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目的 :构建人 β 防御素 3(hBD3)的真核表达载体 ,建立稳定表达hBD3的细胞株。 方法 :用EcoRⅠ酶切含有hBD3全长基因的pGEM hBD3重组质粒 ,获得其编码区全长序列 ,将其连接入EcoRⅠ预处理过的pcDNA3中 ,转化大肠杆菌 ,酶切鉴定筛选出插入方向正确的转化子。采用脂质体转染法将重组pcDNA3 hBD3真核表达载体导入COS 7细胞 ,用G4 18进行抗性筛选 ,用RT PCR和Western印迹检测目的基因hBD3的mRNA和蛋白表达水平。结果与结论 :构建的pcDNA3 hBD3真核表达载体转染COS 7细胞后可稳定表达hBD3的mRNA和蛋白 ,且蛋白主要以分泌形式存在于培养上清中。
OBJECTIVE: To construct an eukaryotic expression vector of human β-defensin 3 (hBD3) and establish a cell line stably expressing hBD3. METHODS: The pBEM hBD3 recombinant plasmid containing the full-length hBD3 gene was digested with EcoRⅠ to obtain the full-length sequence of its coding region. The recombinant plasmid was ligated into EcoRⅠ-pretreated pcDNA3 and transformed into E. coli. The recombinant plasmid was digested with restriction endonucleases Turn. Recombinant pcDNA3 hBD3 eukaryotic expression vector was transfected into COS 7 cells by lipofectamine. The recombinant plasmid pcDNA3 hBD3 was screened by G4 18. The expression of hBD3 mRNA and protein was detected by RT PCR and Western blotting. RESULTS AND CONCLUSION: The constructed hBD3 eukaryotic expression vector transfected COS 7 cells stably expressed hBD3 mRNA and protein, and the protein mainly exists in secreted form in the culture supernatant.