摘要
目的构建抗恶性疟原虫富含组氨酸蛋白Ⅱ(Histidine-rich protein Ⅱ,HRP-Ⅱ）单链抗体库，并筛选出阳性克隆。方 法用噬菌体抗体库技术构建抗恶性疟原虫ＨＲＰ－Ⅱ单链抗体库，并以ＨＲＰ－Ⅱ为靶抗原对该库进行了三轮“亲和吸附 一援救一感染扩增”的富集，挑取单菌落筛选并鉴定阳性克隆。结果获得目的基因并成功构建抗恶性疟原虫 ＨＲＰ－Ⅱ的 单链抗体库，库容为 １０６，并从中筛选出８株阳性克隆。结论 噬菌体抗体库技术具有高效的筛选性能，抗 ＨＲＰ－Ⅱ单链 抗体的制备为其在恶性疟的免疫快速诊断方法中的应用奠定了基础。
Objcetive To construct phage dispaly single-chain antibody fragments(scFvs)library against histidine-rich protein Ⅱ (HRP-Ⅱ)of Plasmodium falciparum and selectspecific scFvs of anti-HRP-Ⅱ for the purpose of malaria diagnosis.Mehtod The genes of variable fragments of heavy chain(V)and light chain(V)were gained from the spleen cells of BALB/c mice immunized with HRP-Ⅱ protein,The Vand V genes were then assembled by the method of splicing overlapping extension and cloned into phagemid vector pCANTAB 5e.The scFv phage antibodies were expressed at the surface of the phage after the rescue by helper phage M13K07.HRP-Ⅱ protein was used as antigenic reagent for panning and screening.Results the total RNA from the spleen cells was isolated, and cDNA obtained and Vand V gene regions amplified using PCR.The V and V gene regions were combined with a flexible linker ligated into the pCANTAB 5E phagemid vector, and transformed into TG1Escherichia coli,The tepertoire of the phage antibody was about 10.After panning and screening,8 positive clones expressed scFv antibodies which were specific for HPR-Ⅱ as demonstrated by ELISA.Conclusion Phage display technology can be used as a powerful tool in making scFv antibodies which have the potential to be uses as reagents in the diagnosis and therapy of malaria.
出处
《第一军医大学学报》
CSCD
北大核心
2001年第4期241-244,共4页
Journal of First Military Medical University
基金
国家自然科学基金!(39600127)
关键词
噬菌体抗体库
单链抗体
恶性疟原虫
富含组氨酸蛋白Ⅱ
phage antibody library
single-chain antibody fragments(scFv)
Plasmodium falciparum
histidine-rich protein
