HBV靶向核糖核酸酶真核表达载体的构建及其在2.2.15细胞内的表达

Construction of HBV targeted ribonuclease and its expression in 2.2.15 cell line

目的 构建人嗜酸性粒细胞来源的神经毒素(hEDN)与乙肝病毒核心蛋白 (HBVc)的融合真核表达载体(该融合蛋白即为构建的靶向核糖核酸酶 ) ,抑制乙肝病毒在细胞内的复制。方法 分别从HL 6 0细胞和 2 2 15细胞中提取总RNA ,用RT PCR特异性扩增hEDN及HBVc编码基因 ,将hEDN和HBVc克隆入真核表达载体pcDNA3 1(- ) ,hEDN克隆入原核表达载体 pGEX4T 1,并以纯化的原核表达产物免疫Balb/c小鼠 ,制备特异性抗体。用免疫荧光法检验 pcDNA3 1(- ) /HBVc -hEDN在转染 2 2 15细胞内地表达。结果 成功地构建了hEDN和HBVc的真核融合表达载体 ,并在 2 2 15细胞中较高效地表达。结论 pcDNA3 1(- ) /HBVc hEDN的构建和在 2 2 15细胞内的表达 。

Aim To construct human eosinophil derived neurotoxin(hEDN) and HBV core protein(HBVc)eukaryotic fusion expression vector, which will be used to inhibit HBV replication. Methods The total RNA were extracted from HL 60 cells and 2 2 15 cells,respectively. The gene encoding hEDN and HBVc amplified by RT PCR were cloned into eukaryotic expression vector pcDNA3 1(-). The gene encoding hEDN was cloned into pGEX4T 1 and was expressed in E coli .The purified hEDN protein was used to immunize the Balb/c mice for preparing specific antibody.After transfection in 2 2 15 cells, pcDNA3 1(-)/HBVc hEDN expression was identified by indirect immunofluorescence staining. Results hEDN and HBVc fusion eukaryotic expression vector are successfully constructed. pcDNA3 1(-)/HBVc hEDN was expressed in 2 2 15 cells efficiently. Conclusion Construction and expression of pcDNA3 1(-)/HBVc hEDN provide a new thread of thought for inhibiting HBV replication.