可表达人呼吸道合胞病毒融合蛋白辅助病毒依赖型腺病毒载体的构建与制备

Construction and preparation of helper-dependent adenoviral vector expressing human respiratory syncytial virus F gene

摘要:

构建可表达A亚型人呼吸道合胞病毒（human respiratory syncytial virus, RSV) 融合蛋白（fusion protein, F）的辅助病毒依赖型腺病毒载体(helper-dependent adenoviral vector，HDAd)，并完成大量制备、纯化和F蛋白的体外表达鉴定。将带有CMV启动子序列的F基因亚克隆至克隆载体pSC11，鉴定正确后，克隆至HDAd质粒pSC15B，构建pSC15B/F HDAd重组质粒，PmeⅠ消化pSC15B/F去除原核复制元件及抗性基因，获得HDAd/F DNA分子，经磷酸钙共沉淀法转染293Cre4细胞，16h后感染辅助病毒，收获HDAd/F载体粗提液，随后以HDAd/F粗提液及辅助病毒连续共感染293Cre4细胞直至HDAd/F达到复制极限，同时以可表达β-半乳糖苷酶（β-galactosidase, LacZ）报告基因的HDAdLacZ载体作为平行对照监测载体复制过程。与辅助病毒共感染293Cre4细胞进一步扩增HDAd/F、CsCl梯度法超速离心制备大量纯化的HDAd/F载体，体外感染293细胞，RT-PCR检测到F基因有转录，Western blot分析表明F蛋白有特异性表达。总之，成功构建HDAd/F载体并在真核细胞中实现表达，为体内免疫学效力试验奠定基础，为研制RSV疫苗提供了一种新方法。

Abstract:

To construct a helper-dependent adenoviral vector expressing human respiratory syncytial virus (RSV) subgroup A F gene, and finish large scale preparation, purification and identification of the vector. F gene under the control of CMV promoter was subcloned into a shutle vector pSC11, and then cloned into HADd plasmid pSC15B. The HDAd/F genome was liberated by removing the bacterial sequences from the resulting plasmid pSC15B/F digested with restriction enzyme PmeⅠ, and then the linear HDAd/F DNA was transfected into 293Cre4 cells with calcium phosphate transfection method. The cells were infected by helper virus 16 hours after transfection. HDAd/F was amplified by serial coinfection of 293Cre4 cells by helper virus and the crude lysates from previous passage until it reached plateau of amplification by BFU staining of parallel amplified control vector pSC9A. HDAd/F was purified by CsCl gradient ultracentrifugation and expression of F protein was identified by RT-PCR and Western blot. HDAd/F was constructed, purified successfully. The expression of F protein was detected. The successful construction and preparation of HDAd/F is the foundation for the further investigation of potential immune protection in vivo and opens a new window for the RSV vaccine research.