构建携带人/鼠IFN-γ重组腺病毒体外转染人脐带间充质干细胞的实验研究

Construction of Recombinant Adenovirus Vector with Human/Mouse Interferon Gene and Its Transfection into Human Umbilical Cord Mesenchymal Stem Cells

目的构建人/鼠干扰素(IFN-γ)的重组腺病毒,体外转染人脐带间充质干细胞(human umbilical mesenchymal stem cells,HUMSCs),为研究IFN-γ在预防和治疗移植物抗宿主病(graft-versus-host disease,GVHD)中的作用提供实验依据。方法采用PCR法从含有人/鼠IFN-γ基因的质粒中扩增出目的基因片段IFN-γ,然后定向克隆到穿梭质粒载体pDC316中,构建pDC316-IFN-γ,经酶切、PCR及测序鉴定后,再将IFN-γ定向克隆至腺病毒骨架载体,从而构建携带IFN-γ的重组腺病毒载体,并转染人胚肾细胞系293细胞,进行重组腺病毒(Ad-IFN-γ)的包装、生产和纯化;用半数组织培养感染量(50%tissue culture infective dose,TCID50)法检测重组腺病毒滴度。将Ad-IFN-γ及空病毒(Ad-null,作为载体阴性对照)分别转染HUMSCs,在荧光显微镜下观察细胞绿色荧光蛋白(GFP)的表达,采用Western blot与ELISA法鉴定IFN-γ蛋白的表达。结果成功构建了携带IFN-γ的重组腺病毒,鼠IFN-γ(Ad-mIFN-γ)的滴度为1.0×1010 IU/mL,人IFN-γ(Ad-hIFN-γ)的滴度为1.6×1010 IU/mL,Ad-GFP的滴度为1.0×109 IU/mL。Ad-IFN-γ转染HUMSCs后24h开始观察到绿色荧光,72h时该荧光表达更强;Western blot和ELISA法均证实HUMSCs表达IFN-γ蛋白。结论成功构建了携带人/鼠IFN-γ基因的重组腺病毒载体,并证实其可有效转染HUMSCs。

Objective Construction and identification of recombinant adenovirus vector with human/mouse interferon（IFN-γ）and effectively transfection into human umbilical cord mesenchymal stem cells（HUMSCs）.Methods IFN-γgene of human/mouse were amplified from the plasmid by polymerase chain reaction（PCR）and then inserted into the plasmid pDC316 to generate pDC316-IFN-γ.After being confirmed by restriction enzyme digestion and DNA sequencing,the DNA encoding IFN-γin the new structure were inserted into the vector of recombinant plasmid adenovirus and confirmed by restricition enzyme digestion.Then the human embryonic kidney cell line 293 were transfected with confirmed Ad-IFN-γ,and the recombinant adenovirus were amplified,and the virus titer were detected using 50% tissue culture infective dose（TCID50）assay.The expression of the green fluorescent protein（GFP）and IFN-γwere detected by fluorescent microsope and Western blot and ELISA after the recombinant adenovirus transfected HUMSCs.Results The recombinant adenovirus Ad-hIFN-γ were constructed successfully,and amplified with titer of 1.6×10^10 IU/mL.The titer of Ad-mIFN-γ was 1.0×10^10 IU/mL and the titer of Ad-GFP was 1.0×10^9 IU/mL.The green fluorescence proteins could be observed under fluorescent microscope in HUMSCs 24 hafter transfection and with a stronger degree after 72 h,and IFN-γexpression in HUMSCs were confirmed by Western blot and ELISA.Conclusion Construction and identification of recombinant adenovirus vector of human/mouse IFN-γand effectively transfection of HUMSCs were successful.