Cell Penetrating Peptides Screening For The Design Of Bacterial Vector Vaccine

It is an attractive strategy to develop a multivalent vector vaccine by expressing exogenous protective antigen in bacteria which could induce multiple immune protection. It is fundamental to establish a safe and effective antigen delivery system for the multiple immune protection of bacterial vector vaccine. In our previous work, a recombinant Escherichia coli vector vaccine has been designed which contains a double-plasmid expression system, one controls the expression of exogenous antigen effectively in vitro and another one expresses lysis protein E, thus establishing an in v/vo-inducible lysis system. The system could release protective antigen and confer biological containment by mediating iron-regulated lysis in host. However, lots of antigen couldn’t been delivered into cells because of the behavior only depending on phagocytosis of macrophages.In the study, we aimed to express the fusion antigen of CPPs and protective antigen in recombinant E.coli vector system which could release fusion proteins in vivo, so that CPPs would deliver fusion proteins inside cells. This system could achieve the cell entry for exogenous antigen. In this work, firstly, we designed29recombinant plasmids by fusing15different CPPs with the C or N-terminus of EGFP which were constructed and transformed into E. coli strain BL21(DE3) to express CPPs-EGFP and EGFP-CPPs fusion proteins for screening CPPs. Then,13fusion proteins were purified to examine the cell uptake after incubation of macrophages J774A.1and EPC. Six efficient CPPs were screened for further work. The flgD gene, encoding the protective antigen FlgD from the fish pathogen Edwardsiella tarda EIB202, was fused with upstream of the CPPs to construct recombinant plasmids. Cytokines were tested after incubation of J774A.1with FlgD-CPPs fusion proteins. Finally, the recombinant E. coli vector vaccines carrying flgD-CPPs fusion gene were evaluated in zebrafish. Over50%of the fish vaccinated with BL21(pUTaAE, pETFTA) survived when challenging with E. tarda EIB202.In this work, we evaluated the delivery tool CPPs for the design of fish vector vaccine for the first time, which turns out to be a potential system for the application of CPPs in V. anguillarum and E. tarda in the future.