Prokaryotic Expression Of Extracellular Region Of Porcine PD-1and Its Ligand PD-l1and Preliminary Analysis Of Its Activity

Programmed death1(PD-1) was originally cloned from a thymic T cell line treated withapoptosisinducing reagents. It blongs to the CD28family and its expression is induced on T cells, B cellsand activated monocytes. Upon interaction with either of its two ligands, PD-L1or PD-L2, it can inhibit thefuction of antigen-specifitic T and B cells. Blockade of the interaction between PD-1and its ligands PD-L1showed that impaired T cells are able to restore their ability to proliferate, secrete cytokines and killinfected cells. Moreover, the expression level of PD-1can to some extent reflect the progression of theinfection. Many researchers have show that the blockade of the PD-1/PD-L1pathway is a promisingtherapeutic method for persistent viral infections. However, just a little functional analysis of thesemolecules has been reported for pig diseases, mainly because of the lack of materials for study the role ofPD-1/PD-L1pathway in swine immunosuppressive. In this study, we expressed the PD-1and PD-L1protein to provide materials.The primers were designed according to the extracellular region of porcine PD-1sequence inGenBank, and the porcine PD-1or PD-L1extracellular region gene was cloned by PCR from porcinePBMC. The PCR products were subcloned to pMD18-T simple vector and transformed into DH5α, therecombinant cloning plasmid was determined by PCR and sequencing. pMD-PD1, pMD-PDL1orpET-32a(+) was digested by restriction endonucleases, the digestion production connected with each otherusing T4DNA ligase enzyme and transformed into DH5α. The recombinant plasmids were determined bysequencing in the E.coli Rosetta（DE3） and than induced with IPTG. The expression products weredetected by SDS-PAGE and Western-blot. The purified recombinant protein was inoculated on rabbit todevelopment polyclonal antibody. In order to verify the bioactivity of recombinant protein, we test thebinding effect by using polyclonal antibody.Results showed that the PD-1and PD-L1extracellular region genes were successfully amplifiedand cloned to expression vector of pET-32a(+). After identification by PCR, enzyme digestion andsequencing analysis, the pET-PD1and pET-PDL1recombinant expression vector have been constructedsuccessfully. The vectors were transformed into Rosetta (DE3) and then induced by IPTG for prokaryoticexpression. The expression products were analyzed by SDS-PAGE and western-blot. The expressionproducts of PD-1and PD-L1were specific expression in form of inclusion body with a relative molecular weight of33.0kDa and45.0kDa, respectively. Western-blot indicated that the expressed protein wasrecognized by His-tag antibody and the PD-1was also recognized by anti-human PD-1monoclonalantibody. The concentration of two proteins was0.9mg/mL and1.5mg/mL, respectively, after recyclingand His column affinity purification. After immunization on rabbit the polyclonal antibodies (pAb) of PD-1and PD-L1were successfully obtained and used to identify the reactivity to PD-1and PD-L1on PBMC byflow cytometry. The obtained pAb of rabbit anti-porcine PD-1and PD-L1can recognize the natural PD-1and PD-L1porteins on PBMC. It is suggested that the recombinant proteins of porcine PD-1and PD-L1have bioactivity，and can be used to develop the monoclonal antibody or prepared as a soluble molecule toresearch the immunosupression function of swine.