gst-ucp2-n 和gst-ucp2-c原核表达载体的构建表达及多克隆抗体 .pdfVIP

() 46 　2 Vol.46, No.2 　　　JOU RNAL OF NANJING UNIVERSIT Y 　　　　 2010 3 Mar.2010 (NA TU RA L SCIENCES) ， GST-UCP2-N GST-UCP2-C ＊ 白　蕊, 郭亚楠, 卞　桢, 刘丹青, 曾　科＊＊ (, , 210093) 　:　pMD18-T-ucp2 , PCR ucp2cDNA N C , pGEX-5X-1 , PC R、、, IPT G , BL21 GST-UCP2-N(C), GST . ucp2 ,w estern blot . pGEX-5X-1-UCP-N (C), .., UCP2 . :　2(UCP2), 　, :　Q 786 Prokaryotic expression vector construction, expression and polyclonal antibody preparation of fusion protein GST-UCP2-N (C) B ai R ui, Guo Ya -N an, B i an Z hen, L iu D an -Qing , Z eng K e (Sc ool of Life Science, Nanjing University, Nanjing, 210093, C ina) Abstract:　According to sequence of ucp2, we designed two pairs of primers and insert BamH Ⅰ and X o Ⅰ restrictive endonuclease sites into 5′termital and 3′termital respectively.Using ucp2 clonal vector as template w e obtain Ext ro-cellular fragment of gene DNA at 5′termital (82-249)and 3′termital (706-927), w ic are named as ucp2-N and ucp2-C, t en inserted into expressive vector and transform into E.co li.T e recombined vectors were identified by PCR, restrictive endonucleases and sequencing. BL21 expression strain w as propagated in lura brot .A series of met ods (low temperature induction, s orten of induction time, etc.)w ere tried to induce soluble GST-UCP2-N(C)fusion proteins but wit out successes.Fusion proteins were mainly expressed as including body.It as s ow ed t at t e most ig -level expression was after 4 ours induced by 0.5 mmol/L IPTG at 28 ℃for 4 ours.Pellet w as resuspended in STE buffer wit