Construction, identification and expression of PDX1 and NKX6.1 double-gene adenovirus expression vectors

唐小龙; 张荣波; 汪雪峰; 张文

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Construction, identification and expression of PDX1 and NKX6.1 double-gene adenovirus expression vectors

更新时间：2026-01-09

- Construction, identification and expression of PDX1 and NKX6.1 double-gene adenovirus expression vectors
- Journal of Army Medical University Vol. 32, Issue 4, Pages: 364-368(2010)
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  CLC： R373;R394-33;R394.2
- Published：2010
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唐小龙,张荣波,汪雪峰,张文.PDX1与NKX6.1双基因表达腺病毒载体的构建、鉴定与表达[J].陆军军医大学学报(原第三军医大学学报),2010,32(04):364-368.

　Tang Xiaolong,Zhang Rongbo,Wang Xuefeng,Zhang Wen.Construction, identification and expression of PDX1 and NKX6.1 double-gene adenovirus expression vectors[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(04):364-368.
唐小龙,张荣波,汪雪峰,张文.PDX1与NKX6.1双基因表达腺病毒载体的构建、鉴定与表达[J].陆军军医大学学报(原第三军医大学学报),2010,32(04):364-368. DOI：

　Tang Xiaolong,Zhang Rongbo,Wang Xuefeng,Zhang Wen.Construction, identification and expression of PDX1 and NKX6.1 double-gene adenovirus expression vectors[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(04):364-368. DOI：

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。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；将PDX1克隆至pShuttle-CMV-GFP/CMV-NKX6.1上，替换掉GFP，得到pShuttle-CMV-PDX1/CMV-NKX6.1；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，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。结果分别酶切重组人PDX1与NKX6.1基因表达穿梭质粒与腺病毒载体质粒，阳性克隆均获得与理论相一致的目的条带；重组腺病毒感染人胎肝间充质干细胞比例达90%以上；RT-PCR 结果表明目的基因在人胎肝间充质干细胞内能稳定表达；免疫细胞化学与间接荧光检测目的PDX1与NKX6.1蛋白分子均特异在细胞核表达。结论应用pADxsi系统重组技术成功构建了人PDX1与NKX6.1双基因表达腺病毒载体。

Abstract

Objective To construct the human PDX1 and NKX6.1 doublegene adenovirus expression vector using the pADxsi system. Methods pShuttle-GFP-CMV-NKX6.1 was constructed by cloning the target gene NKX6.1 into pShuttle-GFP-CMV. pShuttle-CMV-PDX1/CMV-NKX6.1 was constructed by cloning PDX1 into pShuttle-CMV-GFP/CMV-NKX6.1 to replace GFP. The pADxsi-CMV-PDX1/CMV-NKX6.1 was constructed by transferring CMV-PDX1/CMV-NKX6.1 to ADxsi backbone plasmid vector from pShuttle-CMV-PDX1/CMV-NKX6.1. Then

the pADxsi-CMV-PDX1/CMV-NKX6.1 adenovirus vector was amplified in 293 cells and viral titer was measured. Human placental liver mesenchymal stem cells were infected with adenoviruses. Results Restriction enzyme analysis and PCR showed that the PDX1 and NKX6.1 double-gene adeno-virus expression vector was successfully constructed

which could infect the human placental liver mesenchymal stem cells and was specifically located in nuclei. Conclusion Human PDX1 and NKX6.1 double-gene adenovirus expression vectors can be successfully constructed using recombinant techniques of the pADxsi system.

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