Display Of CSF Virus E2 Antigen On The Surface Of Bacteriophage T4 And Study On Its Immunogenicity

The T4 phage display is a newly developed system for displaying of exogenous peptides or protein domains by fusion expression on the T4 surface. In this system the displayed proteins maintain their relatively independent conformation and biological activities. This process is initiated by fusing a foreign gene to the T4 surface capsid accessory protein gene soc (small outer capsid protein) or hoc (highly antigenic outer capsid protein).In the present study, two forms of protective antigenic E2 gene of classical swine fever virus(CSFV), mE2 (encoding 132aa most antigenic peptide at N termianl of E2 ) and E2 (encoding entire 328aa E2 protein without transmembrane domain) genes were separately fused in frame to 3' end of soc in display vector. The soc/mE2 and soc/E2 fusion genes were then respectively integrated into the soc site of the genome of soc-deleted T4 phage mutant by homologous recombination leading to the display of mE2 or E2 protein on the surface of T4 phage.In addition, the E2 gene was also fused in frame to the 5'end of hoc gene in the plasmid vector which resulted in expression of a soluble E2/Hoc fusion protein in E.coli after transformation and IPTG induction. Subsequent infection of the E2/Hoc expression E.coli with the T4 phage, which has amber mutation of hoc gene and already displays mE2 in fusion with SOC on its surface, lead to the surface display of E2 in fusion with HOC during the assembly of the progeny viral particles. This study showed the first trial in construction of the chimeric T4 phage spontaneously displaying two distinct proteins separately on Soc and Hoc sites on its capsid surface. The reactivity with a panel of CSFV-specific polyclonal antisera in an ELISA assay demonstrated that two bound peptides were displayed in a form accessible for antibodies. In western blot the correct bands corresponding to the molecular weights of fusedSOC/mE2 and E2/HOC proteins were visualized and the electromicroscopy applying immunogold staining showed the displayed proteins were located on the surface of T4 phage particles.Inoculation of mice with those chimera displaying foreign proteins either singly in SOC or doubly in SOC and HOC showed a significant immune response as indicated in ELISA using intact CSF virus or E.coli expressed E2 protein as antigens. The detectable antibody level persisted for 56 days of a complete immunization trial. The antibody titer elicited by chimeric soc/E2 T4 phage was higher than that elicited by chimeric soc/mE2 T4 phage indicating the display of full E2 is more antigenic than partial E2. Immune response elicited by directly using the concentrated chimeric phage culture was stronger than that elicited by using E.Coli expressed E2 protein. In the study the cellular immune response of mice against CSFV E2 was also detected after inoculation with chimeric phage. All experiments in the study indicated that the phage T4 display system is an attractive tool to display exogenous proteins or peptides on its capsid surface and may emerge as a powerful system for engineering of multicomponent vaccines.