机构地区:[1]昆明医学院附属昆明市第一人民医院,云南省昆明市650011 [2]昆明医学院附属昆明市第一人民医院肝胆胰一科及云南省器官移植研究所肝移植研究中心,云南省昆明市650011
出 处:《中国组织工程研究与临床康复》2009年第24期4717-4720,共4页Journal of Clinical Rehabilitative Tissue Engineering Research
基 金:云南省科技厅社会发展项目基金资助项目(2007CA007)~~
摘 要:背景:研究提示,半乳凝素9在介导细胞分化、凋亡、黏附、细胞间聚集、炎症反应的调控方面发挥作用,但其作用机制尚不明确。目的:克隆大鼠半乳凝素9基因片段,构建pDC316-GFP-Galectin-9真核表达质粒,以进一步了解半乳凝素9各种功能和反应机制,设计、时间及地点:单一样本观察,于2008-11/2009-01在北京本元正阳基因技术有限公司完成。材料:SPF级雄性Lewis大鼠1只。方法:采用反转录聚合酶链反应技术,从大鼠肝脏组织中扩增出大鼠半乳凝素9基因片段,通过基因重组技术将该基因片段重组到pDC316-GFP真核表达载体上,构建pDC316-GFP-Galectin-9重组质粒,通过用PCR扩增、酶切电泳分析及DNA测序的方法对重组DNA进行鉴定。主要观察指标:重组质粒的鉴定结果。结果:总RNA经RT-PCR扩增后,经琼脂糖凝胶电泳,在1kb下方可见一条明显扩增的条带,与预计的半乳凝素9-cDNA长度相符。重组质粒经NotⅠ/HindⅢ双酶切和未作酶切的重组质粒pDC316-GFP-Galectin-9一起经琼脂糖凝胶电泳,前者出现了980bp和5.8kb2条带(980bp为Galectin-9-cDNA产物,5.8kb为pDC316-GFP线状质粒),后者出现6.7kb条带(重组质粒pDC316-GFP-Galectin-9),初步表明重组质粒构建成功。测序结果用NCBI上的BLAST进行比对分析,其核酸序列与GeneBank中的半乳凝素9的mRNA的编码序列(NM_012977)同源性完全相符,进一步证实所克隆的基因为大鼠半乳凝素9-cDNA。结论:成功克隆了半乳凝素9基因并构建成其真核表达质粒。BACKGROUND: Galectin-9 is considered to play a key role in cell differentiation, apoptosis, adhesion, cell aggregation and regulation of inflammatory response. However, its mechanism is still not clear. OBJECTIVE: To clone rat galectin-9 gene fragment and construct its eukaryotic expression vector pDC316-GFP-Galectin-9, further investigate the function and reaction mechanism of galectin-9. DESIGN, TIME AND SETTING: Single sample observation was performed in the Beijing AGTC Gene Technology Company LTD between November 2008 and January 2009. MATERIALS: One male Lewis rat of SPF grade. METHODS: Rat Galectin-9 gene was amplified by RT-PCR method from rat liver tissue and reconstructed into eukaryotic expression vector pDC316-GFP using gene reconstruction technique to construct pDC316-GFP-Galectin-9 recombinant plasmid. The recombinant DNA was identified by PCR amplification, enzyme digestion and DNA sequencing. MAIN OUTCOME MEASURES: Identifications of recombinant plasmid. RESULTS: After RT-PCR amplification and agarose gel electrophoresis, an obvious band was observed at 1 kb, which accorded with the predicted galectin-9-cDNA length. The recombinant ptasmid pDC316-GFP-Galectin-9 digested with Not Ⅰ/HindⅢ or without digestion were presented with two bands (980 bp: Galectin-9-cDNA product, 5.8 kb: pDC316-GFP plasmid) or one band (6.7 kb: recombinant plasmid pDC316-GFP-Galectin-9), respectively. This was a preliminary marker of successful construction. Sequencing results were analysis by pairs using NCBI BLAST, its nucleotide sequence completely matched the galectin-9 gene mRNA coding sequence (NM_012977) in GeneBank, which was then confirmed as rat galectin-9-cDNA. CONCLUSION: Galectin-9 gene is successfully cloned and its eukaryotic expression vector pDC316-GFP-Galectin-9 is constructed.
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