人Aβ42重组抗原的融合表达及Aβ抗体的检测

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). CONCLUSION: GST-Abeta(42) fusion protein may serve as a substitute for the expensive Abeta(42) peptide for detecting Abeta antibody.