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  • Title: [Construction and expression of eukaryotic expression plasmid containing rat TCR Vbeta8.2 gene].
    Author: Li Y, Ma LP, Zhao WM, Liu ZL.
    Journal: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi; 2006 Sep; 22(5):560-3. PubMed ID: 16948894.
    Abstract:
    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.
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