Expression, Purification Of Ad-5 Knob Protein And Preparation Of Its Monoclonal Antibody

Replication-deficient adenovirus type 5 (Ad5) is an efficient and versatile gene delivery vector that has been widely used for a variety of gene therapy applications in vitro and in vivo. Ad5 efficiently infects a broad range of target cells, including dividing and quiescent cells. The Ad5 cellular entry mechanism is composed of two separate and uncoupled events. First, the virus binds to the host cell through a high-affinity interaction between the trimeric carboxy-terminal knob domain of the viral fiber proteins and CAR displayed on the cell surface. This primary interaction, which dictates the infectivity of the virus, is followed by the association of RGD sequences in the penton base with integrins on the cell surface, thereby activating internalization of the virus.We constructed an efficient prokaryotic expression plamid encoding knob protein and the recombinant Ad5-knob protein mainly existed as a soluble protein. After one step purification with Ni2+-NTA affinity chromatography, the protein was purified to nearly homogeneity.We also express and purify car protein.The data show that the two proteins have obvious combinability.The expression, purification of knob and car protein lay foundation for further study on the adenovirus retargeting.