广西巴马小型猪血清淀粉样P物质基因真核载体构建及细胞转染

Eukaryotic Vector Construction and Cell Transfection in Bama Mini-pig Serum Amyloid P Component Gene

血清淀粉样P物质(Serum Amyloid Pcomponent,SAP)是一种在进化上高度保守的血清糖蛋白,它可与各种类型的原纤维结合,在免疫应答和炎症反应等多种免疫疾病中发挥作用。以广西巴马小型猪肝组织总RNA为模板,利用RT-PCR技术扩增出相应cDNA片段,连接到克隆载体pMD18-T上进行检测,测序结果为675 bp,与GenBank所提供相关序列同源性为100%,并成功构建pEGFP-N1-SAP重组真核表达载体,利用脂质体(Lipofectamine 2000)介导法将重组质粒导入到NIH-3T3细胞中培养,经转染24 h后,置于倒置荧光显微镜下观察,发现含有重组质粒的NIH-3T3细胞中表达出绿色荧光,为进一步研究SAP基因的功能特点及在试验动物相关疾病模型的应用提供条件。

Serum Amyloid Pcomponent（SAP） is a kind of seroglycoid which has conserved translation in evolution.SAP link with various kinds of fibril in order to play effect on immune response,inflammatory reaction and related immune diseases.In our test,RT-PCR was performed to gene amplification,that cDNA fragment was using the total RNA of Bama Mini-pig liver tissue as a template.The amplified fragment connected to pMD18-T vector and checked.The sequence result showed 675 bp.The homology was 100% compared with the sequence GenBank reported about the cloned pig SAP gene.And then successfully constructed the pEGFP-N1-SAP recombined eukaryotic plasmid.To test its perfect,pEGFP-N1-SAP transfected to NIH-3T3 cells culture by the Lipofectamine 2000.After 24 hours,the expression of the exogenous gene in cells was analyzed by inverted microscope.The results showed that the NIH-3T3 cells containing the recombined plasmid presented a green fluorescent.It could be used in next step,research SAP gene function and set up experimental animal related diseases model operations.