Study On The Activity Of Nonspecific Cytotoxic Cell And Expression Of Its Receptor In Red Snapper, Lutjanus Sanguineus

Red Snapper Lutjanus sanguineus, which is one of the most ubiquitous speciescultured marine fish in south of China, was studied in the paper. Rabbit antiserum againstfusion protein of nonspecific cytotoxic cell receptor NCCRP-1was prepared, effectiveNCC isolation method was preliminarily established, the activity of NCC and inhibitoryaction of rabbit antiserum to the activity were studied, recombinant of eukaryoticexpression vector PGBKT7-NCCRP was successfully constructed, and gene expression ofNCCRP-1was studied.The fusion protein of NCCRP-1was purified, and the anti-NCCRP-1serum wasobtained by immuning New Zealand pedigree white rabbits with the purified fusion proteinof NCCRP-1. The titer of anti-NCCRP-1serum was1:32000assayed by the ELISAmethod. The analysis of immunohistochemical mathed using the prepared antiserumshowed that only the specific cells from head kidney, spleen and thymus expressedNCCRP-1.In order to lucubrate inhibitory action of anti-NCCRP-1serum to the activity of NCC,firstly, the head-kidney NCC in Red snapper were isolated, the human cell line K562,which is one of the specific action object of NCC, was used as target cell. NCC againstK562was examined by flow cytometry. The serial diluted antiserum was included in thecultures as the effector cells (NCC) and target cells (K562) mixed with a certain proportion,and the cytotoxic activity of NCC was examined by flow cytometry. The results indicatedthat with a48%Percoll density gradient would obtain the best NCC under the conditionswhich centrifuged at400×g for30min. The proportion of NCC in the total leucocytes was(44.8±0.8)%which examined by flow cytometry. When effector cells (NCC) and targetcells (K562) mixed with a certain proportion, the cytotoxic activity of NCC was examinedby flow cytometry. We found that NCC killed target cells strongly. And this activity didn’tchange as the change of the ratio of two types of cells. The serial diluted anti-NCCRP-1serum was included in the cultures as the effector:target ratio was1:1, the cytotoxicactivity of NCC cells was inhibited in the presence of the anti-NCCRP-1serum. And theinhibition rate changed as the amount of the anti-NCCRP-1serum added. Recombinant of eukaryotic expression vector PGBKT7-NCCRP was successfullyconstructed using PGBKT7vector, which established the foundation for the study on thefunction of NCCRP-1.After injection of lipopolysaccharide (LPS) about twenty-four hours, we found theexpression of NCCRP-1could be detected in head kidney, spleen, thymus, liver, heart,brain, muscle, and intestine by fluorescent real-time quantitative RT-PCR technology. TheNCCRP-1gene was most highly expressed in head kidney and spleen second, following inliver, brain, muscle, thymus and intestines, and the lowest in heart. After stimulating withLPS, phenol and CuSO4, the peak of expressed NCCRP-1was different in the organizationat different times as infection time increasing. When the head kidney tissue was as a modelorganization, the result of RT-PCR showed that expression patterns of NCCRP-1inLutjanus sanguineus were similar after stimulating with LPS, phenol and CuSO4. Theexpression of NCCRP-1increased gradually as time increasing. The maximum levels ofexpressed NCCRP-1were52,30and24times higher than normal level at24h,9h and12h,respectively. Then started to drop.This paper preliminarily studied the activity of the NCC and the basic functions of itsreceptor NCCRP-1in innate immune, which supplied theoretic base for the prevention ofLutjanus sanguineus diseases. Furthermore, the paper established the foundation for thestudy on the biologic characterestic of NCCRP-1.