柔嫩艾美耳球虫第二代裂殖子表面抗原基因(λMZ5-7)在大肠杆菌中的表达

Expression of Surface Antien λMZ5-7 Gene From the Second Generation Merozoite of Eimeria tenella

将重组克隆质粒 (PGEM -λMZ)用EcoRI酶切后 ,电泳回收目的片段 ,克隆到经EcoRI酶切、CIAP处理的表达载体 pET - 2 8a中 ,转化大肠杆菌JM 10 9感受态细胞 ,得到的转化子经PCR鉴定和酶切分析 ,筛选出符合正确阅读框的重组子 ,构建成重组表达质粒 (PET -λMZ) ,并在大肠杆菌BL2 1(DE3)表达菌中成功地表达了含目的蛋白的融合蛋白 ,融合蛋白的分子量约 34KDa ,加入IPTG诱导 6h后 ,蛋白表达接近最高水平。表达产物经Ni-NTA亲和层析柱纯化 ,SDS -PAGE检测为单一条带。经Western -blotting杂交实验 ,纯化出的目的蛋白能与兔抗E .tenella第二代裂殖子抗血清发生反应 ,说明MZP蛋白是E .tenella第二代裂殖子抗原蛋白 ,具有一定的免疫原性 。

The EcoRI fragment encoding the merozoite λMZ5-7 gene was excised from the positive clone PGEM-λMZ and purified by agrose gel fraction method.Then the fragment was subcloned into the PET-28a expressing vector digested by EcoR I restriction enzyme and calf intestinal alkaline phosphatase. The recombinant expressing plasmids (PET-λMZ)were identified by PCR and restriction enzymes analysis. The results indicated the fragment was correctly inserted into the PET-28a expressing vector and conformed to a reading frame. The fusion protein was expressed in E.coli BL21(DE3) host with a predicted molecular weight of 34KDa. Peaks of target protein was achieved at 6h after adding IPTG. The target protein purified by Ni-NTA metal chelate affinity preented one major band in the SDS-PAGE and reacted with rabbit serum raised against the second generation merozoites of Eimeria tanalla in westen blotting ,suggested that the protein could be used as one of candidated antigenes. It provided the basis for further studying recombinant vaccine against coccidiosis.