Aim: To express the growth-associated protein-43 (GAP-43)in prokaryotic cells and prepare monoclonal antibody( mAb)against GAP-43.
Methods: Full length sequence of GAP-43 gene was amplified from the plasmid containing pGAP-43cDNA and was cloned into the expression vector pGEX-4T-I.GST-GAP-43 fusion protein was expressed in E. coil under IPTG induction. Expressed fusion protein was purified by glutathine agarose chromagraphy, Using purified protein as an immunogen, mAb against GAP-43 was prepared.
Results: The recombinant plasmid containing the target gene was constructed successfully. The fusion protein was expressed in E. coli in soluble form. The titer of anti-GAP-43mAb in ascites was 1: 10'. The Ig subclass and subtype of the mAb was IgG2a and K type, respectively. Specificity of the mAb was confirmed by ELISA, Western blot and immunofluorescence technique.
Conclusion: The anti-GAP-43 mAb obtained has strong specificity and high titer, which provides an useful reagent for further studying the function of GAP-43 in nervous system.