[Construction and expression of prokaryotic expression vector of GAP-43 and preparation of monoclonal antibody against GAP-43].
OBJECTIVE
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.


CONCLUSIONS
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.
Duan
Xiao-li
XL
Institute of Neuroscience, Fourth Military Medical University, Xi'an 710032, China.
Huang
Wen-jin
WJ
Wang
Bai-ren
BR
Song
Jun-feng
JF
Jin
Wei-lin
WL
Guo
Xiang
X
Ju
Gong
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D019922:GAP-43 Protein / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D019922:GAP-43 Protein; D011993:Recombinant Fusion Proteins