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噬菌体表面呈现抗人红细胞血型A抗原单链抗体的研究 |
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王长军 唐家琪 李先富 潘秀珍 操敏
摘 要 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性。结果：用M13KO7援救XL-1 Blue转化菌中的pCANTAB5E重组噬菌体，得到滴度 为4×109 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。
关键词 噬菌体表面呈现技术 单链抗体 ABO血型
中国图书分类号 R392.11
Expression of anti-RBC blood group A substance ScFv by using phage display tech nology
WANG Chang-Jun TANG Jia-Qi LI Xian-Fu
（Institute of Military Medicine, Nanjing Command,Nanjing 210002）
Abstract 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 hybrido ma 50A which secrets antibody specifity binding to human blood group A substa nce was performed. By RT-PCR for VH and VL genes assembly of ScFv genes and cl o ning into phagemid pCANTAB5E, transformation of E.coli XL-Blue cells and rescui n g with M13KO7 helper phage. The recombinant phages were panned by whole red bloo d cells over three rounds. Finally, the positive recombinant phages were sequenc ed and identified by immuno-blot assay. Results:A recombinant phage ScFv libra ry with titer of 4×109 pfu/ml was established. The result of immuno-blot in dicated that the phage-displayed 50A-ScFv retained the affinity and specificit y of the original intact antibody to blood group A substance.Conclusi on: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.
Key words Phage display technology Single-chain F v antibody Blood gr[1] [2] 下一页
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