Cloning And Prokaryotic Expression Of LEAP-2 Gene ORF From Salmo Trutta

Brown trout(Salmo trutta)is native to Europe,North Africa,and Western Asia.It is only distributed in the Yadong region of Tibet in China.Due to overfishing and man-made damage to habitats,its wild resources have drastically decreased.LEAP-2(liver-expressed antimicrobial peptide-2)is a type of antibacterial peptide mainly expressed in the liver.It has strong antimicrobial activity and can help the body resist the invasion of foreign microorganisms.In this study,brown trout was used as the research object.The ORF sequence of the antibacterial peptide gene LEAP-2 was obtained by homologous cloning,and the sequence characteristics were analyzed.Real-time PCR was used to detect its expression in different tissues of brown trout.The prokaryotic expression vector of the mature peptide of the brown trout LEAP-2 gene was constructed and expressed by E.coli to optimize the expression conditions,which laid the foundation for further research on the function of LEAP-2.The main findings are as follows:1.Brown trout LEAP-2 gene ORF sequence is 291 bp in length,encoding 96 amino acids,relative molecular weight is 10.78 KDa,isoelectric point is 9.19,and has 5positive charges.Multi-sequence amino acid comparative analysis showed that brown trout LEAP-2 was composed of a signal peptide,a precursor peptide,and a mature peptide.There were four highly conserved cysteine residues on the mature peptide,which could form two disulfide bonds.The analysis of amino acid structure and functional sites showed that brown trout LEAP-2 had 4 glycosylation sites and 7phosphorylation sites.The secondary structure was mainly ?-helix and irregular curl,and the tertiary structure was similar to human LEAP-2 is similar.Phylogenetic analysis shows that brown trout LEAP-2 has high homology with other teleost fishes.Among them,the relationship with Atlantic salmon is the closest,while it has low homology with birds and mammals,and it is on a different branch.2.Quantitative real-time PCR results showed that LEAP-2 was expressed to varying degrees in brown trout tissues,with liver expression being the highest,followed by spleen and intestine,and expression in kidney,heart,muscle,and gills was relativelyhigh.Low,while hardly expressed in the brain and skin.3.The pET32a-m LEAP-2 prokaryotic expression vector was successfully constructed and transferred into E.coli BL21(DE3)to obtain a prokaryotic expression strain.The recombinant protein was analyzed for solubility,the IPTG induction concentration and induction time were optimized,and the recombinant protein was purified.In the pET32a-mLEAP-2 recombinant expression system,the recombinant protein mainly exists in the supernatant as a soluble protein,and a small amount exists in the precipitate in the form of inclusion bodies with a molecular weight of 22.61 KDa.After changing the IPTG induction concentration and induction time,it was found that the expression of recombinant protein was the highest after 4 h induction with 0.25 mmol/L IPTG.