Abstract：Gene recombination technology was used to link AFPⅢ gene and prokaryotic expression vector pET32a(+) and then the recombinant plasmid was transformed into Escherichia coli(BL21)and the positive clones were identified by restriction enzyme digestion, PCR and sequencing. Expression AFPⅢ was induced with IPTG and the expression product was purified, which the purified AFPⅢ was used to immunize mouse to obtain the antiserum.The specificity of the antibodies was examined by Western blotting.The purified antigen was found to have antigenicity by ELISA and the prokaryotic expression vector pET32a-AFPⅢ to be successfully constructed. A fusion protein with a molecular weight of about 26 kD was obtained after induction with IPTG and affinity chromatography.The anti-AFPⅢ antibody was obtained from the immunized mouse.The results of Western blotting indicated that the polyclonal antibody had high specificity to AFPⅢ, providing a foundation for further research on the biological function of AFPⅢ and expression of AFPⅢ in transgenic fish tissues.
收稿日期: 2009-11-25
引用本文:
郝凤霞,胡文革,付伟超,康庄丽,张桂玲. 抗冻蛋白AFPⅢ的原核表达、纯化及多克隆抗体的制备[J]. 水产科学, 2009, 28(11): 671-674.
HAO Feng-xia,HU Wen-ge,FU Wei-chao,KANG Zhuang-li,ZHANG Gui-ling. Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Antifreeze Protein AFPⅢ. Fisheries Science, 2009, 28(11): 671-674.
