Construction And Expression Of Recombinant Adenovirus Containing RSV F Gene

Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract illness (LRI) in infants and children worldwide. And it's also an important pathogen in the elderly and immunodeficiency adults. Although the importance of RSV as a respiratory pathogen has been recognized for long times, there is no effective treatment modality.So the development of RSV vaccine is an urgent need.Fusion protein is an important antigen of RSV. The F protein is highly conserved between the RSV subgroups A and B.There are two important antigenic epitopes in aa190-289.This protein fragment can be recognized by most of F specific neutralizing monoclonal antibodies.Conformational requirements of neutralizing epitopes have been described within this aa sequence, and provided the rationale for eukaryotic expression.In recent years,Adenovirus vector that can induce mucosal immunity has received increasing attention for the expression of foreign proteins , gene-engineered vaccine and gene therapy. In this study, RSV A2 strain was cultured in Vero cells. Firstly, the total RNA of virus was extracted. According to the sequence of RSV F gene in Gene-Bank, designed and synthesized a pair of primers with Kpn I and Hind III restriction endonuclease site in 5'end respectively.In order to eukaryotic expression, the Kozak initiation sequence and stop codon were used.Secondly,the F gene fragment (559-867 bases) was amplified by RT-PCR. Inserted the DNA fragment into pGM-T Easy vector and identified recombinant plasmids by restriction endonuclease, PCR and sequencing. Then, with a series of methods of molecular biology ,this heterogenous gene was cloned into the shuttle vector and named pShuttle-CMV/F. Linearized pShuttle-CMV/F and pShuttle-CMV-lacZ by Pmel. Transformed them into BJ5183-AD-1 electroporation competent cells pretransformed with the pAdEasy-1 plasmid respectively by using electroporate method. Identified homologous recombinant plasmids by restriction endonuclease, PCR and named pAdEasy/F and...