[Construction and expression of eukaryotic expression plasmid containing rat TCR Vbeta8.2 gene].

Publisher: Xi bao yu fen zi mian yi xue za zhi bian ji bu Country of Publication: China NLM ID: 101139110 Publication Model: Print Cited Medium: Print ISSN: 1007-8738 (Print) Linking ISSN: 10078738 NLM ISO Abbreviation: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi Subsets: MEDLINE

Original Publication: Xiʹan : Xi bao yu fen zi mian yi xue za zhi bian ji bu

Eukaryotic Cells/*metabolism ; Genetic Vectors/*genetics ; Peptide Fragments/*genetics ; Plasmids/*genetics ; Receptors, Antigen, T-Cell, alpha-beta/*genetics; Animals ; COS Cells ; Chlorocebus aethiops ; Female ; Gene Expression ; Genetic Vectors/metabolism ; Mice ; Mice, Inbred BALB C ; Muscle, Skeletal/metabolism ; Peptide Fragments/analysis ; Peptide Fragments/biosynthesis ; Plasmids/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Rats ; Rats, Inbred Lew ; Receptors, Antigen, T-Cell, alpha-beta/analysis ; Receptors, Antigen, T-Cell, alpha-beta/biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

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.

0 (Peptide Fragments)
0 (RNA, Messenger)
0 (Receptors, Antigen, T-Cell, alpha-beta)
0 (T-cell receptor Vbeta 8.2)

Date Created: 20060905 Date Completed: 20090501 Latest Revision: 20191210

20240829

16948894