Key Laboratory of Mariculture&Stock Enhancement in North China's Sea,Ministry of Agriculture,Key Laboratory of Fish Applied Biology and Aquaculture in North China,Liaoning Province,Dalian Ocean University,Dalian 116023,China
Abstract: The open reading frame(ORF)of typeⅣantifreeze protein was cloned from Pacific cod Gadus macrocephalus using RT-PCR to investigate the function of typeⅣantifreeze protein.The result showed that the ORF of AFP4 had 375 bp in length,encoding a polypeptide of 125 amino acids.The recombinant pET-32a-AFP4 was constructed by linking the AFP4 ORF with the pET-32a vector,and transformed into Escherichia coli BL21 (DE3).The expressing conditions of the recombinant plasmid were optimized under various concentrations of thermal hysteresis protein(THP),induction time and temperature with the optimal expressing induction conditions of 0.01 mmol/L IPTG,3 h and 37℃.The recombinant protein was purified and used to immunize New Zealand white rabbit.The polyclonal antibody was obtained and tested using indirect ELISA,with the titer of 1∶1 600 000.The specific binding between recombinant protein and Rabbit anti-AFP4 polyclonal antibody indicated that the recombinant protein had high immunogenicity.The findings will provide the foundations for further understanding of AFP4 functions in Pacific cod.
2017, Vol. 32 
