sCD80-Linker-sCD40L融合基因重组腺病毒载体的构建及其在NIH3T3细胞的表达

Construction of recombinant adenoviral vector encoding sCD80-Linker-sCD40L fusion gene and its expression in NIH3T3 cells

目的:构建人sCD80-Linker-sCD40L融合基因重组腺病毒载体,观测其在真核细胞中的表达。方法:以RT-PCR法从人外周血单个核细胞总RNA扩增IL12B亚基信号肽、sCD80和sCD40L编码序列,以重叠PCR法获得带有IL12B亚基信号肽的sCD80-Linker-sCD40L融合基因。使用AdMax腺病毒包装系统构建携带融合基因的重组腺病毒载体。以重组病毒感染NIH3T3细胞,观察融合基因的表达。结果:经测序证实,正确构建了携带sCD80-Linker-sCD40L融合基因的重组腺病毒载体。RT-PCR和Western blot证实融合基因在NIH3T3细胞中表达。结论:成功构建了携带sCD80-Linker-sCD40L融合基因的重组腺病毒载体,并在真核细胞中表达,为后续研究奠定了基础。

Objective： To construct a recombinant adenoviral vector containing sCD80-Linker-sCD40L fusion gene, and to evaluate the expression of the fusion protein in eukaryocyte. Methods： The coding gene of human IL12 B subunit signal peptide, sCD80 and sC D40L were cloned by RT-PCR from human peripheral blood mononuclear ceils. A sCD80-Linker-sCD40L fusion gene containing human IL12 B subunit signal peptide was constructed by overlap PCR, and AdMax Adenoviral Vector System was used to generate a recombinant adenoviral vector containing the sCD80-Linker-sCD40L fusion gene. NIH3T3 ceils were transfected with the recombinant adenovirus. The expression of the fusion gene in NIH3T3 ceils was observed. Results： The recombinant adenoviral vector containing sCD80-Linker-sCD40L fusion gene was constructed correctly, which was confirmed by DNA sequencing. The expression of the fusion gene in NIH3T3 ceils was detected with RT-PCR and Western blot. Concluslon： The successful construction of the recombinant adenoviral vector and the expression of sCD80-Linker-sCD40L fusion protein in eukaryocyte has laid a foundation for further researches.