经点突变的花生主要过敏原Ara h2低致敏原的制备与鉴定

Preparation and characterization of point mutation peanut major allergen Ara h2

目的构建经点突变的Ara h 2表达载体,表达并纯化该蛋白,鉴定其过敏原性。方法将Ara h 2基因进行点突变,并将其序列进行合成,再将合成后的基因连入原核表达载体pET-32a(+)上,然后转入BL21(DE3)宿主表达菌中;IPTG诱导表达;通过Ni2+亲和层析(FPLC)纯化目的蛋白;Western-blotting和ELISA检测该重组蛋白的过敏原性。结果测序结果表明合成后的序列成功转入原核表达载体pET-32a(+)上。重组蛋白纯化后经SDS-PAGE鉴定,目的蛋白大小与理论值相符。Western-blotting和ELISA结果均表明经点突变的Ara h 2蛋白(M-Ara h 2)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显著降低。结论成功构建了经点突变的Ara h 2表达载体,初步的体外实验表明该基因表达的重组蛋白具有低致敏原的潜能。

To express and purify the point mutation peanut major allergen Ara h 2 and preliminarily characterize the allergenicity of purified recombinant M-Ara h 2 protein,the Ara h 2 was mutated and inserted into the expression vector pET-32a（＋）.The vector was transformed into Escherichia coli BL21（DE3） and the protein expression was induced by IPTG.Ni2＋ chelating affinity chromatography was used to purify the recombinant M-Ara h 2 protein.The allergenicity of M-Ara h 2 was examined by Western-blotting and ELISA.The ORF which contained 474 bp and encoded 157 amino acids was authenticated to be M-Ara h 2.The recombinant M-Ara h 2 protein induced by IPTG is consistent with the actual value.The affinity between recombinant M-Ara h 2 protein and IgE antibodies from pooled peanut-allergic patients serum decreased significantly compared with R-Ara h 2 identified by Western-blotting and ELISA.In this research,recombinant M-Ara h 2 protein was obtained with hypoallergenic.