Prokaryotic Expression Of PRAK And Monoclonal Antibody Preparation

Objective Prokaryotic expression of PRAK(p38 regulated/activated protein kinase)recombinant protein,the application of hybridoma technology to prepare PRAK monoclonal antibodies,laying the experimental foundation for its follow-up research.Methods The p ET28a-PRAK prokaryotic high expression plasmid was constructed and transformed into E.coli BL21(DE3).By inducing expression at 16°C and1mmol/L isopropylthiogalactoside(IPTG)for 9-12 hours,the induced recombinant protein was purified with a nickel affinity chromatography column,and then analyzed by SDS-PAGE Western blot method to identify protein expression and its solubility.The purified recombinant protein was used as the antigen to immunize 6-week-old female Balb/c mice.After three immunizations,the serum titer of the mice was detected by indirect enzyme-linked immunosorbent assay(ELISA).The mice with the highest serum titer were injected with an adjuvant-free antigen for booster immunization three days before the cell fusion,and then the spleen cells of the boosted mice were fused with myeloma cells in the logarithmic growth phase,and cultured for 9-11 days.Indirect enzyme-linked immunosorbent assay(ELISA)screened positive hybridoma cells and subcloned 3-4 times until the positive rate reached 100%.The autoclaved liquid paraffin was intraperitoneally injected into 8-10 week-old female Balb/c mice.A week later,the positive hybridoma cell lines that were screened out in good condition were injected into the abdominal cavity of the mice,and the ascites were collected after the mice swelled..Results1.The results of double enzyme digestion and sequencing showed that the p ET28a-PRAK prokaryotic expression plasmid was successfully constructed.2.SDS-PAGE and Western blot(Western blot,WB)identification showed that the recombinant PRAK protein can be expressed as a soluble protein at16 ° C and 1m M isopropylthiogalactoside(IPTG)for 9-12 h.The form of is expressed in large quantities in the supernatant after sonication,and has a high purity.3.Enzyme-linked immunosorbent assay(ELISA)detects the serum antibody titers of mice after immunization,which shows that the prepared recombinant PRAK protein has good immunogenicity and can cause a strong immune response in mice.Conclusions In this study,the PRAK-p ET28 a prokaryotic expression plasmid was successfully constructed,and the recombinant PRAK protein with higher purity was induced to express,and three hybridoma cell lines that could be secreted stably were screened out.