[Fusion expression of human Abeta42 recombinant protein and detection of Abeta antibody].
OBJECTIVE
To clone and express the recombinant amyloid beta-protein (Abeta(42)) antigen and develop a method for detecting Abeta antibody.


METHODS
Two partially complementary fragments of Abeta(42) gene were chemically synthesized for constructing the Abeta(42) gene by PCR. The resultant Abeta(42) gene fragment was subcloned into pGEX-2T expression vector for inducing the expression of GST-Abeta(42) fusion protein, which was purified by affinity chromatography. The antigen specificity and reactivity of the purified GST-Abeta(42) fusion protein to Abeta monoclonal antibody were identified with Western blotting. Using either GST-Abeta(42) fusion protein or Abeta(42) peptide as the coating antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was established for detecting Abeta antibodies in the serum of Abeta(42) polypeptide-immunized SD rats.


RESULTS
GST-Abeta(42) fusion protein was successfully expressed as an soluble protein, which, after purification, was found to have a relative molecular mass of 31 kD. About 800 mg of GST-Abeta(42) fusion protein were obtained from l L cell culture with a purity over 95%. Western blotting demonstrated specific reaction of this purified GST-Abeta(42) fusion protein with Abeta monoclonal antibody. The sensitivity of the indirect ELISA for detecting Abeta antibody was about 2 ng/ml using GST-Abeta(42) fusion protein as the coating antigen. There was no significant difference in the results of Abeta antibody detection using either GST-Abeta(42) or Abeta(42) as the coating antigen (P>0.05).


CONCLUSIONS
GST-Abeta(42) fusion protein may serve as a substitute for the expensive Abeta(42) peptide for detecting Abeta antibody.
Zhang
Ge
G
Department of Molecular Biology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510089, China. ahanggel@sina.com.cn
Wang
Lie-Feng
LF
Wang
Hua-Qiao
HQ
Yao
Zhi-Bin
ZB
D016229:Amyloid beta-Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000906:Antibodies / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
D016229:Amyloid beta-Peptides; D000906:Antibodies; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C075222:amyloid beta-protein (1-42); D005982:Glutathione Transferase