花生过敏原Ara h 1蛋白的原核表达及致敏性分析

Prokaryotic Expression of Peanut Allergen Ara h 1 and Its Allergenicity

从花生中提取总RNA,用反转录聚合酶链式反应得到花生过敏原Ara h 1基因,构建pET-28a-Ara h 1表达载体,转入Rosetta(DE3)宿主表达菌中诱导产物表达,用镍离子亲和层析法纯化得到目的蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示目的蛋白分子质量约为75 kDa,与预计相符;经质谱鉴定为Ara h 1蛋白。用BALB/c小鼠模型评价重组Ara h 1蛋白的致敏性结果显示,重组Ara h 1蛋白致敏小鼠血清中特异性抗体、Th2型细胞因子、组胺含量升高,空肠和肺组织发生病变,表明重组Ara h 1蛋白可以导致小鼠发生Th2型过敏反应,且有与天然Ara h 1蛋白相似的致敏性。同时RBL细胞模型结果显示重组Ara h 1蛋白还可导致RBL细胞脱颗粒,释放β-己糖苷酶,进一步表明重组Ara h 1蛋白具有致敏性。

An Ara h 1 gene was synthesized from total RNA extracted from peanut using oligo primers by reverse transcription polymerase chain reaction(RT-PCR). The gene was cloned into pET-28 a vector and a recombinant expression vector was constructed. Then the recombinant vector was transformed into the bacterial strain Rosetta(DE3) for protein expression, and the target protein was puri?ed by Ni af?nity chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) showed that the molecular mass of the expressed protein was about 75 kDa, which matched with the theoretical value. Analysis by mass spectrometry(MS) con?rmed that the protein was Ara h 1. The allergenicity of recombinant Ara h 1 protein was evaluated using an animal model of BALB/c mice. The results showed that recombinant Ara h 1 protein had a similar allergenicity to the native one. The contents of specific antibody, Th2 type cytokines and histamine in the serum were increased, and in?ammation occurred in the lung and jejunum of mice sensitized to recombinant Ara h 1. Meanwhile, recombinant Ara h 1 could trigger degranulation of RBL cell, resulting in release of β-hexosaminidase.Accordingly, recombinant Ara h 1 could cause Th2 type allergic reaction.