZαA-V H-C H-V L-C L,库容量为105。结论成功构建了分泌型N-糖基化毕赤酵母人全长抗体库。
Objective To construct a recombinant human full-length antibody library based on the N-glycosylation partial modification of Pichia pastoris. Methods The pPICZαA-C H-C L vector was constructed based on the yeast secretory expression vector pPICZαA. The vector was verified by gene sequence analysis and Western blot. Design 44 Degenerate primers were amplified from human peripheral blood mononuclear cells (PBMC) by reverse transcriptase-polymerase chain reaction (RT-PCR) to obtain the light chain variable region V L and the heavy chain variable region VH genome. The full-length antibody expression library pPICZαA-V H-C H-V L-C L was constructed by inserting it into the constant region expression vector by PCR. This library was electroporated into N-glycosylated partially engineered host strain GS115Y. 20 plate clones were randomly selected for bacterial PCR identification, gene analysis, and submitted IgBLAST-imgt database identification antibody library correctness and diversity. Results The expression vector pPICZαA-C H-C L which expressed the heavy chain and the light chain constant region was obtained, and the full-length antibody expression library pPICZαA-V H-C H-V L-C L was obtained. Conclusion The secreted N-glycosylation Pichia pastoris full-length antibody library was successfully constructed.