Construction and characterization of RNAi lentiviral vector targeting rat CD80 gene
- VernacularTitle:大鼠CD80基因RNAi慢病毒载体的构建与鉴定
- Author:
Mei SUN
;
Jindong LI
;
Rui JIANG
;
Nan GAO
;
Chengyan JIN
;
Shuli LUO
;
Rongyou WANG
;
Xingyi ZHANG
- Publication Type:Journal Article
- Keywords:
CD80;
RNA interference;
Lentivirus
- From:
Chinese Journal of Immunology
2009;25(11):1014-1018
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a RNAi lentiviral vector targeting rat CD80 gene and detect its effect of gene silencing in NRK and IEC6 cells.Methods:The effective sequence of siRNA targeting rat CD80 gene was confirmed in our previous work.Oligo-DNA fragment containing short hairpin frame was synthesized and reannealed,and then cloned into pGCSIL-GFP lentiviral expression vector.PCR and sequencing analysis were made for verifying the positive clones.The virus packaging plasmids were transfected into 293T cells to harvest shRNA lentivirus.After infection in NRK and IEC6 cells,Real-time PCR was performed to determine the expressing level of CD80.Results:PCR and sequencing revealed that shRNA plasmids was correctly constructed.Virus with a titer of 4×10~8 TU/ml was successfully packaged.CD80 expression in NRK and IEC6 cells could be knockdown by virus infection as characterized by 66.9% and 63.5% decrease of CD80 mRNA in NRK and IEC6 cells respectively,compared with negative control lentivirus.Conclusion:The recombinant lentiviral shRNA expressing vector targeting rat CD80 gene has been successfully constructed and packaged.CD80 mRNA could be down-regulated availably in NRK and IEC6 cells.
