Study On The Expression,purification And Immune Efficacy Of Fusion Protein HSP65‐PEAⅠ

Pseudomonas aeruginosa isa seriousconcernedopportunistic pathogens in food.In recent years, there are more and more food poisoning caused by Pseudomonas aeruginosa toxin residues.As Pseudomonas aeruginosa has multi-drug resistant, the cure for Pseudomonas aeruginosa poisoning is becoming more and more difficult.At the same time, Pseudomonas aeruginosa is also major pathogens of surface burns infection. The main causative factoris Pseudomonas exotoxin A(PEA). Single-chain protein PEA combines the receptors on cell surface by receptor binding subunits, the toxic moiety is transported to cell pass through the cell membrane, catalysing ribosylation of EF-2 and inhibiting the synthesis of protein, and ultimately the target cell dead. Heat shock protein 65(HSP65)is one of the major chaperone family, involved in protein folding, preventing the denaturation aggregation of protein, promoting denatured protein to refold, and maintaining the structure and function of cell.In this study, use the method of genetic engineering to fusion express the receptor subunit of PEA and the heat shock protein, developing an effective vaccine to fight invasion of Pseudomonas aeruginosa. The vaccine not only has high conservative and immunogenic of PEA, but also has the advantage of enhancing the immunity of heat shock protein. Lay the further foundation of research for prevention of foodborne Pseudomonas aeruginosa infection and anti-burn patients with multiple organ failure.Use genetic engineering to digest gene fragment of HSP65-PEAⅠwith Hind Ⅲenzyme and Not Ⅰ enzyme and then connected to vector pET-28 a to build recombinant plasmid pET-28a-HHP, then transferred to competent E.coli BL21(DE3)to get the expression strain E.coli BL21(DE3)(pET-28a-HHP). After inducing expression of the recombinant protein HHP, explore the purification process to get the best purification conditions, ultimately purity of HHP is 90%. Take HHP as immunogens to establish mice immunity test model, detect antibody titer levels and preliminary evaluate the immunity. Establish the best immunization program and lay the foundation of preparation for multiple organ failure vaccine by burns.PCR and double digestion identification of recombinant plasmid pET-28a-HHP was right and the sequencing results show that gene fragment of HSP65-PEAⅠwas cloned into the vector pET-28 a correctly. The relative molecular mass of recombinant protein is about 97000 Da, expressed as inclusion bodies. Optimization process of washing inclusion bodies, denaturation and refolding, the best condition was washing with 2% TritonX-100 and then 2M Urea, denaturing lysis with 8M Urea, stepwise dialysis urea to a final concentration of about 0.5 M to complete renaturation. Further purification of soluble protein HHP was that sample was uploaded to Anion exchange chromatography Q, SephadexTM G-25 Fine and Metal chelate chromatography Cu2+ stepwise, the concentration of obtained HHP was 0.5 mg / ml and the purity was more than 90%. The specific antibodies was tested in the immunized mice after injecting protein HHP a week, antibody titer levels continued to grow and in the 42 days were up to 1: 210.Among them, the titer of aluminum salt adjuvant group was the highest. Immunized animals poison attack protection experiments showed that the poison attack protection rate of aluminum salt adjuvant group was the best, for 3 × LD50 dose, toxin protection rate up to 100% and for 5 × LD50 dose, toxin protection rate up to 80%. Ultimately determine the best immunization program: 20μg of immunosuppressive agents HHP was mixed with aluminum salt adjuvant at 1: 1(v: v), antibody titers was 1: 212 49 d after immunity. Do the PEA poison attack on test mice, the mice can tolerate toxic amount of 3 × LD50 and the poison attack protection rate of 5 × LD50 was not less then 80%.In the study, the recombinant plasmid pET-28a-HHP was successful expressed protein HHP in E. coli, due to the his-tag, the purification process was simple, time-consuming was short, purity was high, and suitable for pilot scale production. The prepared anti- PEA immune agents showed strong immunogenicity, can protect the body from toxin attack, lay the foundation for treatment and prevention of food poisoning of Pseudomonas aeruginosa and further development of clinical application of anti-PEA immune agents.