Cloning And Expression Analysis Of Natural Killer Enhancing Factor From Japanese Flounder (Paralichthys Olivaceus)

Natural killer enhancing factor (Natural killer enhancing factor , NKEF) is a member of the newly defined peroxiredoxin (PRX) family. It wsa original purified from human red blood cells ,It can enhance the cytotoxic activity of natural killer cells, and it's very important to prevent protein and DNA from being damaged by oxidative stress within living cells. In this study, a method, RACE (rapid amplification of cDNA ends) , was used for cloning the full length cDNA of NKEF from the liver of Japanese flounder Paralichthys olivaceus. Two fragments about 400bp and 800bp were amplified in 5' and 3' RACE-PCR separately, and both of them are very special. Sequence analysis revealed a 1028bp cDNA which contains the 67bp 5' terminal region, 597bp open reading frame (ORF) and 364bp 3' terminal region. The whole ORF encode a NKEF protein of 198 amino acids, it was a 22kD protein, and isoelectric point was 7.2. Japanese flounder NKEF has two high conserved motifs and the amino acids on each side of both Cys-52 and Cys-173 are absolutely conserved with the members of the Prx I subfamily, they were also the functional region of Prx family. RT-PCR analysis showed that Japanese flounder NKEF expressed in various tissues but had very different expression levels, and it can be up-regulated in spleen and head-kidney after LPS challenge, the level of its expression showed no significent change in liver and intestine. The results revealed that although NKEF exist in all tissues, the expression pattern depends on the tissue and stimulation. Japanese flounder NKEF also up-regulated gradully in the process of embryo development.