PDX1与NKX6.1双基因表达腺病毒载体的构建,鉴定与表达.doc

PDX1与NKX6.1双基因表达腺病毒载体的构建、鉴定与表达 唐小龙 HYPERLINK /a/f/dm3/0906031436/htmltool_dm3.htm \l _ftn1 \o \t _blank 1,2，张荣波2，汪雪峰2，张 文 HYPERLINK /a/f/dm3/0906031436/htmltool_dm3.htm \l _ftn1 \o \t _blank 1 （1广州中医药大学第二附属医院(广东省中医院)最好保留检验科, 广州 510120；2安徽理工大学医学院生物工程研究所,安徽 淮南 232001） 最好保留 摘 要：目的 运用pADxsi系统构建带人PDX1与NKX6.1双基因表达腺病毒载体。有测序鉴定。 方法 采用基因克隆技术，将目的基因NKX6.1克隆至pShuttle-GFP-CMV中，得到pShuttle- GFP-CMV-NKX6.1；再将PDX1克隆至pShuttle-CMV-GFP/CMV-NKX6.1上，替换掉GFP，得到pShuttle-CMV-PDX1/CMV-NKX6.1；最后将CMV-PDX1/CMV-NKX6.1从pShuttle-CMV-PDX1/CMV- NKX6.1转移到ADxsi骨架载体上，得到pADxsi-CMV-PDX1/CMV-NKX6.1，然后在293细胞中进行包装与扩增活病毒，病毒滴度测定；体外感染人胎肝间充质干细胞。结果 酶切鉴定和PCR证明重组人PDX1与NKX6.1双基因表达腺病毒载体构建正确，可高效感染人胎肝间充质干细胞，并特异性地定位于细胞核内。结论 应用pADxsi系统重组技术成功构建了人PDX1与NKX6.1双基因表达 有测序鉴定。 关键词：腺病毒；胰腺十二指肠同源框1基因；NK6转录因子相关, 基因座1；间充质干细胞 中图法分类号： 文献标识码：A Construction and expression of PDX1 and NKX6.1 double-gene modified HYPERLINK /a/j/dm3/java_script:showjdsw(showjd_0,j_0) \t _blank adenovirus vectors TANG Xiao-long HYPERLINK /a/f/dm3/0906031436/htmltool_dm3.htm \l _ftn1 \o \t _blank 1,2，ZHANG Rong-bo2，WANG Xue-feng2，ZHANG Wen HYPERLINK /a/f/dm3/0906031436/htmltool_dm3.htm \l _ftn1 \o \t _blank 1 1Clinical Laboratory, Second Affiliated Hospital, Guangzhou University of Traditional Chinese Medicine(Guangdong Provincial Hospital of TCM), Guangzhou 510120；2Research Section of Biomedical Engineering, Medical College, Anhui University of Science and Technology, Huainan 232001, China Abstract：Objective Constructing the PDX1 and NKX6.1 double-gene modified HYPERLINK /a/j/dm3/java_script:showjdsw(showjd_0,j_0) \t _blank adenovirus vectors, to explore the ability and HYPERLINK /a/j/dm3/java_script:void(0) \t _blank mechanism of difference from stem cell to Pancreatic beta-cells induced by PDX1 and NKX6.1. Methods The pShuttle-GFP-CMV-NKX6.1 was obtained by the cloning of the interest gene NKX6.1 into pShuttle-GFP-CMV; pShuttle-CMV-PDX1/CMV-NKX6.1 was constructed when GFP’s instead of PDX1; the CMV-PDX1/CMV-NKX6.1 was transfe