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

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.