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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 被引量:4

Expression of anti-RBC blood group A substance ScFv by using phage display technology
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摘要 构建表达抗人红细胞血型A抗原 5 0A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5 0A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 .coliXL Blue,辅助噬菌体援救构建 5 0A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 1Blue转化菌中的pCANTAB5E重组噬菌体 ,得到滴度为 4×10 9pfu 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 5 0AMcAb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。 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 hybridoma 50A which secrets antibody specifity binding to human blood group A substance was performed. By RT-PCR for VH and VL genes assembly of ScFv genes and cloning into phagemid pCANTAB5E, transformation of E.coli XL-Blue cells and rescuing with M13KO7 helper phage. The recombinant phages were panned by whole red blood cells over three rounds. Finally, the positive recombinant phages were sequenced and identified by immuno-blot assay. Results:A recombinant phage ScFv library with titer of 4×10 9 pfu/ml was established. The result of immuno-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.
出处 《中国免疫学杂志》 CAS CSCD 北大核心 2000年第5期244-247,共4页 Chinese Journal of Immunology
基金 "九五"全军招标课题! (编号 :96M0 2 5 )
关键词 噬菌体表面呈现技术 单链抗体 红细胞 ABO血型 Phage display technology Single-chain Fv antibody Blood group ABO substance
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