NGN3与PAX4双基因表达腺病毒载体的构建与表达  

Construction and expression of NGN3 and PAX4 double-gene modified adenovirus vectors

作　　者：唐小龙[1] 江振友[2] 庞雪云[1] 蓝锴[1] 欧财文[1] 

机构地区：[1]广东省中医院检验科,广东广州510120 [2]暨南大学医学院微生物学与免疫学教研室,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2010年第4期346-351,共6页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家自然基金项目(309736937);广东省教育厅基金项目(83057)

摘　　要：目的:运用pADxsi系统构建带人NGN3与PAX4双基因表达腺病毒载体。方法:采用基因克隆技术,将目的基因NGN3从pEGFP-N1-NGN3质粒上切下连到pShuttle-GFP-CMV上,替换GFP,得到pShuttle-CMV-NGN3;再将PAX4从pEGFP-N1-PAX4质粒上切下连到系统pShuttle-CMV-NGN3上,得到pShuttle-CMV-NGN3/CMV-PAX4穿梭质粒;最后将CMV-NGN3/CMV-PAX4从pShuttle-CMV-NGN3/CMV-PAX4转移到ADxsi骨架载体上,得到pADxsi-CMV-NGN3/CMV-PAX4病毒质粒,然后在293细胞中进行包装与扩增活性病毒,并进行病毒滴度测定。体外感染人脐带间充质干细胞。逆转录-聚合酶链反应(RT-PCR)与免疫印迹(Western blotting)检测目的基因在细胞内的表达情况,免疫细胞化学与间接荧光法检测目的分子在细胞内的定位。结果:酶切鉴定和PCR证明重组人NGN3与PAX4双基因表达腺病毒载体构建正确;RT-PCR与Western blotting结果显示目的基因在细胞持续稳定表达;间接荧光与免疫化学表明重组腺病毒可高效感染人脐带间充质干细胞,且目的基因特异性地定位于细胞核内。结论:应用重组技术成功构建人NGN3与PAX4双基因表达腺病毒载体,且在间充质干细胞内持续而稳定表达。Aim：To construct NGN3 and PAX4 double-gene modified adenovirus vectors using pADxsi system. Methods： By using gene-clone technology,the target gene NGN3 was obtained from the pEGFP-N1-NGN3 plasmid,to replace GFP in the pShuttle-GFP-CMV vector,and thus the pShuttle-GFP-CMV-NGN3 was constructed;second,PAX4 was cut off from pEGFP-N1-PAX4 plasmid and was inserted into pShuttle-CMV-NGN3 to construct the pShuttle-CMV-NGN3/CMV-PAX4 shuttle plasmid;Finally,CMV-NGN3/CMV-PAX4 was obtained from pShuttle-CMV-NGN3/CMV-PAX4 vector and transferred to ADxsi skeleton vector to construct the pADxsi-CMV-NGN3/CMV-PAX4 virus vector.pADxsi-CMV-NGN3/CMV-PAX4 virus was packaged and amplificated in 293 cells for virus titration.Human umbilical cord mesenchymal stem cells were infected by the virus plasmid.The target genes expression in cells were detected by Reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting,and the location of molecules within the cells were showed by immunocytochemistry and indirect immunofluorescence assay.Results： Construction of NGN3 with PAX4 double-gene modified adenovirus expression vector were confirmed by restriction analysis and PCR;RT-PCR and Western blotting results showed that the target genes were expressed continuously in cells.Indirect fluorescence and immunocytochemistry showed that the recombinant adenovirus could infect human umbilical cord mesenchymal stem cells efficiently,and NGN3 with PAX4 were only localized in nucleus.Conclusion：Recombinant human NGN3 and PAX4-gene expression vector was successfully constructed.The target genes can be expressed continuously and stably in mesenchymal stem cells.

关 键 词：腺病毒 神经元素3(NGN3) 成对盒基因4(PAX4) 间充质干细胞(MCS) 

分 类 号：Q78[生物学—分子生物学]