Preparation Of Non-muscle Myosin Heavy Chain IIA C-terminal B Cell Epitope Peptide And Identification Of Pathogenicity

Objective:To predict the B-cell epitope of nonmuscular myosin heavy chain ⅡA(NMMHC ⅡA),to study the biological activity of the synthetic epitope peptide and to prepare and characterize its anti-epitope antibody.Methodology:According to the bioinformatics,combined with bio-software and net protocol,the most likely B-cell epitope was predicted.This epitope peptide were synthesized and coupled with keyhole limpet hemocyanin(KLH),which were used to immune New Zealand male rabbits to prepare epitope-specific antibody.Western blotting and Co-immunoprecipitation were performed with the total cell lysates and anti-peptide rabbit serum.Mass spectrometry was used to analyze the reactive protein bands and confirm the identity of the target antibody with specific antigens.Meanwhile,renal damage of the rabbits was observed by immunofluorescence and renal pathology.We then collected and stored serum samples from 15 patients with membranous nephropathy(biopsy-proven),10 patients with secondary MN,and 10 normal people as controls.The diagnosis of those chosen patients was pathologically confirmed by percutaneous renal biopsy and their serum samples were collected before the renal biopsy was performed.This epitope peptide was synthesized and coupled with bovine serum albumin(BSA),which were sequentially detected anti-NMMHC-ⅡA.antibody in the serum samples of those patients.As a contrast,we performed western blotting analysis of the same serum samples with the use of nonreduced podocyte protein to detect anti-PLA2R antibody,which was identified as reacting to the major target podocyte antigen involved in adult autoimmune IMN.Results:A 14-mer peptide NH2-QVDDERRNAEQYKD-COOH(1850-1863)was the dominant B-cell epitope.Synthesized peptide coupled with KLH was then used to immunize New Zealand male rabbits.Compared with the normal control group(saline-injected group),the epitope-peptide immunized rabbits and induced specific anti-peptide antibodies,and the titer of which was 1:16 detected by the immune double diffusion assay.The epitope-peptide immunized rabbits serum specifically was identified a 227-kD protein by co-immunoprecipitation,which was sequentially identified to be NMMHC-IIA confirmed by mass spectrometry.The epitope-specific antibodies were reactive to the commercial NMMHC-IIA protein(Tagged-His Tag)by western blotting analysis.Furthermore,the immunostaining study of rabbit kidney tissues showed that the rabbit antibody was linear deposits along the glomerular basement membrane,while the renal pathological changes under the electron microscope were foot process fusion and mesangial widening.In all 15 patients with IMN,14 patients(93.3%)were reactive with NMMHC-IIA epitope protein,which was visualized as a 70-kD protein band,while 12 patients(80.0%)were reactive with the 185-KD PLA2R,the location of which was confirmed by polyclonal anti-PLA2R antibody.In 10 patients with secondary MN(8 patients with LN class V,one HBVAN and one gastric cancer associated MN),two(20%)LN only had detectable anti-NMMHC-IIA antibody,three cases had anti-PLA2R antibody,the normal controls had nondetectable antibodies to NMMHC-IIA or PLA2R.Conclusions:Using the synthesized peptides as hapten,we have successfully prepared epitope-specific antibody against NMMHC-ⅡA,a podocyte protein,and its antibodies can be detected in a majority of patients with IMN,indicating that NMMHC-IIA is a major auto-antigen in this disease.It is meaningful for further study on the pathogenesis of IMN.[Abstract]Objective:To investigate the renal clinical and pathological features in type 2 diabetes mellitus with or without diabetic renal damage.Method:One hundred and five cases of type 2 diabetes mellitus with proteinuria were enrolled by renal biopsy.The clinical and laboratory data as well as histological characters were analyzed retrospectively from January 2008 to April 2011.According to with or without diabetic renal damage,the patients were divided into two groups.Results:(1)Fifty one cases of type 2 diabetes mellitus with non-diabetic renal damage(NDKD)accounted for 48.57%in the whole cases of type 2 diabetes mellitus(T2DKD).In general,such as duration of diabetes,duration of hypertension,metabolic disorders and kidney damage in NDKD were less severe than that in type 2 diabetic kidney disease group(T2DKD)significantly(P<0.01).Pathological types of NDKD group were mainly primary glomerular diseases in type,including focal segmental glomerulosclerosis(25 cases,49.02%),IgA nephropathy(16 cases,31.37%),mesangial proliferative glomerulonephritis(8 patients,15.69%),IgM nephropathy(2 cases,3.92%).Vascular damage was also lighter,hyaline degeneration of small arteries(only 8%)was significantly lower than T2DKD group(50%)(P<0.01).(2)There were 54 patients in T2DKD group,accounting for 51.43%.According to the extent of the damage of renal pathology,it was divided into mild and severe groups.With the degree of severity of the pathology increasing,the more severe clinical manifestations showed.Conclusions:There was significantly different between T2DKD group and NDKD group in the clinical manifestations,including general condition,metabolism disorder,kidney damage,and renal pathological changes.As the severity of pathological conditions differing in T2DKD group,clinical manifestations and laboratory parameters were significantly different.