A Preliminary Study On The Immunological Methods Targeted To Clec8A And XCR1 Receptors Of Pig Dendritic Cells

Dendritic cells(DCs)are professional antigen presenting cells that act as a bridge between innate and adaptive immune responses.Clec8 A is a kind of C-type lectin receptor expressed by mature or immature dendritic cell and plays an important role in recognition and capture of antigen.Antigen binding to Clec8 A receptor can promote DCs maturation,and stimulate T lymphocyte proliferation and promote the production of pathogen-specific antibodies.XCR1 is the corresponding receptor of type C chemokine.Antigen targeting to DCs by antibody or ligand of surface receptors can enhance humoral and/or cellular immunity against specific pathogen.Therefore,this study aimed to develop the single domain antibodies of Clec8 A and XCR1 receptors,and explored the potential method for the antigen targeting to DC by antibody or ligands of DC surface receptors.These researches will make a good foundation for the study of antigen-targeted vaccines against infectious disease of animals.Single-domain antibody library of Clec8 A and XCR1 were prepared previously by immunizing camel with ectodomain of Clec8 A and XCR1 expressed in E.coli.Biotinylated ectodomain were used to screen this library for single domain antibodies against DC surface receptors Clec8 A and XCR1.The yeast cells expressing the positive single domain antibody were enriched by magnetic bead absorption and flow cytometry staining.The proportion of positive cells of Clec8 A increased from 2.19% to 30.82%,and the proportion of XCR1 positive yeast cells increased from 0.49% to 17.19% after three rounds enrichment.One positive yeast cell clone expressing single domain antibody of Clec8 A was screened out from 40 clones after three round enrichment,We identified this clone by flow cytometry staining and sequenced the coding sequence of it.Through this study,we established the methods of screening single domain antibody yeast library by immunomagnetic beads and flow cytometry in our laboratory,which laid the foundation for the follow-up study.In this study,pig XCL1 was fused with a multi-epitope protein of type A FMDV to explore its immune effect.First,we designed an antigen A10 containing three major strains of type A FMDV.The XCL1 and A10 are linked together by peptide linker,then we obtained XA10 protein.In addition,two cysteines in XCL1 were mutated and fused with A10 to obtain DXA10 protein.Three recombinant proteins were expressed in E.coli and purified to emulsified six kinds of vaccines agonist for pig immunization.The protection rate of XA10 alone group was 80%(4/5),groups vaccinated with A10,DXA10 with or without addition of CpG were all completely protected(5/5)after challenge.The results showed that the antigen fusion with porcine XCL1 chemokine can enhance the cellular immune response.