鼠IL-35真核表达载体的构建及在Hepa1-6细胞中的表达

Construction of the Eukaryotic Expression Vector Containing Mouse Interleukin-35 Gene andExpression in Hepa1-6 Cells

目的 构建小鼠IL-35的真核表达载体并使其在小鼠肝癌细胞株Hepa1-6中表达.方法 采用PCR方法从含有小鼠IL-35基因的pET-30a-mIL-35中扩增目的 基因片段,将其克隆到真核表达载体pcDNA3.1/V5-HisB中,经菌落PCR、双酶切鉴定及测序正确后,用脂质体转染方法转染到Hepa1-6细胞中,48h后收集细胞,SDS-PAGE和Western Blotting检测目的 蛋白的表达.结果 经双酶切和测序鉴定证实重组表达载体构建成功,转染入Hepa1-6细胞经SDS-PAGE电泳和Western Blotting证明均得到与预期相对分子量大小相符的蛋白条带.结论 成功构建小鼠IL-35真核表达载体并在Hepa1-6细胞中表达,为进一步研究mIL-35的生物学功能提供了条件.

Objective To construct eukaryotic expression vector of mouse interleukin-35 gene and observe its expression in mouse hepatoma cells Hepa1-6. Methods The gene containing mouse mIL-35 was amplified by PCR from the pET30a-IL-35 and cloned into the eukaryotic expression vector pcDNA3, 1/V5-HisB. The recombinant plasmid was transfected into Hepal-6 cells by the lipofeetion method. The cells were collected after 48h. The expression of the target molecule was identified with SDS-PAGE and Western Blotting. Results The recombinant expression vector was identified and confirmed by digestion of restriction enzyme and DNA sequencing. SDS-PAGE electrophoresis and Western Blotting were proved to have a size protein bands consistent with the expected molecular weigtht. Conclusion Mouse raiL-35 can expresse in Hepal-6 cells. It provides the conditions for the further study of roIL-35 in tumor cells.