Construction and screening of phage display single chain antibody library against histidine-rich protein Ⅱ of Plasmodium falciparum
10.3321/j.issn:1673-4254.2001.04.001
- VernacularTitle:抗恶性疟原虫富含组氨酸蛋白Ⅱ单链抗体库的构建及筛选
- Author:
Yun-Xia HOU
1
;
Wen-Qi DONG
;
Wei-Wen XU
;
Ping WANG
;
Bai-Hong CHEN
;
Ming LI
Author Information
1. First Military Medical University
- From:
Journal of Southern Medical University
2001;21(4):241-244
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct phage display single-chain antibody fragments (scFvs) library against histidine-rich protein Ⅱ (HRP-Ⅱ) of Plasmodium falciparum and select specific scFvs of anti- HRP-Ⅱ for the purpose of malaria diagnosis. Method The genes of variable fragments of heavy chain (VH) and light chain (VL) were gained from the spleen cells of BALB/c mice immunized with HRP-Ⅱ protein. The VH and VL 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 VH and VL gene regions amplified using PCR. The VH and VL gene regions were combined with a flexible linker ligated into the pCANTAB 5E phagemid vector, and transformed into TG1 Escherichia coli. The repertoire of the phage antibody was about 106. 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 used as reagents in the diagnosis and therapy of malaria.
