含弓形虫复合抗原基因的真核表达质粒在哺乳动物细胞中的表达

EXPRESSION OF TOXOPLASMA RECOMBINANT PLASMID IN MAMMALIAN CELLS

目的研究弓形虫复合抗原真核表达质粒pcDNA3.1-P30-P22-CTXA2/B在哺乳动物细胞中的表达情况。方法利用脂质体介导的转染技术,将真核表达质粒pcDNA3.1-P30-P22-CTXA2/B和空载体pcDNA3.1分别转染He-la细胞,400μg/mlG418加压筛选和200μg/mlG418维持筛选,获得稳定转染的Hela细胞。采用SDS-PAGE和West-ern-blot方法对复合基因P30-P22-CTXA2/B的表达产物进行鉴定。结果SDS-PAGE结果显示,重组质粒转染Hela细胞后的表达产物分子质量单位为64ku,Western-blot显示此蛋白条带能被抗P30抗体识别。结论构建的真核表达质粒pcDNA3.1-P30-P22-CTXA2/B能在哺乳动物细胞中成功表达插入基因所编码的融合蛋白,为进一步动物实验提供了实验依据。

Objective To study the eukaryotic expression of the recombinant plasmid pcDNA3.1-P30-P22-CTXA2/B in mammalian cells. Methods The eukaryotic expression plasmids pcDNA3. 1-P30-P22-CTXA2/B and pcDNA3. 1 were transfected into Hela cells respectively with liposome according to the manufacturers protocol. In order to generate stable transfectants, after a 48 h incubation period in regular Dulbecco＇s Modified Eagle＇s Medium （DMEM） supplemented with 10% heat inactivated fetal calf serum, cells were selected by neomycin （400μg/ml of G418） and maintained with neomycin （200 μg/ml of G418）. G418-resistant colonies, which became visible after 2 weeks, were isolated and screened for expression of the vector encoding protein. The control transfections were performed with the pcDNA3.1 vector without recombinant. The expression product of the insertional genes were analysed by SDS-PAGE and Western-blot. The cells transfected by pcDNA3.1 were used as the negative control, Results The expression plasmid showed one protein band at 64 ku site compared with the negative control by SDS-PAGE. The result of Western-blot proved that the compound protein can be identified by P30 antibody. Conclusion The eukayotic expression plasmid pcDNA3.1-P30-P22-CTXA2/B can express fusion protein product which were encoded by insertional gene P30-P22-CTXA2/B in mammalian cells.