用噬菌体呈现技术表达抗人红细胞血型B抗原的单链抗体

Expression of Anti-RBC Blood Group B Substance ScFv by Using Phage Display Technology

本研究的目的是构建表达抗人红细胞血型B抗原5D12杂交瘤细胞的单链抗体(ScFv)。应用重组噬菌体抗体技术，分离与构建5D12McAb单链抗体基因，将其克隆入噬粒pHEN-1中，转化E．coliTGl，M13KO7辅助噬菌体援救构建5D12噬菌体单链抗体库；用完整虹细胞亲和富集法淘选阳性重组噬菌体，重组噬菌体鉴定及序列测定分析；免疫杂交试验检测重组噬菌体单链抗体的特异性抗原活性。经M13K07援救E．coli TG1转化菌中的pHEN-1重组噬菌体，得到滴度为3×10^9pfu／m1噬菌体单链抗体库；免疫杂交试验证实表达产物保留了亲本单抗对人红细胞血型B抗原的特异性亲和力。本研究成功地构建5D12McAb单链噬菌体抗体库，为进一步研制；与特异性和高亲和力的基因工程原核血型检定抗体试剂奠定了基础。

Antibody library technology coupled with guide selection is one of the efficient methods to generate antigen-specific antibodies. In this paper, the recombinant phage display technique was used to construct, clone screen, and express single chain antibody of 5D12 hybridoma. The variable region genes of antibody were amplified by PCR from a hybridoma cell line 5D12 which secreted group B substance. The ScFv gene fragments were successfully cloned into phagemid vector pHEN-1. Rescuing with M13KO7 helper phage, a recombinant phage ScFv library with titer of 3 × 109 pfu/ml was established. Panned by whole blood group B cell over three rounds, the positive recombinant phages were sequenced and identified by immunol-blot assay. The result of immunoblot indicated that the phage-displayed 5D12-ScFv antibody retained the affinity and specificity of the original intact antibody to blood group B substance, which would be potentially useful in constructing engineering antibody fragments as blood grouping reagent.