TRAF-6真核表达载体的构建和转染牙龈上皮细胞

Construction of recombinant pEGP-N3 TRAF-6 plasmid

目的:构建TRAF-6与绿色荧光蛋白融合的真核表达载体,为研究其在口腔疾病发病中的作用奠定基础。方法:应用基因重组技术,将反转录PCR得到的TRAF-6基因,克隆入荧光真核表达载体pEGFP-N3中,用含卡那霉素LB培养基平板筛选转化菌,阳性重组子经HindⅢ和KpnⅠ双酶切分析、PCR鉴定。重组质粒酶切鉴定正确后,以脂质体转染牙龈上皮细胞。分别由间接免疫荧光和免疫组织化学检测目的蛋白的表达。结果:获得的TRAF-6基因重组真核表达质粒,经间接免疫荧光和免疫组化检测,TRAF-6成功转染上皮细胞。结论:成功克隆TRAF-6基因,构建了融合有TRAF-6基因的真核表达质粒,对其在体外进行了表达,并转染牙龈上皮细胞,为进一步的研究奠定了基础。

AIM:To construct a recombinant expression vector fused with TRAF-6 gene and green fluorescent protein,and then introduced into gingival epithelium cells.METHODS:TRAF-6 gene was obtained by RT-PCR and cloned into fluorescent eukaryotic expression vector pEGFP-N3.The positive recombinants on LB plates were screened and identified by HindIII and KpnI restriction enzyme analysis and PCR amplification.Then the plasmid was transfected into gingival epithelium cells.The expression of TRAF-6 was detected by indirect immunofluorescence and immunohistochemistry.RESULTS:The eukaryotic expression plasmid containing TRAF-6 gene was constructed correctly.After transfection, the expression of TRAF-6 was detected in gingival epithelium cells.CONCLUSION:The eukaryotic expression plasmid containing TRAF-6 gene (TRAF-6/pEGFP-N3) has been successfully constructed. The expression plasmid has been transfected into gingival epithelium cells.