摘要
目的 观察HBV靶向核糖核酸酶 (targetedribonuclease,TR)的体外抗病毒作用。方法 将包含HBV核心蛋白 (HBVc)和人类嗜酸性粒细胞来源的神经毒素 (hEDN)编码基因克隆入pcDNA3.1(- ) ,构建HBVTR重组真核表达载体p TR。构建包含HBV核心蛋白、人类嗜酸性粒细胞来源的神经毒素以及突变失活的HBVTR编码基因的重组真核表达载体p HBVc、p hEDN、p TRmut (hEDN的Lys113→Arg ,失去其RNase活性 )作为对照。脂质体转染p TR、p HBVc、p hEDN、p Trmut、pcDNA3.1(- )进入 2 .2 .15细胞。转染后 ,RT PCR证实转染基因的表达 ,通过观察转染细胞的形态核MTT法检测转基因表达的细胞毒性。用固相放免法测定转染细胞培养上清HBsAg浓度。结果 转基因在2 .2 .15细胞内都有表达 ,且对细胞无毒性。与空转染组相比 ,pcDNA3.1(- ) TR转染组HBsAg含量下降 5 8% ;对照各质粒无此变化。结论 TR在体外可以抑制HBV复制 ,同时对宿主细胞无毒性。TR可以作为一种新型的抗病毒制剂治疗乙肝病毒感染。
Objective To study the effect of a novel targeted ribonuclease (TR) on the HBV replication in vitro. Methods The gene encoding the targeted ribonuclease was cloned into pcDNA3.1(-) to form a recombinant eukaryotic expression vector p/TR. Control plasmids, including p/hEDN, p/HBVc, p/TRmut in which a Lys113→Arg mutation was introduced by sequential PCR to inactivate ribonuclease activity of hEDN, were also constructed. Liposome-mediated transfection of 2.2.15 cells by p/TR, p/TRmut, p/hEDN, p/HBVc, pcDNA3.1(-), or mock transfected was performed. After transfection, RT-PCR was done to verify transgenes expression. Morphology of the transfected cells was observed in microscope and MTT assay was performed to detect the cytotoxicity of transgenes expression. Concentration of HBsAg in the supernatants of the transfected cells was measured with solid-phase radioimmunoassay. Results Transgenes were expressed in 2.2.15 cells. The expression of transgenes had on cytotoxicity on the 2.2.15 cells. Transfection of 2.2.15 cells by p/TR reduced the HBsAg concentration by 58% while all control transfections had no such an effect. Conclusion The targeted ribonuclease can inhibit HBV replication in vitro while has no cytotoxicity to host cells. The targeted ribonuclease may be a candidate of novel antiviral agent against human HBV infection.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2003年第6期484-488,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目 ( 3 0 10 0 15 7)
全军医药卫生科研基金 ( 0 1MA184)
第四军医大学创新工程项目 (CX990 0 5 )
