Screening Of Porcine DC Cell Targeting Peptides And Their Expression In Human Adenovirus System

As an excellent antigen delivery tool,adenovirus has been widely used in vector vaccine research.Although the adenovirus vector represented by HAd V-5 has many advantages in the field of animal vaccines,such as high safety,high immunogenicity,high viral production and high stability,the autoimmunity of the vector and lack of targeted immune cells are the two main factors affecting the immune efficacy of the adenovirus vector vaccine.This paper is mainly aimed at many types of adenovirus receptors,especially the defects of DC cell targeting.Screening of porcine DC cell receptor（CD205,CD209）targeting peptides by phage display peptide library,and trying to screen the porcine DC cell receptor targeting peptide expressed in fusion with adenovirus p IX,and the porcine DC cells targeting recombinant human adenovirus vector were constructed,in order to improve the safety and efficacy of the adenovirus vector vaccine.This study consists of the following three parts:1.Cloning and expression of extracellular domain of DC cell surface receptor CD205and CD209.Firstly,the nucleotide sequence of CRD-5 in the whole extracellular domain of CD205and complete extracellular domain of CD209（DC-SIGN）was synthesized.The 5th CRD fragment of CD205 and the complete CRD fragment of CD209 were amplified by PCR,and the two fragments were ligated to the p ET32a vector respectively,and the vectors p ET32a-CD205-5 and p ET32a-CD209 expression vectors were constructed.2.Screening and identification of targeting peptides in the extracellular domain of CD205 and CD209The fusion protein CD205 and CD209 were used as bait,and the phage random12-peptide library was screened.After deducting phage monoclones that only bind to Trx label proteins,The high binding short peptide obtained by phage panning was specifically identified by phage ELISA assay,DC cell immunofluorescence assay and flow cytometry assay.It was determined that the DC receptor CD209 targeting peptide was screened and named CD209A/CD209B/CD209C.3.Construction and identification of recombinant human adenovirus expressing DC targeting peptide.The receptor binding domain（RBD）of S gene of PEDV was amplified from pFPAV3-S_RBD by PCR,flanking two homologous arms at each terminus forrecombination with the PH5L backbone to construct a vector p H5L-S_RBD.On the basis of p H5L-S_RBD,the specific short peptide CD209A/CD209B/CD209C screened from the phage library was cloned into the C-terminal of p IX in the vector of p H5L-S_RBD and fused with p IX to construct the shuttle plasmid pH5L-S_RBD-p IX/CD209s which can express both S_RBD and p IX/CD209s.The vector p H5L-S_RBD and the targeting vector p H5L-S_RBD-p IX/CD209s were co-transfected into 293A cells with the genomic backbone vector p H5R for viral packaging,respectively.The packaged recombinant human adenoviruses were designated HAd V5-S_RBD（control）,HAd V5-S_RBD-p IX/CD209B and HAd V5-S_RBD-p IX/CD209C,respectively.The targeting recombinant viruses had been successfully constructed by PCR,Western blot and real-time fluorescence quantitative PCR identification.In the future,we will further verify the display of DC targeting peptide on the surface of adenovirus and the DC targeting ability of adenovirus with DC targeting peptide.S_RBD was used as antigen to verify whether the DC targeted peptide could enhance the immune effect of vecotored vaccine based on human adenovirus in pigs,and lay the foundation for later development of high-efficient human adenovirus vecotors for vaccine delivery by targeting porcine DC cells strategy.