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Journal of Southern Medical University ; (12): 2175-2178, 2009.
Artigo em Chinês | WPRIM | ID: wpr-325154

RESUMO

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>


Assuntos
Animais , Humanos , Coelhos , Anticorpos , Alergia e Imunologia , Especificidade de Anticorpos , Escherichia coli , Genética , Metabolismo , Soros Imunes , Alergia e Imunologia , Proteínas de Membrana , Genética , Alergia e Imunologia , Proteínas do Tecido Nervoso , Genética , Alergia e Imunologia , Plasmídeos , Genética , Proteínas Recombinantes de Fusão , Genética , Alergia e Imunologia
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