Study On Anti-infective Immunity Of MHC Ⅰα And Histone In Paralichthys Olivaceus

Paralichthys olivaceus is a kind of hard bone fish with high nutritional value and economic value.Recently,due to the expansion of cultivation scale,the deterioration of environment and the decline of disease resistance of flounder,the disease has occurred continuously and caused serious economic losses.Edwardsiella tarda is a common and important pathogen of Paralichthys olivaceus diseas.The major histocompatibility complex(MHC)is a highly polymorphic region of the vertebrate genome that plays a critical role in initiating immune responses towards invading pathogens.It well known that MHCI moleculars play a central role in the immune response to virus.However,rare literatures were reported the role of MHC I in the resistance to intracellular bacteria.Histone proteins are not only structurally important for chromosomal DNA packaging but also involved in the regulation of gene expression and the immune response of host against pathogens.Here,the following studies focused on the anti-infective immune mechanism of MHC Ⅰα(PoMHC Ⅰα)and histone H3.3(Po H3.3)in Japanese flounder(Paralichthys olivaceus):Study on anti-infective immunity of PoMHC Ⅰα as following:1.Cloned c DNA sequence of PoMHC Ⅰα by RT-PCR.Results showed that PoMHC Ⅰαcontains 1011 bp from opening read frame.PoMHC Ⅰα possesses conserved structural characteristics of MHC Ⅰα proteins,including signal peptide,MHC＿I domain,IGc1 domain,transmembrane region was performed with SMART tool,also contains one N-glycosylation site.The results indicated that the biological functions of PoMHC Ⅰα may be similar to those of other vertebrates.2.Expression of MHC Ⅰα in Japanese flounder tissues was detected by RT-q PCR.Results showed that the highest expression was found in blood for PoMHC Ⅰα and was also highly expressed in immune tissues such as spleen,kidney and liver.Stimulation to Japanese flounder with extracellular and intracellular bacterial pathogens and viral pathogen up-or down-regulated the expression of MHC Ⅰα and four histones m RNAs in a time-dependent manner.3.PoMHC Ⅰα recombinant protein(r PoMHC Ⅰα)was expressed and purified by prokaryotic expression in vitro.Results showed that r PoMHC Ⅰα not only promotes the resistance of HLKs to E.tarda infection,but also promotes the m RNAs expression of its related immune factor such as IL-10、TNFα、TCRα and CD8α.4.Through the experiment of flow cytometry,it was confirmed that r PoMHC Ⅰα had antigen presenting function and activated PBLs proliferation of flounder.5.We constructed the PoMHC Ⅰα plasmid,PoMHC Ⅰα was overexpressed in flounder tissues.The infection experiment showed that PoMHC Ⅰα drive significantly enhanced the resistance of flounder to E.tarda.Study on anti-infective immunity of Po H3.3 as following:1.In this stduy,we also studied the related immunity of four histones gene of Paralichthys olivaceus,multiple sequence alignment of four histones of Paralichthys olivaceus with known histone sequences of other fishes or mammals showed that H1 has medium overall sequence homology(38.46-49.30%),while H2 AX,H3 and H3.3 have high overall sequence homology(more than 80%).The results indicated that the biological functions of Paralichthys olivaceus histone may be similar to those of other vertebrates.2.Expression of four histones(H1、H2AX、H3、H3.3)in Japanese flounder tissues was detected by RT-q PCR.Results showed that four histones were highly expressed in the blood and also in immune tissues such as the spleen,kidneys and liver.Stimulation to Japanese flounder with extracellular and intracellular bacterial pathogens and viral pathogen up-or down-regulated the expression of four histones m RNAs in a time-dependent manner.3.Po H3.3 recombinant protein(r Po H3.3)was expressed and purified by prokaryotic expression in vitro.Bacterial binding experiments showed that r Po H3.3 had extensive binding ability to gram-positive bacteria and gram-negative bacteria,and significantly inhibited the growth of bacteria after binding.4.We constructed the Po H3.3 plasmid,Po H3.3 was overexpressed in flounder tissues.The infection experiment showed that Po H3.3 drive significantly enhanced the resistance of flounder to E.tarda.This research revealed that MHC Ⅰα and histones play an important role in the immune response of pathogenic microorganisms of Paralichthys olivaceus.This study will contribute to understanding the immunology of Paralichthys olivaceus and offered new insights for the immunological function of teleost.