Expression And Purification Of Protein Kinase R(PKR) And Preparation Of Polyclonal Antibody And Monoclonal Antibody

Protein kinase R also known as the double-stranded RNA-activated protein kinase(PKR), is an important antiviral effector of host innate immune system and is activated by the interferon and double-stranded RNA induced by viral infection. However, many viruses are able to antagonize the antiviral effect of PKR through the function of virus-encoded proteins, for example, the influenza virus NS1 protein. In this study, we prokaryotically expressed and purified the PKR protein of human origin and prepared its polyclonal antibody with the aim to create conditions and convenience for the study of interaction between PKR and viral proteins.1. Prokaryotic expression and purification of protein kinase R(PKR) and preparation of polyclonal antibodyThe coding sequence of PKR protein was amplified by RT-PCR, and cloned into the GST tagged prokaryotic expression vector p GEX-6p-1. After sequencing validation, the positive recombinant was transformed into E. coli BL21(DE3). The recombinant protein induced with 0.5 mmol?L-1 IPTG was highly expressed and identified by the SDS-PAGE. The recombinant protein was purified on Glutathione-sepharose 4B affinity chromatography, processed to remove the GST tag with enzyme and further purified with ion-exchange and size exclusion columns on the AKTA Explorer purification system. Afterwards, the purified PKR protein was used to immunize New Zealand rabbits for preparing the polyclonal antibody reacting with PKR protein. The titer of the rabbit antiserum against PKR was determined by dot blot hybridization with a value of over 1:50000 and its specificity were validated by western blot and indirect immunofluorescence assay(IFA).2.Preparation of the anti-PKR monoclonal antibodyThe purified PKR protein was uesed as immunogen inoculate to SPF female BALB/C mice at age about 8 weeks. Need 4 times of inoculation, 3 days after 4th of inoculation,spleen cells were isolated from immune mice by aseptic measures, and fused with the logarithmic phase of SP2/0 myeloma cells. Positive fused cell were detected with indirect ELIAE method,and via messures of limited dilution method for 4 times to attain hybridoma cell lines that permanently produce monoclonal antibody specific PKR. Ultimately achieve sustained secrete 3 lines of monoclonal hybridoma cell and named 2E8, 4A7, 5A4. Respectively detect the valence of antibody for their culture supernatant were 1:3200, 1:3200 and 1:6400. Enormous monoclonal antibodies were produced from ascites of SPF BALB/C mice about 10 weeks immuned with hybridoma 5A4 which had the highest valence, after 7 days of immuned to collecte ascites. Determination of ascites valence of 1:512000 and further purified with affinity chromatography. Analyzing the monoclonal antibody subtype demonstrated that all of the attained monoclonal antibodies were belonged to Ig G1 subtype and its specificity for PKR were validated by indirect immunofluorescence assay(IFA). Undergoing many passages of 3 hybridoma cell lines and reviving of those stored cells could substainable produce monoclonal antibody, which was validated via indirect ELISA assay for antibody titer of their culture supernatant. In conclusion, monoclonal antibody specitic for PKR was prepared successfully.