Construction of retrovirus expression vector containing GLUT-2 gene and its identification / 第三军医大学学报

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".