Cloning And Expression Analysis Of NCCRP-1 Gene In Crimson Snapper (lutjanus Sanguineus )

Nonspecific cytotoxic cell (NCC)in fish is the evolutionary precursor of natural killer (NK) cell in mammalian. The function of NCC and NK cell are identical. Furthermore, the evolution of the immunocyte is conserved from fish to mammalian. NCC mainly exists in blood and lymphoid organs, as the first barrier of defense to protect the animal against virus, bacteria and protozoa parasitism. NCC plays an important role in nonspecific immunity in fish. NCCRP-1 was expressed in NCC membranes, and it maybe participate in innate immunity against bacteria by granule exocytosis during inflammatory responses in fish. NCCRP-1 is the molecular basis of NCC function and provides an important role in target cell recognition and activation of cytotoxicity.Crimson Snapper Lutjanus sanguineus,which is one of the most ubiquitous species cultured marine fish in south of China, was studied in the paper. NCCRP-1 was cloned and expressed. All of the known NCCRP-1 gene sequences from fish were compared in bioinformatic method. The highly conserved domains in NCCRP-1 were selected to design primers. Total RNA was extracted from headkidney in Lutjanus sanguineus. The cDNA sequence of NCCRP-1 gene was obtained by RT-PCR and RACE techniques. Results of sequence analysis showed that NCCRP-1 gene was 980 bp in length with an ORF of 702 bp which coded for 233 amino acids, 42 bp 5′UTR and 236 bp 3′UTR. The result of phylogenetic analysis showed that the NCCRP-1 in Lutjanus sanguineus has the highest identity to NCCRP-1 in Sparus aurata, which is 84%. But the identity of NCCRP-1 in Lutjanus sanguineus and in mammals was very low.In order to attain the high expression of NCCRP-1 gene in E.coli BL21, the NCCRP-1 gene of Lutjanus sanguineus was cloned into pET21a vector. Then the recombinant vector pET21-NCCRP was transformed into E.coli BL21. The NCCRP-1 gene was most highly expressed with 0.8 mmol/L IPTG for three hours. The protein was successfully obtained and proved by the corresponding molecular mass to the presumptive mass. The fusion protein was expressed as inclusion body and purified by HisTrap HP affinity column. The western-blot analysis showed that the corresponding antibody could specifically combined with the recombinant protein, so the expressed protein was definitly confirmed to the aim protein. The results of Real-time PCR for Lutjanus sanguineus infected Vibro alginolyticus about four hours showed that NCCRP-1 gene was most highly expressed in head kidney, lower in spleen, liver, muscle, gill, thymus and heart, and lowest in gut. And that NCCRP-1 gene was most highly expressed in 22℃and with 20 salinity.The study supplied theoretic base for the prevention of Lutjanus sanguineus diseases and enriched the research of fish molecular immunity. Furthermore, the paper established the foundation for the study on the biologic characterestic of NCCRP-1 and the function of NCCRP-1 in fish innate immunity.