Preparation of tag-free NT-proBNP recombinant protein and its polyclonal antibody
XU Wen-chun · Chinese Journal of Clinical Laboratory Science · 2012
Objective To express tag-free N-terminal pro-B-type natriuretic peptide(NT-proBNP) by prokaryotic expression system and prepare polycolonal antibodies against the recombinant NT-proBNP.Methods NT-proBNP gene sequence was obtained from pET-32a(+)-NT-proBNP and inserted into pCold TF vector.The recombinant pCold TF-NT-proBNP was transferred into E.coli BL21(DE3) and the recombinant NT-proBNP was expressed by IPTG induction.Subsequently,the recombinant NT-proBNP was purified by Nickel affinity column,and further digested with HRV 3C protease.After purification with Nickel affinity column again,tag-free recombinant NT-proBNP was obtained and its antigenicity was analyzed with the commercial kit employed in clinical test.Polyclonal antibodies against the recombinant tag-freeNT-proBNP were prepared by immunizing rabbits and the specificity of antibodies was tested by Western blot.Results Highly purified tag-free NT-proBNP protein was prepared and could be detectable by Roche NT-proBNP test kit.The polyclonal antibodies against recombinant tag-free NT-proBNP with high titer and specificity were also prepared.Conclusion Highly purified tag-free recombinant NT-proBNP protein and its specific polyclonal antibodies were successfully prepared for clinical research of NT-proBNP and widespread application in diagnosis.