Study On Effects Of Leptin And LepROT From Rana Amurensis And Rana Dybowskii During Anti-infection

In order to obtain information about the roles and their mechanisms of Leptin（LEP）,Leptin receptor overlapping transcription protein（LepROT）and Nuclear transcription factor Kappa B（NF-κB）signaling pathway in amphibians anti-infection,RT-PCR is used to clone Leptin and LepROT genes from Rana amurensis and R.dybowskii for bioinformatics analysis.The infection models of Aeromonas hydrophila（Ah）and Lipopolysaccharides（LPS）are constructed,and the characteristics of histopathological and anatomical changes after infection are obtained.The dynamic expression of Leptin,LepROT and Nuclear factor-Kappa B（NF-κB）is determined by quantitative reverse transcription polymerase chain reaction（qRT-PCR）.Then immunofluorescence assay is used to obtain protein expression changes of Leptin,LepROT and NF-κB,and the main results are as follows:1.This study clones the full-length ORF sequences of the Leptin and LepROT genes from R.amurensis and R.dybowskii.Obtain the length of Leptin gene products of R.amurensis and R.dybowskii are 857 bp and 845 bp,respectively,and encoding 173 amino acid residues,nucleotide homology of the two species reach 96.36%.The length of gene products of LepROT of R.amurensis and R.dybowskii are 396 bp,and encoding 131 amino acids,and nucleotide homology of the two species is 96.72%.LepROT gene has a higher conserved rate among species,while Leptin gene has a lower conserved rate.2.A recombinant expression vector of Leptin gene peptide binding region from R.amurensis is constructed in this study,which with a recombinant protein size of 37 KD and a multiclonal antibody titer of 1:20000 after immunization.3.HE staining is performed on the constructed inflammation model of R.amurensis.The heart,liver,spleen,lung,kidney,skin,muscle and stomach showed pathological changes with the prolongation of infection time.4.Quantitative real-time PCR is used to detect the m RNA relative expression levels of Leptin,LepROT,NF-κB,IKKα and IKKβ in different tissues of R.amurensis and R.dybowskii.5.Immunofluorescence analysis shows that under Ah and LPS stress,the expression of Leptin,LepROT and NF-κB in lung,kidney,muscle and stomach of R.amurensis and R.dybowskii are basically consistent with the expression levels of qRT-PCR at each time point.