Cloning And Expression Of Extracellular Region Gene Of Chicken PD-1and Its Ligand PD-l1and Preparation Of Their Polyclonal Antibodies

PD-1（Programmed cell death1）is one of immunoglobulin superfamily, which is discovered in thestudy of the hybridoma cell apoptosis regulation. Ahmed and his colleague confirmed that PD-1is therejection capability receptor on the surface of T cell in the model of LCMV chronic infection on the mouse.PD-1and PD-Ls mediated signaling pathways that play an important regulating role in T cell immuneinhibitory signals activation, T cell proliferation and balance process of immunity mediated tissue damage.There are two types for PD-1’s ligand which are PD-L1and PD-L2, respectively. Interdicting PD-1and itsligand binding can make CD8+T cell which is dysfunction and function failure when virus persistentlyinfect recovery function. PD-1and its ligand PD-L1become a hot focus in recent years in theimmunosuppression disease study of chronic persistent infection, immune tolerance disease, tumor etc. asPD-1:PD-L1pathways pass immune tolerance and immunosuppression signals in the central immune andperipheral immune response. At present, they have confirmed that chicken T cell surface exists PD-1molecules, but the role of the biological functions and the related research for PD-1and PD-L1moleculesin chickens and other poultry immune inhibitory disease caused by virus infection or virus persistentinfection diseases has just started. This study took chicken PD-1and its ligand PD-L1extracellular regionas the research object. The activity chicken PD-1and PD-L1extracellular region prokaryotic expressionrecombinant proteins were obtained by cloning, expression and purification technology etc. It is expectedthat the gene expression product can be used to make the specificity monoclonal antibody. This study laiedthe research foundation to explore the biological function for the PD-1and PD-L1in poultry immuneinhibitory related diseases.In order to amplify PD-1and PD-L1extracellular region gene by PCR, we designed their extracellularregion primers according to PD-1and PD-L1gene in GenBank by bioinformatics comparation ofmulti-clone restriction enzyme sites of prokaryotic expression vector. The pET-28a-PD-1andpET-32a-PD-L1recombinant expression plasmids were constructed respectively. The recombinantexpression vectors were induced in Rosetta(DE3) after identified by restriction enzymes and sequenced. Itwas used to analyse expression status and expression form of recombination proteins and identifyantigen-specific of recombination protein by SDS-PAGE and Western-blot. The purified products ofrecombination proteins were obtained by recycling and His column affinity, meanwhile The purified products were used to immunize rabbit for preparaton of anti-serum. The titer and immunological activityof anti-serum were identified respectively by ELISA and Western-blot.Results showed that the extracellular region target genes of PD-1and PD-L1were successfulyamplified and cloned to expression vector of pET-28a(+) and pET32a(+). After identification by PCR,enzyme digestion and sequencing analysis, the pET-28a-PD-1, pET-32a-PD-L1recombinant expressionvector were constructed successfully. The vectors were transformed into Rosetta (DE3) and induced byIPTG. The expression products were analyzed by SDS-PAGE. SDS-PAGE showed that we had expressedtarget proteins respectively with a relative molecular weight of20.0kDa and44.0kDa which were thesame with prospective proteins.Western-blot indicated that the recombinant proteins of PD-1and PD-L1extracelluar region were identified respectively by His monoclonal antibody, meanwhile both of therecombinant proteins had good reactionogenicity. Two proteins concentration were0.8mg/mL and1.5mg/mL respectively by purifying recombination proteins of PD-1and PD-L1. The titer and immunologicalactivity of anti-serum were identified respectively by ELISA and Western-blot It will lay a foundation forpreparation of monoclonal antibody for blocking PD-1/PD-L1signal pathway and studying the functionand effect in chicken immunerepress diseases.