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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。
Objective To construct the mutant Ara h 2 expression vector, express and purify the protein and identify its allergenicity. Methods Ara h 2 gene was mutated and sequenced. The synthesized gene was inserted into prokaryotic expression vector pET-32a (+) and then transformed into BL21 (DE3) host. The recombinant plasmid was induced by IPTG The protein was purified by Ni2 + affinity chromatography (FPLC). The allergenicity of the recombinant protein was detected by Western-blotting and ELISA. Results The sequencing results showed that the synthesized sequence was successfully transferred into prokaryotic expression vector pET-32a (+). The recombinant protein was purified by SDS-PAGE identified, the size of the target protein consistent with the theoretical value. Both Western-blotting and ELISA results showed that the IgE in mixed serum of peanut-allergic patients was significantly lower than that of recombinant Ara h 2 (R-Ara h 2) in the Ara h 2 protein (M-Ara h 2) . Conclusion The Ara h 2 expression vector was successfully constructed. The preliminary in vitro experiments showed that the expressed recombinant protein has the potential of being hypoallergenic.