Screen The Autoimmune Target Protein That Relevant Chronic Nephropathy

This study based on the modle of chronic nephropathy that was caused by ischemia reperfusion of rat double kidneys?use immunology?proteomics and other experimental methods to try to find the autoimmune target antigen that associated with chronic nephropathy.The first(1)use this experiments of double kidneys ischemia reperfusion to establish the rat model of chronic nephropathy that clamp bilateral renal artery 75 min.Monitoring rerult found that the disease group appeared the renal function damage after 40 weeks,the proteinuria(urine protein/urine creatinine,g/mmol)level reached(0.94±0.15)?(1.67±0.50)in 48 and 52 weeks,and it has extremely significant difference compared with the normal control group(< 0.3),immunofluorescence tests showed the kidney of disease group have IgG antibody deposition,HE staining showed the disease group kidney has fibrosis change,this prompt the animal model of chronic nephropathy were established.(2)Extracted the total protein from normal rat kidney,and then took elisa and westen blot experiments with the first antibody of model rat serum to validate the antigen antibody reaction that the kidney total protein with the serum of disease rats.Elisa experiments have showed that serum of disease rats and kidney total protein of normal rat have antigen antibody reactions,there were significant difference in disease group(average OD value was 0.5011±0.047)and control group(average OD value was 0.2370±0.019),and the westen blot experiments that were taked by first antibody of dsease group serum and rats kidney protein have appeared immunoblotting stip.The next(3)sparate extracted total kidney protein of the disease and control group to take the coimmunoprecipitation assay(COIP)experiments,use A/G agarose magnetic-rabbit anti rat IgG to capture the antigen antibody reaction in the kidney,after was precipitated?purificated?eluted and electrophoresis,preliminary confirmed the trget protein is the 37 KDa band which was only displayed in the disease group,(4)cut trget protein gel strips that sparated by SDS-PAGE gel electrophoresis to take mass spectromentric analysis,selected the potential target proteins that the amino acid sequence matching rate ranked first 10.(5)the amino acid sequence matching that ranked top 2 are gamma globul in IgG2a(the matching rate of amino acid sequence reached 96.3%)and tropomyosin 3(tropomyosin alpha 3 chain,TMP3)(the matching rate of amino acid sequence reached 76.4%),then use anti lgG2 a and anti TMP3 commercialization antibody which be known by us and total protein of normal rats kidney to take one-dimensional gel westen blot experiments.Determine the target protein that the molecular weight that conform 37 KDa was TMP3,and the stip reacted with IgG2 a that the molecular weight were 25 KDa and 50 KDa.The last(6)use the total protein of normal rats took two-dimensional gel electrophoresis,respectively using the known of TMP3 antibody?the serum of desiase group rats of chronic nephropathy?the serum of control group to take westen blot experiments,further identification TMP3 is the autoimmune target protein that is related to chronic nephropathy,and the expression level of TMP3 of disease kidney was higher than that of normal kidney.(7)Detected the antibody levels of anti TMP3 and anti Vimentin(Vim)in serum of rats with chronic nephropathy by elisa experiments,and then compared the immunogenicity of the two protein that existed in chronic nephropathy.Experiments showed that the antibody levels of anti TMP3 and anti Vim have reacted,but the level of TMP3 antibody in the disease group was higher than the control group(P(27)0.05),and the level of Vim antibody was no significant difference compared with the control group(P>0.05).This study based on the modle of chronic nephropathy that was caused by ischemia reperfusion of rat double kidney,for the first time screened out the autoimmune target protein TMP3 that relat to chronic nephropathy,it has strong immunogenicity and chronic nephropathy relativity.The research results have psitive significance for the diagnosis and treatment of chronic nephropathy.