抗转铁蛋白受体单链抗体原核表达载体的构建和表达

Construction and Expression of a Single-chain Antibody against Transferrin Receptor

目的 构建抗转铁蛋白受体 (TfR)单链抗体原核表达载体 ,为进一步研究其效应奠定基础。方法 从抗TfR单克隆抗体重链和轻链可变区基因的克隆载体 pGEM T VH和 pGEM T VL中扩增重链可变区 (VH)和轻链可变区 (VL)基因 ,用重叠延伸PCR的方法 ,在VH和VL基因间引入连接短肽 (Linker) ,构建VH Linker VL的单链抗体 (singlechainFv ,scFv)基因。经NcoⅠ和NotⅠ酶切后亚克隆到原核分泌型表达载体pUC19/119上 ,转化和筛选后 ,阳性细菌经IPTG诱导表达。间接免疫荧光法 (IFA)及抗体封闭试验鉴定其抗体活性。结果 凝胶电泳可见重叠延伸PCR扩增产物约 70 0bp条带 ,SDS PAGE鉴定表达产物的分子量为 2 7kD左右 ,与scFv的理论值一致 ,IFA及抗体封闭试验证明表达产物有抗人TfR的活性。结论 本研究成功地构建并表达了抗人TfRscFv ,为抗人TfRscFv的应用奠定了基础。

Objective To construct a single chain antibody (scFv) against transferrin receptor (TfR) for further studing its effects. Methods The VH-linker-VL, namely scFv gene was prepared by amplifying the VH and VL genes of plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension PCR (SOE PCR). After modified by Nco I and Not I, the scFv gene was subcloned into the secretory expression vector pUC19/119 labeled by 6 His and c-myc. The vector was transformed into E.coli. TG1. The expressing bacterial colonies were screened by colony PCR and their expression was induced by isopropyl β-D-thiogalactopyanoside (IPTG). The scFv activity was identified by immunofluorescence assay (IFA) and antibody blocking test. Results Agarose gel electrophoresis identified the product of SOE PCR was 700 bp; the expression product displayed a 27 kD band in SDS-PAGE. IFA and antibody blocking test demonstrated the expression product could specifically react with TfR. Conclusion The anti-human TfR scFv have been successfully prepared.