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


CONCLUSIONS
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.
Li
Yun
Y
Department of Immunology, Capital University of Medical Sciences, Beijing 100069, China. ly23338@sohu.com
Ma
Li-ping
LP
Zhao
Wen-ming
WM
Liu
Zhen-long
ZL
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005057:Eukaryotic Cells / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018482:Muscle, Skeletal / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D010446:Peptide Fragments; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta; C109168:T-cell receptor Vbeta 8.2