Research On The Molecular Structure And Biological Properties Of GILT In Guinea Pigs And Tilapia

Antigen-presenting based on type II histocompatibility antigen complexes need processing natural protein into short peptide to adapt to the combination of MHC and the identification of T cell receptors.Antigen-presenting cell antigen processing involves the antigen of degeneration,to fold,disulfide bond protein hydrolysis,reduction and the reduction of disulfide bond is crucial step.GILT(the full name is gamma-interferon induced lysosome thiol reductase)has the activity of eduction of disulfide bond,so it can promote the natural protein antigen to folding and further protease.Under the condition of low pH,GILT has higher activity in the specialized antigen-presenting such as B cells and macrophages,and proved plays an important role in antigen processing and immune table a dominant display.GILT also has a role in the neutralization of extracellular pathogens and removes pieces of infected cells.Recent studies have shown that the absence and expression of GILT in cells will affect the immune esponse to the virus,tumor,bacteria,antigen,and may affect the development of autoimmune diseases.For example,the expression of GILT in T cell will reduce the sensitivity of T cells and reduce their own immune response.GILT is the key factor of the liszt fungus host,which can cause fish,birds and mammals pathogenesis.All of these show GILT can be applied in the field of medical research and treatment of autoimmune diseases,so could provide reference value for new prevention and treatment of diseases.Lysozyme is a kind of alkaline globulin,the proportion of the activity.center of an enzyme is glutamic acid and aspartic acid.Lysozyme is one kind of hydrolases which has a special role in microbial cell wall,so it is called package murein enzyme or N-acetyl murein chitosan hydrolysis enzyme,it single-mindedly acted on beta 1-4 glycosidic bond between N-acetyl acid cell wall and N-acetyl glucosamine,and then destroys bacteria cell walls,eventually dissolve dead bacteria.Lysosome is a part of the congenital immune system,mainly produced by mononuclear macrophages,plays a non-specific defense role in the body.It has a variety of action such as antibacterial,anti-inflammatory,antiviral.As a kind of non-toxic protein in the human body,lysozyme is a kind of safe food antistaling agent and preservatives,has become a hot research topic in the field of scientific research.Section one:Cloning and expression of GILT and its active research in guinea pigs and Oreochromis niloticus.Through the RT-PCR method,we cloned GILT respectively from the guinea pigs and Oreochromis niloticus.we obtained 705 bp of guinea pig GILT(cpGILT)and 756 bp of Oreochromis niloticus GILT gene(onGILT).CpGILT gene CDS area code 234 amino acids,on-line predict the relative molecular mass is 25.85 kDa.OnGILT gene CDS area code 252 amino acids,online prediction of its corresponding protein molecular weight of about 27.8kD,isoelectric point theoretical value PI = 5.86.Through the software to predict the three-dimensional structure,onGILT and cpGILT are similar with the structure of the rat GILT.Both the characteristics of the GILT protein contains a GILT protein sequence:CXXC and CQHGX2ECX2NX4C,and evolution analysis shows that the GILT originated from a common ancestor with other species and Real-time quantitative PCR results show that both the expression of GILT has tissue specificity,and after LPS stimulation expression amount raised in the spleen and kidney tissue.By building fusion expression vector,we efficient expressed recombinant protein PET28a-cpGILT and PET28a-onGILT,and carries on the SDS-PAGE and western blot.Then we used human IgG as substrates to determine the thiol reductase activity of cpGILT and onGILT.Finally,through the cell transfection and immunofluorescence technique,we used subcellular localization technology to determine cpGILT/onGILT and hGILT position.Experimental results show that the cpGILT and onGILT can restore the disulfide bond,plays an important role in the body's natural immune protection animals,the subcellular localization of cpGILT/onGILT is similar to hGILT,and their orientation is consistent with antigen-presenting cells,that providing reference and basis for further inquiry GILT in the new role of fish and mammals.Section two:The cloning,expression and its biological activity analysis of rabbit LYZ.First,we extract the RNA from the Oryctolagus cuniculus kidney tissue,which in turn amplification of rabbit LYZ gene(hereinafter referred to as ocLYZ)by RT-PCR.The cDNA sequence of ocLYZ contains 432 bp,which encoding 144 amino acids,protein molecular weight is about 16.17 kDa.OcLYZ protein contains LYZ common characteristic,such as signal peptide sequence,and the structure and evolution analysis indicated that ocLYZ originated from a common ancestor with other species.By constructing prokaryotic expression vector,we expressed PET28a-ocLYZ recombinant protein with His label in BL21(DE3),after the purification,we examined it by SDS-PAGE and Western Blot.Finally,we measured the bacteriostatic activity of ocLYZ by using bacteriostatic ring experiment,and we also studied the optimum temperature and optimal pH conditions of lysozyme.These results suggest that ocLYZ has bacteriostatic action,it plays an important role in the body's natural immune protection,it also provide new research basis for further research which enable us to more fully use of lysozyme.