鼠抗寻常性天疱疮抗原噬菌体抗体库的构建与筛选

The construction and bio-panning of mouse ScFv phage-displayed antibody library against pemphigus vulgaris antigen

目的 :应用重组噬菌体抗体库技术制备抗寻常性天疱疮抗原 (PVA)的单链抗体。方法 :用PVA免疫小鼠 6w后 ,抽取小鼠脾脏细胞RNA ,逆转录为cDNA。设计针对小鼠抗体重链可变区 (VH)和轻链可变区 (VL)的特异性引物 ,PCR扩增出VH 和VL 片段。用带有VH3′端、连接肽和VL5′端序列的引物修饰VL 得VL′后 ,与VH 进行重叠延伸拼接 (splicingbyoverlapex tension ,SOE) ,连接成VH—Linker—VL(ScFv) ,并大量扩增。此ScFv经限制性内切酶Sfi1和Not1酶切后 ,以噬粒pCANTAB 5E为载体 ,构建重组质粒 ,转导TG1 感受态菌 ,建成库容为 1 5× 10 6 的噬菌体抗体库 ,用PVA筛库。结果 :构建了抗PVA的特异性单链抗体库 ,筛选出一高丰度表达单链抗PVA抗体的细胞株。结论 :从小鼠噬菌体抗体库中获得特异性的、单克隆的。

Objective:To construct mouse ScFv against pemphigus vulgaris antigen (PVA) through phage displayed antibody technology Methods:Total RNA was isolated from the spleen cells of mice immunized with recombinant pemphigus vulgaris antigen (rPVA) after six weeks, and then reverse transcripted into cDNA Primers specific for mouse V H and V L were synthesized to amply the V H and V L by ploymerase chain reaction (PCR) V L gene was modified and amplified with linker primer (which contain 3′ region of V H, linker, and 5′ region of V L), the resultant gene called V L′ Purified V L′ and V H were subjected to SOE (splicing by overlap extension) ligation, and resultant ScFv gene was digested with SfiI and NotI, cloned to the identically digested pCantab 5E Library of TG1 transfected with recombinant phagemid were derived with diversity of 1 5×10 6 Results:The pemphigus vulgaris phage antibody library was constructed, and one positive TG1 clone was obtained, which can highly expresses phage displayed antibody Conclusion:Specific genetic antibody against PVA can be made from phage displayed antibody library