摘要
目的构建小鼠pcDNA3.1/CXCL14真核表达载体,观察其在293T细胞中的表达,为进一步探讨CXCL14对脂肪细胞因子、氧化应激及胰岛素信号转导通路的影响提供工具。方法从C57/BL胚鼠肝脏组织中抽提RNA,RT-PCR扩增CX-CL14基因片段,并将其插入pcDNA3.1进行测序,鉴定正确构建pcDNA3.1/CXCL14后转染293T细胞,用RT-PCR法和Westernblot法分别从mRNA水平和蛋白质水平分别检测CXCL14的表达情况。结果重构质粒经PCR、限制性核酸内切酶NheⅠ和XhoⅠ酶切以及DNA序列分析鉴定,表明真核表达载体构建正确;以脂质体法转染293T细胞后,RT-PCR、Western blot能检测到目的蛋白表达。结论成功构建了小鼠pcDNA3.1/CXCL14真核表达载体并能在真核细胞中进行表达。
Objective To construct the recombinant eukaryotic expression vector pcDNA3. 1 （ - ） CXCL14 and express it in 293Tcells, thus to provide tools for further study of fat ceils factor, oxidatie stress, and the effect of insulin signaling pathways. Methods The CXCL14 was amplified from C57/BL rat liver by reverse transcnptase polymerase chain reaction （ HT - PCR）. Then recombined eukaryotic expression vector pcDNA3.1/CXCL14 was constructed. After the reconbinant plasmid was proved to be constructed correctly by cndonucleases digesting and DNA sequencing, we trasfected it into 293T cells through liposome inducing. The expression of CXCLI4 in transfectant 293T was detected by RT - PCR and Western blot. Results Estriction enzyme digestion,PCR amplifying and DNA sequencing confirmed that pcDNA3. I/CXCL14 had been constructed suecessfully. Both on the mRNA Ievel and the protein level,the expression of CXCL14 was expressed obviously in the transfected 293T cells detected by RT - PCR and Western blot respectively. Conclusion The recombinant eukaryotic expression vector pcDNA3.1/CXCLI4 was constructed,and it could be expressed in 293T cells.
出处
《医学研究杂志》
2013年第5期82-86,共5页
Journal of Medical Research
基金
湖北省自然科学基金资助项目(2011CDB487)
