title: [Prokaryotic expression, purification of human LINGO-1(aa76-319) and preparation of its polyclonal antibody].
abstract: OBJECTIVE
  To express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb).
  
  
  METHODS
  The 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting.
  
  
  RESULTS
  The prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity.
  
  
  CONCLUSIONS
  The fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.
authors.affiliation: Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. lvjun0000@126.com
authors.forename: Jun
authors.initials: J
authors.lastname: Lv
authors.forename: Xin
authors.initials: X
authors.lastname: Lu
authors.forename: Xiao-Dan
authors.initials: XD
authors.lastname: Jiang
authors.forename: Chang-Chen
authors.initials: CC
authors.lastname: Hu
authors.forename: Ying-Qian
authors.initials: YQ
authors.lastname: Cai
authors.forename: Mou-Xuan
authors.initials: MX
authors.lastname: DU
authors.forename: Yu-Xi
authors.initials: YX
authors.lastname: Zou
authors.forename: Ling-Sha
authors.initials: LS
authors.lastname: Qin
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
chemical_list: D000906:Antibodies; D007106:Immune Sera; C482293:LINGO1 protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins