[Prokaryotic expression, purification of human LINGO-1(aa76-319) and preparation of its polyclonal antibody].
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.
Lv
Jun
J
Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. lvjun0000@126.com
Lu
Xin
X
Jiang
Xiao-Dan
XD
Hu
Chang-Chen
CC
Cai
Ying-Qian
YQ
DU
Mou-Xuan
MX
Zou
Yu-Xi
YX
Qin
Ling-Sha
LS
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*
D000906:Antibodies; D007106:Immune Sera; C482293:LINGO1 protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins