噬菌体表面呈现人红细胞血型McAb 50A单链抗体的研究

目的 构建表达抗人红细胞血型A抗原50A杂交瘤细胞的单链抗体(ScFv)。方法 应用重组噬菌体抗体技术,从50A杂交瘤细胞中分离、构建单链抗体基因,并将其克隆入噬粒pCANTAB5E中,转化E.coli XL-Blue,辅助噬菌体援救构建50A噬菌体单链抗体库;采用完整红细胞亲和富集法淘选阳性重组噬菌体,鉴定重组噬菌体并进行序列测定分析;免疫印迹试验检测重组单链抗体的特异性抗原活动。结果 用M13KO7援救XL-Blue转化菌中的pCANTAB5E重组噬菌体,得到滴度为4×10~9 pfu/ml噬菌体单链抗体库;免疫印迹试验证实表达产物保留了亲本单抗对人红细胞血型A抗原的特异性亲和力。结论 成功构建50A McAb单链噬菌体抗体库,为进一步研制高特异性、高亲和力的基因工程原核血型检定抗体试剂奠定了基础。

Expression of McAb 50A anti-RBC blood group A substance ScFv by using phage display technology

Objective To construct a phage-displayed ScFv antibody of 50A hybridoma anti-blood group A substance. Methods Based on recombinant phage display techniques,the construction,screening and expression of functional single-chain Fv fragment (ScFv) from murine hytridoma 50A which secrets antibody specifically binding to human blood group A substance was performed . By RT-PCR VH and VL genes were assembled with ScFv genes and cloned into phagemid pCANTABSE ,then to transformate E. coli XL-Blue cells and rescue with M13KO7 helper phage. The recombinant phages were panned by whole red blood cells over three rounds. Finally, the positive recombinant phages were se-quenced and identified by immunol-blot assay. Results A recombinant phage ScFv library with liter of 4X109 pfu/ml was established. The result of immunol-blot indicated that the phage-displayed 50A-ScFv retained the affinity and specificity of the original intact antibody to blood group A substance .Conclusion Single chain antibody fragment 50A-ScFv displayed on the surface of filamentous phage was successfully produced ,which would be potentially useful in constructing engineering antibody fragments as blood grouping reagents.