有linker插入的靶向核糖核酸酶抑制乙肝病毒复制  

Optimizing HBV targeted ribonuclease by introduction of linker

作　　者：宫卫东[1] 刘军[1] 丁劲[1] 赵亚[1] 李英辉[1] 薛采芳[1] 

机构地区：[1]第四军医大学基础部病原生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2003年第21期1956-1959,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金 (30 1 0 0 1 57)

摘　　要：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)之间插入有linker(Gly4Ser) 3 的融合真核表达载体 (该融合蛋白即为靶向核糖核酸酶 ) ,以优化分子折叠 ,并将其应用于抑制HBV复制的体外研究 .方法 :以第四军医大学病原生物学教研室已构建的pcDNA3.1 (- ) /TR为基础 .首先合成linker序列 ,经过退火形成双链并克隆入 pcDNA3.1 (- ) /TR生成pcDNA3.1 (- ) /HBc linker质粒 ;随后hEDN片段经PCR扩增后克隆入 pcDNA3.1 (- ) /HBc linker质粒生成 pcDNA3.1 (- ) /TNL质粒 ,其中效应分子 (hEDN)与靶向分子 (HBVc)之间为linker序列 .应用间接免疫荧光法检测 pcDNA3.1 (- ) /TNL在细胞的表达 ,并应用放免法测定其抗HBV活性 .同时 ,应用MTT比色法检测细胞的代谢活性 .结果 :成功构建了有linker (Gly4Ser) 3 介入的hEDN和HBVc真核融合表达载体 ;并在HepG2 .2 .1 5细胞中得到有效表达 .转染质粒 pcDNA3.1(- ) /TNL与 pcDNA3.1 (- ) /TR相比可明显地降低HepG2 .2 .1 5细胞上清中HBsAg的含量 (P <0 .0 5 ) .MTT比色分析表明细胞代谢活性未受到影响 (P >0 .0 5 ) .结论AIM: To construct human eosinophil derived neurotoxin (hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly 4Ser) 3 inserted to optimize the molecular folding, which was used to inhibit HBV replication in vitro . METHODS: Previously constructed pcDNA3.1(-)/TR was used as the template. Firstly linker sequence was synthesized and annealed to form ds linker, which was cloned into pcDNA3.1(-)/TR to produce plasmid pcDNA3.1(-)/HBc linker. Then the hEDN fragment was PCR amplified and inserted into pc DNA3.1 (-)/HBc linker to form pcDNA3.1(-)/TNL in which the effector molecule and the targeting molecule were separated by a linker sequence. pcDNA3.1(-)/TNL expression was identified by indirect immunoflurescence staining. Radioimmunoassay was used to analyse anti HBV activity of pcDNA3.1(-)/TNL. Meanwhile, metabolism of cells was evaluated by MTT colorimetry. RESULTS: hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly 4Ser) 3 inserted was successfully constructed. pcDNA 3.1(-)/TNL was expressed in HepG2.2.15 cells efficiently. Compared to pcDNA 3.1(-)/TR, a significant decrease of HBsAg concentration from pcDNA3.1(-)/TNL transfectant was observed ( P <0.05). MTT assay suggested that there were no significant differences between groups ( P >0.05). CONCLUSION: Linker introduction may enhance the inhibition effect of HBV targeted ribonuclease significantly.

关 键 词：乙肝病毒 LINKER 核衣壳导向的病毒灭活 靶向核糖核酸酶 抑制效应 

分 类 号：R373.21[医药卫生—病原生物学]