ABSTRACT
<p><b>OBJECTIVES</b>To obtain prokaryotic expressed IA-2 recombinant protein and to identify its immunological activity.</p><p><b>METHODS</b>The complimentary DNA (cDNA) coding for the intracytoplasmic part of IA-2 (IA-2ic) was amplified from human fetal brain RNA, and was subcloned into the PinPoint Xa-1 T vector to construct recombinant expression plasmid, and was then expressed in E. coli JM109 cells as a fusion protein with a biotinylated peptide sequence at the aminoterminus. The biotinylated fusion protein was then purified by affinity chromatography and was subsequently dialyzed. Finally, its immunogenicity was evaluated by enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The purified IA-2ic fusion protein resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single Coomassie brilliant blue stained band with a molecular weight of 59 kDa and its immunogenicity was confirmed by ELISA.</p><p><b>CONCLUSIONS</b>E. coli expressed IA-2ic fusion protein has immunological activity. It can be used for detection of IA-2 autoantibodies (IA-2A) and for further studies on type 1 diabetes in future.</p>
Subject(s)
Animals , Humans , Rabbits , Autoantigens , DNA, Complementary , Diabetes Mellitus, Type 1 , Allergy and Immunology , Escherichia coli , Genetics , Membrane Proteins , Genetics , Plasmids , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases , Genetics , Receptor-Like Protein Tyrosine Phosphatases, Class 8 , Recombinant Fusion Proteins , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVES</b>To identify differences in gene expression in renal and visceral adipose tissue in type 2 diabetic rats using cDNA representational difference analysis (RDA) and to explore the molecular pathogenesis of type 2 diabetes and its chronic vascular complications.</p><p><b>METHODS</b>A rat model of type 2 diabetes was generated by administration of a high fat and calorie diet combined with a low dose of streptozocin (STZ) injected into the tail vein. The difference bands were generated by cDNA representational difference analysis (cDNA RDA). The final difference products were ligated into the pUC-18 vector and sequenced. A bioformatics analysis was performed on the obtained expressed sequence tags (ESTs), and then the expression levels of known and novel genes were verified by semi-quantitative reverse transcription-PCR (RT-PCR). At the same time, full-length cDNA of a novel gene was cloned in silico.</p><p><b>RESULTS</b>The type 2 diabetic rats in this experiment experienced hyperglycemia, lipidemia, lower insulin sensitivity and normal body weight. We obtained 9 novel ESTs and 2 novel genes from renal tissue of rats and 6 novel ESTs and 1 known gene, the rat lipoprotein lipase (LPL) gene from their visceral adipose tissue. The 2 novel genes (RS91 and RS2) from the renal tissue were both very similar to serine (or cysteine) proteinase inhibitor, clade F and eukaryotic translation initiation factor 3 and subunit 5 (EIF-3 epsilon). The expression of both novel genes and the LPL gene were upregulated in renal and visceral adipose tissue of type 2 diabetic and fat-enriched rats. Full-length cDNA of the novel gene RS91 was cloned in silico.</p><p><b>CONCLUSIONS</b>(1) The rat model of type 2 diabetes generated in this study was ideal because the disease in the animals closely mimicked type 2 diabetic patients. (2) cDNA RDA is a flexible, inexpensive, more accurate, sensitive and highly effective technique for identifying differences in gene expression. (3) Six novel ESTs and 1 known gene were obtained from rat visceral adipose tissue. The LPL gene was upregulated in adipose tissue of type 2 diabetic and fat-enriched rats, a result possibly related to the diabetic animals' high fat and calorie diet, lipidemia, insulin resistance (RI) and molecular pathogenesis. (4) Nine novel ESTs and 2 novel genes were obtained from the renal tissue of rat. We believe the 2 novel genes to be the serine proteinase inhibitors clade F and EIF-3 epsilon in rats. The upregulation of the 2 novel genes in renal tissue of type 2 diabetic rats and may have been related to their renal impairment.</p>
Subject(s)
Animals , Male , Rats , Adipose Tissue , Metabolism , Cloning, Molecular , Diabetes Mellitus, Type 2 , Metabolism , Expressed Sequence Tags , Gene Expression Profiling , Kidney , Metabolism , Oligonucleotide Array Sequence Analysis , Plasmids , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , VisceraABSTRACT
Using the technique of fluorescent-labled mRNA differential display, new apoptosis related gene 2ass-bnip3 of type 2 diabetic cardiomyopathy was found, the sequence of 1594 bp with coding 187 amino acids was obtained by the full-length clone, and its structural and functional predictions were performed. 2ass-bnip3 may play an important role in the development of diabetic cardiomyopathy via a regulatory pathway of calcium regulation and apoptosis.
