可复制型抗乙肝免疫基因治疗剂pSVK-HBVE的构建与表达  

Construction and expression of a replicable immune agent for gene therapy of hepatitis B

作　　者：侯沙沙[1,2,3] 高燕[1] 王宇[1] 武帅[1] 朱晓明[1] 郭炳冉[2] 张巍[1] 徐元基[1] 阎瑾琦[1] 于继云[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]曲阜师范大学生命科学学院,273165 [3]北京交通大学理学院,100044

出　　处：《免疫学杂志》2014年第9期816-820,共5页Immunological Journal

基　　金：国家自然科学基金(31100655)

摘　　要：目的构建能同时表达抗体小分子靶向干扰素(dsFvα)和人白介素12(hIL-12)的可复制型抗乙肝免疫基因治疗质粒pSVK-HBVE。方法将pVAX-HBVE质粒双酶切,回收dsFvα-IRES-hIL12片段;将dsFvα-IRES-hIL12片段克隆入早期构建的pSVK载体,得到重组质粒pSVK-HBVE。将重组质粒瞬时转染到293T细胞,ELISA法检测目的基因表达;提取重组质粒pSVKHBVE,注射乙肝转基因小鼠腿部肌肉并采用电穿孔递送,定量PCR检测注射前后转基因小鼠体内HBV基因拷贝数的变化。结果pSVK-HBVE经酶切和测序分析与预期设计完全一致,显示重组质粒构建成功;ELISA检测显示dsFvα和IL-12基因在细胞上清中获得表达;注射重组质粒pSVK-HBVE 30μg仅1次即可使转基因小鼠体内的HBV基因拷贝数降低2个数量级。结论成功构建能够同时表达抗体靶向干扰素(dsFvα)和hIL-12的可复制型抗乙肝免疫基因治疗质粒,为慢性乙肝免疫基因治疗提供新的备选方案。This study is performed to construct a new replicable anti-hepatitis B therapeutic plasmid which can express both hepatitis B surface antibody targeted interferon（dsFvα） and human interleukin 12（hIL-12）, thus provide new alternatives for gene therapy aimed at chronic hepatitis B. pVAX-HBVE plasmid was digested to obtain dsFvα-IRES-hIL12 fragment, then clone the fragment into the replicative vector pSVK to construct recombinant expression plasmid pSVK-HBVE. Subsequently, the recombinant plasmid was transiently transfected into 293 T cells, and the target gene expression was detected by ELISA; the recombinant plasmid pSVK-HBVE was extracted and injected into the leg muscles of HBV transgenic mice with electroporation delivery, then the change of HBV gene copy number was detected by quantitative PCR. Data indicated that only one injection of recombinant plasmid pSVK-HBVE（30 μg） could reduce HBV gene copy number of transgenic mice by 2 orders of magnitude. In conclusion, this study successfully constructs a new replicable anti-hepatitis B therapeutic plasmid expressing both hepatitis B surface antibody targeted interferon（dsFvα） and interleukin 12（hIL-12）, which provides new alternatives for gene therapy of chronic hepatitis B.

关 键 词：乙肝 复制型 dsFv IFN-Α hIL-12 

分 类 号：R512.62[医药卫生—内科学]