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为了获得鼠源抗HFRSV衣壳蛋白McAbF3 株轻链和重链可变区基因 ,由连接肽体外连接获得单链抗体基因 ,在大肠杆菌中表达。从鼠源抗HFRSV衣壳蛋白McAbF3株细胞中分离总RNA ,以oligo(dT) 18为引物逆转录成cDNA ,通过PCR扩增出抗体的轻链 (VL)和重链可变区 (VH)基因 ,由连接肽体外连接获得单链抗体 (ScFv)基因。将此单链抗体 (ScFv)基因插入原核表达载体PET2 8a ,转化大肠杆菌BL2 1(DE3) ,经IPTG诱导后表达。结果构建的鼠源抗HFRSV衣壳蛋白McAbF3株单链抗体 (ScFv)基因 ,全长 741bp ,在大肠杆菌中获得了表达。结论为表达产物经SDS—PAGE及Western印迹分析 ,此单链抗体分子相对分子量为 2 9,0 0 0 ,与兔抗 鼠IgG产生特异反应。
In order to obtain murine anti-HFRSV capsid protein McAbF3 strain light chain and heavy chain variable region genes, single chain antibody gene was obtained by in vitro linkage of linker peptides and expressed in E. coli. Total RNA was isolated from mouse anti-HFRSV capsid protein McAbF3 cells and oligo (dT) 18 was used as a primer to reverse transcribe into cDNA. The light chain (VL) and heavy chain variable region (VH) of the antibody were amplified by PCR. Gene, a single chain antibody (ScFv) gene is obtained by in vitro linkage of the linker peptide. This ScFv gene was inserted into prokaryotic expression vector PET2 8a and transformed into E.coli BL21 (DE3), which was induced by IPTG. Results The constructed murine anti-HFRSV coat protein McAbF3 single-chain antibody (ScFv) gene, full-length 741bp, was expressed in E. coli. Conclusion The expressed product was analyzed by SDS-PAGE and Western blotting. The relative molecular weight of the scFv antibody was 2900000, which reacted specifically with rabbit anti-mouse IgG.