Construction And Preparation Of Helper-dependent Adenoviral Vector Expressing Human Respiratory Syncytial Virus F Gene

Object: Human Respiratory syncytial virus (RSV), the widely spread virus around theworld, is the most common etiological agent in lower respiratoy infection among thebabies and infants. Till now, there are no safe and effective vaccines to prevent RSVfrom infection. The two key proteins of fusion protein F and attachment protein G,which are all expressed on the surface of glycoprotein, play important roles in inducingneutralization antibodies, and contribute a lot to the researches on vaccines development.Compared with protein G, the protein F owns more epitopes and is well conserved with89% amino-acid identity between groups A and B, which is the most important targetantigen in vaccines researches, not only stimulates humoral immunity and cellullarimmunologic response but also elicits cross-protective immunity against differentgroup's virus. Having been used as the gene therapy vectors many years,Helper-dependent adenoviral vector (HDAd) steppes later in the vaccines researches. Todate, there are still no reports on HDAd used as gene delivery system aiming atmucosal immunity in vaccines investigation. HDAd can infect respiratory epithelia ashigh efficiently as wild adenovirus and express transgene lasting for a long time. Here,we focus on constructing an HDAd expressing group A of RSV F gene and performinglarge scale preparation, purification and identification of the vector.Methods: F gene with CMV promoter was subcloned into a shutle vector pSC11, andthen cloned into HADd plasmid pSC15B. The HDAd/F genome was liberated by removing the bacterial sequences from the resulting plasmid pSC15B/F digested withrestriction enzyme Pmeâ… , and then the linear HDAd/F DNA was transfected into293Cre4 cells with calcium phosphate transfection method. The cells were infected byhelper virus 16 hours after transfection. HDAd/F was amplified by serial coinfection of293Cre4 cells by helper virus and the crude lysates from previous passage until itreached plateau of amplification by BFU staining of parallel amplified control vectorpSC9A. HDAd/F was purified by CsCl gradient ultracentrifugation and expression of Fprotein was identified by RT-PCR and Western blot°.Result: HDAd/F expressing RSV F was successfully construced, prepared and purified.The resultant HDAd/F showed typical adenovirus morphology with a diameter about 70nm under transmission electron microscope. The concentration of vector particles in thepurified preparation is determined spectrophotometrically as: 7.4×10¹² viral particles/ml.After infecting 293 cells, the transcribed F gene and expressed F protein were detectedby RT-PCR and Western blot assay, respectively.Conclusion: The successful construction and preparation of HDAd/F sets thefoundation for the further investigation of potential immune protection in vivo andopens a new window for the RSV vaccine research.