鼠源抗HFRSV衣壳蛋白McAb F3株单链抗体基因克隆构建及表达

Construction and expression of mouse anti-HFRSV capsid protein single-chain antibody

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。

Constructing recombinant mouse anti hemorrhagic fever with renal virus syndrome(HFRS) single chain antibody gene,and expressing it in E.coli.The total RNA was separated,the cDNAs of variable heavy chain(V H) and variable light chain(V L) region were synthesized by PCR,the DNAs of heavy and light chain were jointed together via a linker to get mouse anti HFRSV ScFv gene,and then the ScFv gene was inserted into plasmid PET28a.The ScFv gene of mouse anti HFRSV is 741 bp,the obtained recombinant plasmid was transformed into E.coli BL21(DE3),and expressed by IPTG inducing.The expressed production was analyzed by SDS PAGE and Western blotting.Its molecular weight is 29 000 ,the expressed product could specifically react with rabbit anti mouse IgG.