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目的制备花生主要过敏原Ara h 2三聚体重组蛋白并检测其过敏原性。方法利用分子生物学的方法将3分子的Ara h 2依次串联起来,并将其整合到原核表达载体pET-32a(+),再转化到感受态Origami中;然后利用IPTG诱导其表达;通过Ni2+亲和层析纯化三聚体重组蛋白;Western-blotting和ELISA检测目的蛋白的过敏原性。结果测序结果表明Trimer成功整合到pET-32a(+)上。三聚体重组蛋白纯化后经SDS-PAGE鉴定,蛋白大小与理论值相符。Western-blotting和ELISA结果表明Trimer与重组的Ara h 2(r-Ara h 2)蛋白相比,结合花生过敏病人混合血清中IgE的能力有所降低。结论成功制备花生主要过敏原Ara h 2三聚体重组蛋白,初步的体外实验表明该重组蛋白具有低致敏原的潜能。
Objective To prepare Ara h 2 trimer recombinant protein which is the major allergen of peanut and test its allergenicity. Methods Three molecules of Ara h 2 were put in series by molecular biology method and integrated into the prokaryotic expression vector pET-32a (+) and then transformed into competent Origami. The expression was induced by IPTG. Purified trimer recombinant protein was purified by affinity chromatography. The allergenicity of the target protein was detected by Western-blotting and ELISA. Results Sequencing results showed that Trimer was successfully integrated into pET-32a (+). Trimer recombinant protein purified by SDS-PAGE identified, the protein size and theoretical value. Western-blotting and ELISA showed that Trimer had a reduced ability to bind IgE to mixed peanut allergy patients compared to recombinant Arah2 (r-Arah2) protein. Conclusion Ara h 2 trimer recombinant protein, a major peanut allergen, has been successfully prepared. Preliminary in vitro experiments show that the recombinant protein has the potential of being hypoallergenic.