抗乙酰胆碱受体单链抗体基因克隆与表达

目的重建含有抗乙酰胆碱受体(AChR)单链抗体1929#(single-chain Fv antibody 1929#,scFv1929#)基因的载体并在大肠杆菌(E.coli)进行表达,提高scFv1929#的可溶性表达量。方法用聚合酶链反应法(PCR)扩增载体pHEN1中的scFv1929#结构基因,将扩增产物定向克隆至载体pET32a(+)的多克隆位点中构建新的载体pET32a(+)-scFv1929#,经EcoR v和NotⅠ双酶切后进行鉴定,并将所构建载体转化入表达宿主E.coli BL21(DE3)plysS菌株内诱导表达后集菌并裂菌,将诱导前菌体、诱导后菌体,以及裂菌后所得诱导后上清、诱导后沉淀进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,并采用Western blot(WB)法进行鉴定。结果鉴定结果显示载体pET32a(+)-scFv1929#中所插入的scFv1929#基因片段大小为730bp,与预计相符,经测序确定该基因序列无误。SDS-PAGE电泳结果显示37℃下诱导前菌体、诱导后菌体、诱导后上清、诱导后沉淀中均可在相对分子质量(Mr)接近44 000处见到蛋白条带,其中诱导后菌体及诱导后沉淀中的条带较粗大。经WB法证实融合蛋白scFv1929#具有较高特异性。结论 scFv1929#表达载体pET32a(+)-scFv1929#被成功构建,并可在大肠杆菌中高效表达,其表达产物具有较好特异性。

The cloning and expression of single-chain Fv antibody gene fragments against acetylcholine receptor

Objective To reconstruct the vector inserted the gene fragments of single-chain Fv antibody 1929#(scFv1929#) against acetylcholine receptor(AChR),express the gene fragments in Escherichia coli(E.coli) and obtain the soluble products. Methods The scFv1929# gene fragments in the primary vector pHEN1 with the restriction site of EcoR v and Not Ⅰ were amplified by polymerase chain reaction(PCR) and cloned into prokaryotic expression vector pET-32a(+).The accuracy of the inserted gene was detected by two enzymes digestion technology.Then E.coli BL21(DE3) plysS cells were transformed with the recombinant expression vector pET-32a(+)-scFv1929# and the scFv1929# was expressed.The induced cells were collected and split.The cells before induction and after induction,and culture supernatants and sediment after splitting were analyzed by SDS-PAGE.The specificity of scFv1929# was tested by Western Blot(WB) technique. Results The length of the scFv1929# gene fragments inserted into pET-32a(+) was 730 bp and the DNA sequences were verified by sequencing.The 44 000 expected band in the cells before induction and after induction,and in culture supernatants and sediment after splitting could be found on SDS-PAGE gels.The objective bands in the cells after induction and sediment after splitting were larger.The specificity of scFv1929# was certified by WB. Conclusions It was suggested that the prokaryotic expression vector of anti-AChR scFv fragment-pET32a(+)-scFv1929# was reconstructed successfully and the scFv1929# was expressed in E.coil.The specificity of the expression products was good.