Construction And Screening Of The Head-kidney CDNA Library From Japanese Sea Bass(Lateolabrax Japonicus) And The Research On The NCC Of The Mid-kidney

This paper consists two parts,one is the construction and screening of immune-related genes of the head-kidney cDNA library from Japanese sea bass;the other is the research on the NCC of the mid-kiney.Japanese sea bass(Lateolabrax japonicus) is a marine fish with great commercial value especially in Asia.In breeding farms,unfavorable conditions or poor management practices may put stress on fishes,which will cause reduction of growth rate,immune suppression and various infectious diseases.To date,genetic knowledge on sea bass immune relative genes responsible for resistance to pathogens is poor. Progress in these research fields will have significant value in the near future.The head kidney of teleosts serves as main immune organ and plays an important role in specific and non-specific immune defences.A cDNA expression library from head kidney of Japanese sea bass was constructed. The first-strand cDNA was synthesized with Moloney Murine Leukaemia virus reverse transcriptase with 5μg mRNA and the double-stranded cDNA was digested by XhoⅠenzyme.Size fractionation was performed on CHROMA SPIN-400 columns. cDNA fragments longer than 500 bp were ligated into theλZAPExpress vector.The recombinant DNA was packaged in vitro with GigapackⅢgold packaging extract. The titers of the primary and amplified library were 1.7×10~5 pfu/mL and 5.0×10~9 pfu/mL respectively.To characterize the constructed cDNA library,15 phage plaques were selected randomLy to test the inserted fragments.The results showed that the inserts were mostly longer than 500 bp.Small-scale sequence and PCR with specific primers for immune-related genes were conducted for the screening of the cDNA library.76 EST sequences was obtained from the screening.8 immune-related genes(Ig heavy chain,TLR,MyD88, Mx,Gig2 and TNF-α,CC chemokine and CSF1-2) were obtained and showed 15.1% of knowned genes,10.9%of all the obtained EST sequences.The homology and phylogenetic analysis on the main genes showed that they all had the highest identity to the species of Perciformes,which was consistent with the systematic classification. The results indicated that the cDNA library was constructed successfully,and could be used for the screening of the immune-related genes.Japanese sea bass is a euryhaline fish,is able to live in waters with a wide range of salinity and maintain nearly constant ion concentrations of body fluids irrespective of the external salinity,in patieular,displaying a remarkable plasticity when it comes to adjusting ion transport in response to changes in environment salinity.NCC(Na-Cl cotransporter) is a protein with 12 transmembrane-spanning segments,and apieally located in the epithelial cells,and is one of the members of the electroneutral, cation-chloride eotransporters family,which play a important role of ion regulation.The NCC was cloned from the mid-kidney of the Japanese sea bass according to the specific primers designed by the known species.Based on the PCR combination and the 3'-RACE PCR,a cDNA,3346bp long,was obtained from the mid-kidney. The homology and phylogenetic analysis were conducted about the NCC sequence. On the other hand,time-course changes in the mRNA,following transfer of Japanese sea bass from seawater to freshwater for 0,3,7 and 14 days were examined in the tissue of gill,intestine and mid-kidney.The results showed that the NCC mRNA in all the 3 tissues displayed the similar trend.The mRNA in the 3 tissues all reached the maximum on 3 days acclimation and then decreased gradually.The abundance of the NCC on 14 days accilimation in gill and mid-kidney was higer than that of initial abundance;and the abundance in intestine was lower than that of initial abundance. The results implied that the NCC may participate in the cousrse of fast response of the organism to the environment.In order to uncover the location of the NCC in mid-kidey,the preparation of polyclonal antibody was conducted against the 645bp fragment(coding for 215 Aa fragment) located in the carboxyl termini.The pET-28a vector was chosen for the expression of the fusion protein.Induced by 1mM IPTG,the fusion protein was expressed in inclusion bodies.After purification,the protein was isolated by the SDS-PAGE electrophoresis.Then,the part of the protein was cut for immune New Zealand male rabbits for 1month.The anti serum was obtained and used for the immunohistochemistry of NCC.The reults showed that the NCC of Japanese sea bass was located in the distal ducting cells and collecting cells.