| The hypercoagulable rat model induced by experimental hypertriglyceridemia had been established in our lab. Suppression subtractive hybridization (SSH) was applied to screen, identify and clone hypercoagulability-related novel genes in aorta of rat.We established endogenous hypertriglyceridemia (HTG) of rat by feeding rats with high starch (carbohydrate is 80% of total calories) diet. The activity of clotting and fibrinolytic system in HTG rats had been detected. The results showed that the clotting activity increased and the same time, the fibrinolytic activity decreased in HTG rats. That is to say, the HTG rats were hypercoagulable. This indicated that hypercoagulable rat model had been successfully established by feeding rats with high starch diet.SSH requires 0.5~2ug mRNA. The concentration of total RNA in aorta of rat is very limited; it is not enough for the isolation of mRNA for SSH. Therefore, we used SMART technique for synthesizing dscDNA from little amount of total RNA. We chose aorta of hypercoagulable rats as tester and aorta of normal rats as driver. Total RNA was isolated from tester and driver, respectively. Total RNA was then transcript into single-strandcDNA (sscDNA). LD PCR was used to synthesize double-strand cDNA (dscDNA). After purification by column chromatography, digestion by RsaI and precipitation of ethanol, the purified dscDNA for SSH had been prepared.Then, SSH was performed to construct the subtractive library. The subtracted product was ligated to pMD18-T vector and transformed into E. coli JM 109. The hypercoagulable rat cDNA subtractive library had been successfully constructed. The amplified library contained about 1000 white clones. Small scale of cultivation of randomly selected 150 white clones was performed. Plasmid were isolated from them and analyzed with EcoRI and Hindlll. It showed that large percentage of them (53.3%) were recombinant clones. Twenty recombinant clones with 400~600bp insert were sequenced. The results of sequencing were compared with nr and est database in GenBank. It showed that these clones represented 19 genes, 8 of them were known genes that were the first found to be possibly related to hypercoagulability, and 11 are novel genes (ESTs) that had deposited to GenBank. These new ESTs provided an important clue for cloning novel genes of hypercoagulability. At the same time, 5 ESTs were identified with semi-quantitative RT-PCR. HC002, HC057 and HC067 were differentially expressed ESTs and HC019 and HC039 were negative clones.SMART RACE technology was applied to obtain the full-length cDNA of HCR2, a hypercoagulability-related gene. Gene specific primers and nested primers were designed according to HC002 EST sequence. In2order to amplify its 5'and 3' ends, 5'-RACE PCR and 3'-RACE PCR were performed. 5'-RACE product of 500bp and 3'-RACE product of 800bp were obtained. After cloning and sequencing, the 5', 3'-RACE product and EST were spliced into a complete 1275bp sequence. It is the full-length cDNA of HCR2 (GenBank accession number AY234417) with single exon. HCR2 was mapped to rat chromosome 4qll with bioinformatics. The putative protein encoded by this gene was 78 amino acids with a theoretical molecular weight of 8841.7 and isoelectric point of 8.59. It had no signal peptide and transmembrane sequence. It was probably a nuclear protein. The sequence has N-glycosylation site, Protein kinase C phosphorylation site and Casein kinase II phosphorylation site. It shared no significant homology with any known protein. Northern hybridization was applied to study the expression of HCR2 in multiple tissues of rat. This provided necessary experimental data for further study of its function.This subtractive library is highly efficient and has laid a foundation for screening and cloning hypercoagulability-related novel genes of rat. HC002, HC057 and HC067 were suggested to be involved in the molecular process of hypercoagulable state. The full-length cDNA of HCR2 had provided important data for the further study of mechanism of it in t... |
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