[1]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].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 Third Mil Med Univ,2010,32(11):1207-1209.
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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(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
32卷
期数:
2010年第11期
页码:
1207-1209
栏目:
论著
出版日期:
2010-06-15

文章信息/Info

Title:
Construction of retrovirus expression vector containing GLUT-2 gene and its identification
作者:
方芳凌贤龙
第三军医大学新桥医院消化内科
Author(s):
Fang FangLing Xianlong
Department of Gastroenterology,Xinqiao Hospital,Third Military Medical University, Chongqing, 400037,China
关键词:
葡萄糖转运蛋白-2逆转录病毒载体PA317细胞包装鉴定
Keywords:
glucose transporter-2retroviral vectorPA317 cellpackagingidentification
分类号:
R394-33;R394.3
文献标志码:
A
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7.1×105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 vector 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 7.1×105 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”.

参考文献/References:

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].第三军医大学学报,2010,32(11):1207-1209.

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更新日期/Last Update: 2010-06-04