Construction of retrovirus expression vector containing GLUT-2 gene and its identification

方芳; 凌贤龙

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Construction of retrovirus expression vector containing GLUT-2 gene and its identification

更新时间：2026-01-09

- Construction of retrovirus expression vector containing GLUT-2 gene and its identification
- Journal of Army Medical University Vol. 32, Issue 11, Pages: 1207-1209(2010)
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  CLC： R394-33;R394.3
- Published：2010
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方芳,凌贤龙.葡萄糖转运蛋白-2逆转录病毒表达载体构建/包装及其鉴定[J].陆军军医大学学报(原第三军医大学学报),2010,32(11):1207-1209.

　Fang Fang,Ling Xianlong.Construction of retrovirus expression vector containing GLUT-2 gene and its identification[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(11):1207-1209.
方芳,凌贤龙.葡萄糖转运蛋白-2逆转录病毒表达载体构建/包装及其鉴定[J].陆军军医大学学报(原第三军医大学学报),2010,32(11):1207-1209. DOI：

　Fang Fang,Ling Xianlong.Construction of retrovirus expression vector containing GLUT-2 gene and its identification[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(11):1207-1209. DOI：

摘要

目的构建葡萄糖转运蛋白-2（glucose transporter-2，GLUT-2）基因逆转录病毒表达载体及其稳定产毒细胞系，为新型“人工胰岛β细胞”的构建奠定基础。方法质粒pCB7/GLUT2经EcoRⅠ、BamHⅠ双酶切，克隆至逆转录病毒载体PLXSN，构建成重组逆转录病毒表达载体pL-GLUT2-SN，经酶切及测序鉴定；转pL-GLUT2-SN至包装细胞系PA317，G418抗性筛选，检测上清病毒滴度，挑选滴度较高的稳定产毒细胞克隆行PCR及RT-PCR鉴定。结果建立GLUT-2基因逆转录病毒表达载体pL-GLUT2-SN，经酶切及测序证实目的基因插入位点及读码框架正确；所建稳定产毒细胞克隆PA317/GLUT2，其最高病毒滴度达7.1×10

CFU/ml，PCR及RT-PCR证实GLUT-2基因整合入其中并稳定表达。结论成功构建GLUT-2基因逆转录病毒表达载体及其稳定产毒细胞系，为“人工胰岛β细胞”的构建奠定了基础。

Abstract

Objective To establish the retrovirus expression vector containing glucose transporter-2 (GLUT-2) gene and its stable virus producing cell line，for the construction of new“artificial islet beta cells”. Methods Plasmid pCB7/GLUT2 was digested by EcoRⅠ/BamHⅠ and cloned to retroviral vector PLXSN for the construction of recombinant plasmid pL-GLUT2-SN

which was identified by enzyme digestion and sequencing

and then transfected to packaging cell line PA317.Titer of the recombinant virus was detected. Cell lines with a high titer were cloned and identified by PCR and RT-PCR. Results The retrovirus expression vec

tor pL-GLUT2-SN was established

in which the insertion site and reading frame of target gene were confirmed by enzyme digestion and sequence analysis. The highest titer of virus producing cell line PA317/GLUT2 was 7.1×10

CFU/mL. PCR and RT-PCR showed that the GLUT-2 gene was integrated into PA317/GLUT2 and stably expressed. Conclusion A retrovirus expression vector containing GLUT-2 gene and its stable virus producing cell line PA317/GLUT2 are successfully constructed

thus laying a foundation for the construction of “artificial islet beta cells”.

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