Related Experiment Video
Updated: Jul 20, 2026
Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs
Published on: September 10, 2017
[Construction and expression of eukaryotic expression plasmid containing rat TCR Vbeta8.2 gene]
Yun Li1, Li-ping Ma, Wen-ming Zhao
1Department of Immunology, Capital University of Medical Sciences, Beijing 100069, China. ly23338@sohu.com
Aim:
To construct the recombinant eukaryotic expression vector pTARGET-TCR Vbeta8.2 and detect its their expression.
Methods:
Gene encoding TCR Vbeta8.2 was amplified by RT-PCR from spleen cells of Lewis rats, and then cloned into eukaryotic expression vector pTARGET. Recombinant clones were identified by blue/white screening on indicator plates after transformed into E.coli strain JM109, and then by bacteria colonies PCR and DNA sequencing. Recombinant plasmid was injected into BALB/c mice intramuscularly. Then the injected skeletal muscle was isolated, and expression of TCR Vbeta8.2 gene was detected by RT-PCR and immunohistochemistry. Immunocytochemical staining was used to detect the expression of pTARGET-TCR Vbeta8.2 gene after the recombinant plasmid was transfected into COS-7 cells by lipofectamine.
Results:
DNA sequencing demonstrated that TCR Vbeta8.2 gene was successfully inserted into pTARGET. RT-PCR demonstrated that TCR Vbeta;8.2 gene was successfully expressed in the injected muscle. Immunohistochemistry staining showed the expression of recombinant plasmid in the transfected COS-7 cells.
Conclusion:
The eukaryotic expression vector pTARGET-TCR Vbeta8.2 was successfully constructed and expressed in vivo and vitro, which would lay foundation for further studies on the protective effects of TCR Vbeta DNA vaccine on CIA.
