| In recent years, there is a newly defined member in peroxiredoxin (Prx) family. Natural killer enhancing factor (NKEF) was purified from human red blood cells in1993and named for its ability to enhance NK cell cytotoxicity against tumour cells in vitro. In order to study immune defense mechanism of NKEF in teleost, in this paper, a method, rapid amplification of cDNA ends (RACE) was used for cloning the full length cDNA of NKEF-A in miiuy croaker. The determined full-length cDNA of NKEF gene is1235bp, including a5’-terminal untranslated (UTR) region of47bp, a597bp open reading frame (ORF) encoding198amino acid and a3’UTR of591bp. Miiuy croaker NKEF-A is a23kDa protein and the amino acids of Cys-52and Cys-173are well conserved within the members of Prx I subfamily. Also they are the functional region of Prx family. RT-PCR analysis showed that miiuy croaker NKEF-A gene was constitutively expressed in ten normal tissues but had very different expression levels. And expressionlevel was up-regulated in liver, spleen and kidney after challenge with Vibrio anguillarum. Finally, the NKEF-A was constructed and expressed in Escherichia coli. Then the purified recombinant pET-NKEF protein was used to produce the polyclonal antibody and the polyclonal antibody against NKEF-A was tested by Western blot analysis. The result indicated NKEF-A serum protein can specifically recognized the recombinant protein. |
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