用无细胞蛋白合成系统重组表达恶性疟原虫蛋白的研究

Expression of recombinant Plasmodium falciparum protein by using cell-free protein expression system

目的探索无细胞麦芽体外蛋白合成系统重组表达恶性疟原虫蛋白可行性,并检测用重组蛋白制备的免疫血清对原虫蛋白的特异性反应。方法将目的蛋白PfRON2的部分片断克隆连接到重组表达载体后,用无细胞麦芽体外蛋白合成系统进行重组表达并纯化,用纯化的重组蛋白免疫小鼠制备免疫血清,并用免疫斑点实验(Western blot)和免疫荧光抗体实验(IFA)检测该血清对恶性疟蛋白的特异性反应。结果成功克隆的重组质粒能在无细胞麦芽体外蛋白合成系统中生成大小相符的GST融合蛋白,并能较好地纯化。免疫斑点实验中,用该重组蛋白制备的免疫血清能检测到重组蛋白和恶性疟目标蛋白,免疫荧光抗体实验显示该免疫血清能成功地标记恶性疟目标蛋白所在部位。结论无细胞麦芽体外蛋白合成系统可应用于恶性疟原虫蛋白的重组表达,获得的免疫血清能特异性识别疟原虫蛋白。

To explore the novel apporach for recombinant Plamodium falciparum protein expression, the fragement of a new P. falciparum gene PfRON2 was cloned into vector pEU-GSTPre and expressed using cell-free wheatgerm expression system, the GST fusion recombinant protein was produced as expected size. Subsequently, the anitserum was raised by mouse immunization using the purified recombinant protein. Western blot and Immunofluorescence assay （IFA） were performed to evaluate the specific reaction of the antiserum against native protein of parasites. The antiserm could detect a 260 kDa band （similar to the putative target protein） from parasite extract, and reacted on the apical end of merozoites. It suggests that the cell-free wheatgerm expression system can be applied for P. falciparum protein expression, and the recombinant protein can be purified and used for further reseach purpose.