Expression Of MHCI Gene In Rana Dybowskii Under Stress Of Aeromonas Hydrophila

Major Histocompatibility Complex(MHC)is a genetic group closely related to immune response,mainly involved in the presentation of antigens.The antigen binding site of MHCI genes α chain is the region with the greatest variation and is closely related to the resistance of disease.The population of amphibian has declined sharply in recent years,and some species are on the verge of endangerment,which microbial infection is one of the important reasons.The study of the role of MHC gene in anti-infection has become a hot topic in amphibian anti-disease research.This study takes Rana dybowskii as the main research object and Rana amurensis as the contrast species to clone the full-length ORF sequences of two frogs MHCI genesα chain,and analyze the MHCI gene characteristics through bioinformatics software.And a recombinant expression vector of MHCI gene peptide binding region was constructed to prepare polyclonal antibody.Aeromonas hydrophila and LPS was used for stress and quantitative real time polymerase chain reaction(q RT–PCR)technology was used to analyze transcription of MHCI.Then the pathological changes and protein expression of Rana dybowskii were observed by hematoxylin-eosin staining and immunohistochemistry,which provided a theoretical basis for the study of immune function of amphibian MHCI genes.The main results were as follows:(1)This study cloned the full-length ORF sequences of the MHCIα gene from Rana dybowskii and Rana amurensis,which were 1047 bp and encoded 348 amino acids.The nucleotide and amino acid homology of two frogs are 88.7% and 97%.Rana dybowskii has one gloyoosylation sites and thirty-three phosphorylation,while Rana amurensis has three gloyoosylation sites and nineteen phosphorylation,both of these two frogs contain an IGc1 functional domain.Evolutionary relationship shows that the Rana dybowskii and Rana amurensis had the highest homology with Rana chensinensis,and had low homology with manmmals and birds.(2)A recombinant expression vector of MHCI gene peptide binding region from Rana dybowskii has been constructed in this study,which with a recombinant protein size of 39 k D and a multiclonal antibody titer of 1:20000 after immunization.(3)q RT-PCR analysis showed that MHCI genes expression distributed in all the examined tissues of Rana dybowskii and Rana amurensis under normal physiological conditions,and the spleen had the highest expression.(4)The MHCI gene transcription levels of the lung,kidney,spleen and skin from Rana dybowskii and Rana amurensis had been more higher under the stress of Ah and LPS,while MHCI genes respond more positively to LPS in liver and muscle.Under the Ah infection model,the liver,spleen and skin of Rana dybowskii reached a peak at 6 h,the lung and muscle tissue reached a peak at 12 h,and the kidney tissue reached a peak at 24 h.MHCI m RNA reached a peak in the liver,spleen,skin and muscle of Rana amurensis at 6 h,while reached a peak in the lungs and kidneys respectively appear at12 h and 24 h after infection.In the LPS infection model,the MHCI gene of Rana dybowskii showed upward expression in the inspected tissues,the lung and skin reached a peak at 12 h,the liver and spleen respectively reached a peak at 24 h and 48 h,and the kidney and muscle tissue reached a peak at 72 h.m RNA expression of MHCI in liver and muscle from Rana amurensis respectively reached a peak at 6 h and 12 h,the kidney and skin reached a peak at 24 h,the spleen and lung respectively reached a peak at 48 h and 72 h,while the overall transcription levels of the MHCI gene in heart from two frogs were low expression.(5)Hematoxylin-eosin staining showed that the stress of Ah and LPS could lead to the aggravation of cell damage in the liver,skin and muscle tissue of Rana dybowskii with the prolongation of infection time.Immunohistochemistry showed that more brown-yellow positive particles appeared in the liver,skin and muscle of Rana dybowskii after infection with Ah and LPS compared with the control group.Among them,the liver and skin of Rana dybowskii showed more brown-yellow particles under the stress of Ah at 6 h,while positive particles increased significantly in muscle tissue at12 h.The liver,skin and muscle MHCI protein expression of Rana dybowskii under the stress of LPS were respectively highest at 24 h,12 h and 72 h.In this study,we had cloned the MHCIα gene of Rana dybowskii and Rana amurensis,and the prokaryotic expression of the MHCI peptide binding region of Rana dybowskii was carried out and polyclonal antibodies were prepared.The changes of the MHCI of the two frogs under the stress of Ah and LPS were analyzed in different tissues.This study provides a theoretical basis for the study of the immune function of amphibian MHCI genes,and also provides new ideas for the study of amphibian evolution,infectious disease resistance and other issues.