Recombinant Expression Of Canine MG53 And Preliminary Analysis Of Its Biological Characteristics

Mitsugumin 53?MG53?is a memberof the tripartite motif protein family?TRIM?,also known as TRIM72,whose distributioninrodents bodyhas amuscle tissuespecificity.MG53 protein can repair the damaged cell membrane by the nucleation of intracellular vesicles;it can also through the insulin signal pathway involved in the regulation of insulin expression levels;it can repair renal tubular epithelial cells,alveolar epithelial cells and cardiomyocytes as well as can effectively resist acute renal injury and lung injury,while in the repair of myocardial cell membrane injury has therapeutic activity;MG53 protein can also repair brain damage through the blood-brain barrier.Currently,the functional study of MG53 protein is mainly focused on human and mouse.Canineas an important model of animals without MG53 protein related research,which lacks support for its biological effect in higher animals.Therefore,this study cloned the MG53 gene and expressed the recombinant protein.The structure and biological function ofMG53 protein in canineswere predicted and the differences in the expression of MG53 protein in canines was preliminary tested.To further study the expression of MG53 gene throughout the body and the biological characteristics of its protein,this study used the gene cloning and protein expression technology to clone the canine MG53 gene composed of 1434 nucleotides for the first time;it is found that the protein is easily degradable by structure and biological function and has four functional domains:RING,B-Box,Coiled-coil and PRY-SPRY,which are distributedwith Cysteamine,2-Amino-1-Ethanethiol,Thioethanolamine,2,3-Deshydrolanthionine,Zn²⁺ and Mg²⁺and other molecular binding sites with protein binding capcity.The predicted results were similar to those related studies,suggesting that the MG53 gene mayplay an important role in the ubiquitination and repair of skeletal muscle cells.In this study,we used the pMAL-c5X prokaryotic expression system to obtain the MG53 gene and its truncated gene recombinant protein.The recombinant protein was sequenced with MBP and His tag.The recombinant protein was purified by Ni²⁺fillers.BALB/c mice were immunized with the purified product as immunogen and the hybridoma cells were prepared.Three positive hybridoma cell lines were obtained by ELISA and Westen blot.In addition,the expression and distribution of MG53 protein in different tissues and organs of canines were also preliminary studied.A total of 15 different tissue samples were collected from adult dogs and young dogs.Total RN A was extracted by TRIZOL.The expression levels of mRN A and differences in the mRN A of MG53 gene in each tissue were determined by fluorescence quantitative PC R?qPCR?.Results show that in adult each organization,MG53 gene mRNA expression of the highest in the small intestine,is significantly higher than other test groups?P<0.05?,while the lowest amount of expression in the brain and myocardial,no significant difference?P?0.05?.In young canines each organization,MG53 gene mRNA expression in the lung volume is the highest,and significantly higher than other test groups?P<0.05?,the second is the expression of diaphragmatic muscle and spleen volume is higher also,no significant difference between them?P?0.05?,but the expression difference between with other testing organizations significantly?P<0.05?;Each test organization of the rest of the expression quantity is low,which in the myocardium and the minimum amount of expression in the brain,but no significant difference between each other?P?0.05?.Canine MG53 genes in the same organization different day age canine comparison showed that the amount of mRNA expression in the in leg muscle,lung,heart,brain and liver of MG53 mRNA expression quantity are young canine than adult,only lung significant difference?P<0.05?in the rest of the test group were not significant?P?0.05?;In in the kidney expression quantity differences embodied in adult higher than the young canine,and no significant difference?P?0.05?.In conclusion,the MG53 gene was successfully cloned and the recombinant protein expression system of MG53 was established in this study.The recombinant protein of MG53 was expressed and purified.The molecular biological function of the protein was predicted and predicted.For canineMG53 gene mRNA expression in different tissues and organs in canines difference has carried on the preliminary test and study;we screen out the three strains hybridoma MG53 protein.The results of this study will provide the experimental material for the preparation of monoclonal antibody of MG53 protein.It provides the basic background and research methods for the future research of canine MG53,and also provides reference for the related research of MG53 protein in other animals.