【摘要】 目的:制备人胎盘酸性铁蛋白(PAF),构建、表达并鉴定抗人PAF单链可变区抗体片段(scFv)。方法:设计以SfiⅠ、NotⅠ为酶切位点、以(Gly4Ser)3为linker的2对引物,从抗人PAF单克隆抗体可变区基因的克隆载体中扩增VH和VL基因,用重叠延伸PCR在VH和VL基因间引入连接短肽,构建VH-linker-VL的scFv基因。经SfiⅠ、NotⅠ酶切后克隆到原核分泌型表达载体pUC19/119上,转化大肠杆菌TG1和筛选后测序验证,IPTG诱导表达,SDS-PAGE鉴定其相对分子质量(Mr)。以人PAF为抗原,scFv为一抗,抗His单克隆抗体为二抗,间接ELISA鉴定其抗体活性。结果:重叠延伸PCR扩增产物经凝胶电泳可见约700bp的条带,DNA序列分析证明2株抗体具有完整的scFv序列,并含有c-myc和His。IPTG诱导阳性菌表达产物经SDS-PAGE鉴定有Mr约27000的显示条带,符合scFv与表达标签融合蛋白的理论值;间接ELISA证明表达产物可与PAF结合。结论:成功构建并表达了抗人PAF单链抗体,为进一步建立检测PAF的间接ELISA奠定了基础。更多还原

【Abstract】 Objective: To construct, express and characterize anti-human placental acidic ferritin scFv. Methods: The VH-linker-VL, namely scFv 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 the scFv was modified by SfiⅠ and NotⅠ, it was subcloned into the expressing vector pUC19/119 labeled by c-myc and His. The vectors were transformed into E.coli TG1. The expressing bacteria colonies were screened by bacteria colony PCR and their expressions were induced by IPTG. Results: Agarose gel electrophoresis identified the product of SOE PCR was 700 bp. The scFv DNA sequences analysis was completed by using IMGT/V-Quest soft. Induced by IPTG, the E.coli TG1 expressed a 27 kD protein, which was observed by SDS-PAGE. The indirect ELISA test showed that that the scFv can specifically bind to the PAF. Conclusion: The anti-human placental acidic ferritin scFv has been successfully prepared, and it affords experiment evidence to further establish indirect ELISA.更多还原