人Aβ42重组抗原的融合表达及Aβ抗体的检测

摘要/Abstract

摘要： 目的原核表达β-淀粉样蛋白(Aβ₄₂)抗原,建立检测Aβ抗体的间接ELISA技术。方法化学合成Aβ₄₂基因两个互补片段,通过PCR构建人Aβ₄₂基因,克隆至pGEX-2T表达载体中,原核表达并亲和纯化GST-Aβ₄₂融合蛋白。Westernblotting检测其抗原特异性。以GST-Aβ₄₂和Aβ₄₂肽分别为包被抗原,间接ELISA法检测Aβ₄₂免疫的大鼠血清中Aβ抗体。结果原核表达的可溶性GST-Aβ₄₂融合蛋白相对分子质量为31000,经亲和纯化后,融合蛋白产量为800mg/L菌液,纯度大于95%;Westernblotting证实纯化的融合抗原与Aβ单抗特异反应;以GST-Aβ₄₂为包被抗原,间接ELISA法检测Aβ抗体灵敏度为2ng/ml;以GST-Aβ₄₂和化学合成的Aβ₄₂分别为包被抗原,间接ELISA法检测Aβ₄₂肽免疫大鼠血清中Aβ抗体滴度,两者比较差异无显著性(P>0.05)。结论原核表达的GST-Aβ₄₂融合蛋白可替代昂贵的化学合成的Aβ₄₂肽抗原,用于检测Aβ抗体。

Abstract: Objective To clone and express the recombinant amyloid beta-protein(Aβ₄₂) antigen and develop a method for detecting Aβ antibody. Methods Two partially complementary fragments of Aβ₄₂ gene were chemically synthesized for constructing the Aβ₄₂ gene by PCR. The resultant Aβ₄₂ gene fragment was subcloned into pGEX-2T expression vector for inducing the expression of GST-Aβ₄₂ fusion protein, which was purified by affinity chromatography. The antigen specificity and reactivity of the purified GST-Aβ₄₂ fusion protein to Aβ monoclonal antibody were identified with Western blotting. Using either GST-Aβ₄₂ fusion protein or Aβ₄₂ peptide as the coating antigen, an indirect enzyme-linked immunosorbent assay(ELISA) was established for detecting Aβ antibodies in the serum of Aβ₄₂ polypeptide-immunized SD rats. Results GST-Aβ₄₂ 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-Aβ₄₂ fusion protein were obtained from l L cell culture with a purity over 95%. Western blotting demonstrated specific reaction of this purified GST-Aβ₄₂ fusion protein with Aβ monoclonal antibody. The sensitivity of the indirect ELISA for detecting Aβ antibody was about 2 ng/ml using GST-Aβ₄₂ fusion protein as the coating antigen. There was no significant difference in the results of Aβ antibody detection using either GST-Aβ₄₂ or Aβ₄₂ as the coating antigen(P>0.05). Conclusion GST-Aβ₄₂ fusion protein may serve as a substitute for the expensive Aβ₄₂ peptide for detecting Aβ antibody.

中图分类号:

Q78
R329.1

张革^1,2, 王烈峰², 汪华侨², 姚志彬². 人Aβ₄₂重组抗原的融合表达及Aβ抗体的检测[J]. 南方医科大学学报, 2005, 25(02): 160-164.

ZHANG Ge^1,2, WANG Lie-feng², WANG Hua-qiao², YAO Zhi-bin². Fusion expression of human Aβ₄₂ recombinant protein and detection of Aβ antibody[J]. Journal of Southern Medical University, 2005, 25(02): 160-164.

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人Aβ₄₂重组抗原的融合表达及Aβ抗体的检测

Fusion expression of human Aβ₄₂ recombinant protein and detection of Aβ antibody

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