Establishment Of Three Cell Lines Of Chinese Sturgeon、Dabry’s Sturgeon And Cloning And Expression Of Four Immune Genes

Chinese sturgeon(Acipenser sinensis)、Dabry’s sturgeon(Acipenser dabryanus)、White sturgeon(Psephuyrus gladius)is the unique sturgeons in the Yangtze River,White Sturgeon has been declared extinct,Chinese Sturgeon and Dabry’s Sturgeon are also in varying degrees of endangered state.Although there are breakthroughs in artificial reproduction,with the expansion of the scale of culture,it is mainly plagued by bacterial diseases in the process of culture.However,a little about the molecular mechanism involved in the immune response against these pathogens.It also increases the difficulty for the research on the protection of sturgeons.As a national protected animal,the population of Chinese Sturgeon and Dabry’s Sturgeon is relatively scarce,and it is even more limited to be used as fish for scientific research.Cell line is one of the effective ways to study immune genes and immune mechanism.In order to solve the above problems,firstly,Chinese Sturgeon(Spleen、Kidney)and Dabry’s Sturgeon(Fin)cell lines were isolated and cultured,and on this basis,the cloning and expression patterns of immune-related genes TLR1,TLR4,NK-Lysin and cathelicidin of Dabry’s Sturgeon were studied.The results are as follows:(1)Establishment of Sturgeon cell lineIn this study,three kinds of sturgeon cells were isolated and cultured continuously,the spleen cells of Chinese Sturgeon reached 25 generations,the kidney cells reached 25 generations,and the fin cells of Dabry’s Sturgeon reached 67 generations.The optimum culture conditions of Sturgeon cells were briefly explored,the optimum temperature is 25℃ and the best medium is M199.The successful culture of cells provide valuable materials for subsequent experiments and achieve the purpose of protecting Sturgeon resources.(2)Cloning and expression pattern of TLR1 and TLR4 of Dabry’s SturgeonTLRs(Toll-like receptors)are important pattern recognition receptors(PRRs)of teleost innate immune system.However,information about TLRs is absent in Dabry’s sturgeon(Acipenser dabryanus),one of the most primitive actinopterygii species.In the present study,the full lengths of TLR1 and TLR4 were cloned from Dabry’s sturgeon using RT-PCR and RACE-PCR.The obtained TLR1 was 2957 bp in length,encoding a putative protein of 767 amino acids and TLR4 c DNA was 2902 bp in length,encoding a putative protein of 830 aa.Both TLR1 and TLR4 possessed several typical TLRs motifs,including signal peptides,leucine-rich repeat(LRR)motifs,a transmembrane domain and a TIR motifs.In addition,TLR4 contained three conserved boxes in its TIR motif,involving in TLRs signal transduction.A proline,important for LPS recognition of mammalian TLR4,was also found in TLR4.Physicochemical features of TLR1 and ada TRL4 were also analyzed.Quantitative realtime PCR showed that both transcripts were ubiquitously expressed in all 11 normal tissues selected,but they exhibited different expression patterns,with TLR1 highly expression in heart and TLR4 highly in skin.Further,TLR1 and TLR4 were up-regulated in the primary head-kidney leucocytes following LPS and Poly I:C stimulation,indicating that both genes involved in the sturgeon immune response to LPS and Poly I:C.To our best knowledge,this was the first report of these genes in sturgeon and these results provided the basis for further elucidating the ligand specificity and signaling pathway of fish TLRs.(3)Cloning and expression pattern of two antimicrobial peptides NK-Lysin and cathelicidinAntimicrobial peptides(AMPs)are a conserved component of the innate immune response in many species.In the present study,the c DNA sequences encoding two AMPs(cathelicidin and NK‐lysin,comprising 1,576 and 606 bp,respectively)were cloned from Dabry’s sturgeon.Phylogenetic analysis demonstrated that the two AMPs were clustered together with homologous protein sequences from other fish.NK‐lysin was highly expressed during early embryonic development,suggesting maternal transmission.Tissue distribution analysis showed that cathelicidin had the highest expression in the liver and NK‐lysin was most abundantly expressed in the spleen.In response to Poly I:C treatment,the expression of cathelicidin was upregulated at 12 and 24 hr post induction(hpi),but downregulated at 72 hpi.NK‐lysin m RNA expression increased after treatment with Poly I:C,reaching a peak at 24 hpi.Lipopolysaccharide treatment also induced the expression of two antimicrobial peptide genes.Lipopolysaccharide treatment significantly upregulated the expression of cathelicidin at 6,24,and 48 hpi,and upregulated NK‐lysin expression at 6 and 12 hpi.These results suggested that two AMPs could participate in the immune response induced by Poly I:C or LPS stimulation.