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。
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 Sfi Ⅰ and Not Ⅰ, it was subcloned into the expressing vector pUC19/ll9 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.
出处
《生物技术通讯》
CAS
2006年第5期685-687,共3页
Letters in Biotechnology
基金
国家重点基础研究发展规划项目(2002CB513109)
