title: [Construction and expression of eukaryotic expression plasmid containing rat TCR Vbeta8.2 gene].
abstract: 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.
authors.affiliation: Department of Immunology, Capital University of Medical Sciences, Beijing 100069, China. ly23338@sohu.com
authors.forename: Yun
authors.initials: Y
authors.lastname: Li
authors.forename: Li-ping
authors.initials: LP
authors.lastname: Ma
authors.forename: Wen-ming
authors.initials: WM
authors.lastname: Zhao
authors.forename: Zhen-long
authors.initials: ZL
authors.lastname: Liu
mesh_terms: 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
chemical_list: D010446:Peptide Fragments; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta; C109168:T-cell receptor Vbeta 8.2