HBV的小鼠模型研究进展

乙型肝炎病毒感染是全球范围内影响人类健康的重要问题,目前人们对HBV及其所致疾?膊 病有了相当深入的认识.但由于缺乏合适的动物模型,乙型肝炎病毒的生物学研究和治疗进展缓慢.小鼠作为一种实验室常用的动物,遗传免疫背景清楚明确,已经成为人们研究乙肝的重要工具.本文简要综述了小鼠模型在乙型肝炎研究中的进展.     

用于大容量人源天然噬菌体抗体库的表达载体的构建及鉴定

目的构建一个应用于大容量Fab段天然噬菌体抗体库的表达载体。方法用定点突变技术将表面呈现噬菌粒载体pDF上的BssHⅡ酶切位点改为BglⅡ酶切位点,然后分别在抗体轻链和重链位置插入自杀基因SacB,构建含自杀基因的噬菌粒载体pDF-D-SacB;利用抗乙肝表面抗原抗体的基因为模板,PCR扩增重链和轻链基因片段。将PCR扩增的轻链基因和重链基因分别插入载体pDF-D-SacB内,利用电转化的方法将其转入Trans1-Blue大肠杆菌,构建2个初级质粒,再利用初级质粒超感染BSl365菌,使其轻链与重链发生重组,获得重组质粒,进一步获得抗乙肝表面抗原抗体的噬菌体。之后利用该噬菌体感染大肠杆菌Trans1-Blue进行扩增,得到大量抗乙肝表面抗原的噬菌体抗体。最后,通过酶联免疫吸附剂测定检测所获得的抗体。结果通过向pDF重轻链区域插入SacB基因,改造抗体基因克隆位点,构建了pDF-D-SacB载体;经检测,pDF-D-SacB可以表达具有功能的Fab噬菌体抗体,可以在分泌Cre蛋白酶的细菌胞内发生预期的Cre-Loxp介导的定点重组。结论所获得的含自杀基因的噬菌粒载体pDF-D-SacB适用于构建大容量噬菌体抗体库。     

基于核酶自剪切机制稳定分泌丙型肝炎病毒的细胞模型

Objective To construct a stable HCV-producing cell model for anti-HCV drug research. Methods The HCV-ribozyme recombinant plasmid pJFHl-Rbz was constructed to generate the exact 5' and 3' ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy. Results HCV core protein was detected in die screened cell clone, and the level of HCV RNA was up to 1 ×107 in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN α. Conclusions We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.     

基于核酶自剪切机制稳定分泌丙型肝炎病毒的细胞模型

Objective To construct a stable HCV-producing cell model for anti-HCV drug research. Methods The HCV-ribozyme recombinant plasmid pJFHl-Rbz was constructed to generate the exact 5' and 3' ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy. Results HCV core protein was detected in die screened cell clone, and the level of HCV RNA was up to 1 ×107 in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN α. Conclusions We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.     

基于核酶自剪切机制稳定分泌丙型肝炎病毒的细胞模型

目的 利用核酶自剪切机制建立稳定分泌HCV的细胞膜型.方法 以HCV感染性克隆的全基因组cDNA重组质粒为基础,在基因组两侧引入核酶序列,构建重组质粒pJFH1-核酶,并转染入HepG2细胞,加入G418溶液筛选整合有该质粒的细胞克隆.用荧光定量PCR、免疫荧光、Westrn blot、透射电镜等技术挑选稳定分泌HCV的单细胞克隆.结果 成功筛选到稳定分泌HCV的单细胞克隆,该细胞克隆能够有效产生HCV相关蛋白,上清液中HCV滴度达到1×107拷贝/ml,电子显微镜观察HCV直径为55 nm,上清液中病毒滴度随干扰素α浓度的升高而降低. 结论 利用核酶自剪切机制可建立稳定分泌HCV的细胞模型,该模型可用于抗HCV药物的筛选.