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| Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Antifreeze Protein AFPⅢ |
| HAO Feng-xia,HU Wen-ge,FU Wei-chao,KANG Zhuang-li,ZHANG Gui-ling |
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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.
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Received: 25 November 2009
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