【摘要】 目的:构建含小鼠细胞因子信号抑制因子-1基因(SOCS1)的重组腺病毒载体(Ad5F35-SOCS1),探讨其介导SOCS1基因在小鼠树突状细胞中的表达。方法:设计含AgeI和NheI酶切位点的SOCS1基因上下游引物,以质粒pEF-FLAG-1/mSOCS1为模版,通过PCR扩增获得SOCS1全部序列,片段回收后经AgeI和NheI酶切,再定向插入到经AgeI和NheI酶切的质粒pDc316-LacZa中,获得重组穿梭质粒pDC316-SOCS1,经AgeI和NheI酶切、PCR及测序等鉴定后,用脂质体将穿梭质粒pDC316-SOCS1与腺病毒骨架质粒pBHGF35共转染293细胞,经位点特异性重组获得重组腺病毒Ad5F35-SOCS1,行PCR鉴定,经293细胞扩增、纯化制备高滴度病毒液,TCID50法测定病毒滴度。用获得的重组腺病毒感染小鼠树突状细胞,以免疫组化检测SOVD1的表达。结果:成功构建了含小鼠SOCS1基因的重组腺病毒载体,病毒感染滴度为1.4×10~9IU/ml,该载体能有效介导SOCS1基因在小鼠树突状细胞中的表达。结论:重组腺病毒载体能将SOCS1基因转入小鼠树突状细胞并有效表达,为基因转染制备耐受性树突状细胞奠定了基础。更多还原

【Abstract】 Objective:To construct recombinant adenovirus vector containing mouse suppressor of cytokine signaling-lgene (Ad5F35-SOCS1),and to investigate the expression of SOCS1 in mouse dendritic cells transfected by Ad5F35-SOCS1.Methods:Full length SOCS1 cDNA was obtained from plasmid pEF-FLAG-1/mSOCS1 by PCR amplification.The PCR product was digested by re- striction endonucleases AgeI and NheI,and then inserted orientationally into plasmid pDC316-LacZa digested by AgeI and NheI.Once constructed,the recombinant shuttle plasmid pDC316-SOCS1 was identified by PCR and sequencing,and then cotransfected with rescue plasmid pBHGF35 into 293 cells by liposome reagent.Ad5F35-SOCS1 was generated by site-specific recombination and confirmed by PCR.Ad5F35-SOCS1 was propagated in 293 cells and purified.The titer of viral stock was determined by TCID50 assay. Ad5F35-SOCS1 was then used to transfect mouse dendritic cells,and the expresssion of SOCS1 was confirmed by immunohistochem- istry.Results:The recombinant adenovirus vector containing mouse SOCS1 gene(Ad5F35-SOCS1)was successfully constructed and the infective titer was 1.4×10~9 1U/ml.The expression of SOCS1 in mouse dendritic cells transfected by Ad5F35-SOCS1 was efficient. Conclusions:Recombinant adenovirus could transmit SOCS1 gene to mouse dendritic cells,and the expression of SOCSl was efficient.It laid the foundation for producing tolerogenic dendritic cells via gene transfection.更多还原