Clone And Expression Of Porcine CD163 Gene And Preparation Of Its Specific Antibodies

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most seriously pathogens for the swine industry. Macrophages, particularly Porcine Alveolar Macrophages (PAMs), are considered to be the primary targets for viral replication. The entry of PRRSV into its host cell is the first crucial step in infection and its interaction with recepter has the potential for altering both innate and adaptive immune response of pigs. Scavenger receptor CD163 is a type I transmembrane glycoprotein mainly expressed on cells of the monocyte / macrophage lineage. It has been recently reported that non-permissive cells transfected CD163 allowed replication of both type I and II PRRSV, indicating the crucial role of this molecule acted as a mediator of uncoating. During PRRSV infection of PAMs, the viral genome is released from the early endosome into the cytoplasm of the host cell depending on scavenger receptor CD163. The role of CD163 in genome release may require its interaction with the GP2 and GP4 glycoproteins and relies on a functional CD163 SRCR domain 5, while deletion of the cytoplasmic domain of CD163 enhances PRRSV replication.Here we constructed prokaryotic expression vector for porcine CD163 SRCR4-5 domain, and induced protein expression in E. coli, and then immunized New Zealand white rabbits and BALB/c mice with it. Finally, we prepared and indentified polyclonal antibodies and monoclonal antibodies specific for porcine CD163, providing a basis for investigating the function and interaction mechanism with PRRSV during the infection of PRRSV. Our results are as follows in detail:1.We constructed the prokaryotic expression vector pGEX-6P-CD163, and induced and expressed it in E. coli BL21 using IPTG. SDS-PAGE and Western-blot analysis showed that we got the fusion protein of right size.2.We purified the porcine GST-CD163 fusion protein by cutting gels of right size. After repeated freezing and thawing, the fusion protein was used as immunogen to immune New Zealand white rabbits. Polyclonal antibodies specifically against porcine CD163 were prepared and identified with FACS and antibody blocking assay.3.We immuned BALB/c mice with the fusion protein. Antigen-stimulated spleen B lymphoblast were fused with the mouse myeloma cell line Sp2/0 using 50% PEG4000 after three regular immune and a boost immune. Then we developed indirect ELISA and Western Blot for testing the cells survived after fusion. Finally, using limited dilution, we got a line of positive hybridoma cells secreting antibodies against GST tag, 3E2F2. The positive hybridoma cells against CD163 is in further screening.In summary, we got polyclonal antibodies for porcine CD163 and a line of hybridoma cells that can stably secrete monoclonal antibodies against GST tag. A line of hybridoma cells secreting CD163 monoclonal antibodies would provide foundation for further investigation on the biological function of the recepter CD163 in PRRSV infection.