The Purification Of Natural F1 And Recombinant Rv270 Antigen Of Yersinia Pestis And Evaluation Of Their Protective Efficacy Against Plague In Monkey

The Purification of natural F1 and recombinant rV270 antigen of Yersinia pestis and evaluation of their protective efficacy against plague in monkeyPurpose: Plague is a zoonotic and deadly infectious diseases caused by the Yersinia pestis, it is contagious,high mortality and universal human susceptibility, untreated bubonic plague mortality was 50% to 70% while the incidence of pneumonic plague rapidly and can spread among people, if not treated, mortality can reach 100%. Vaccine is the most effective way to block the spread of the disease, so in order to protect the lives of the people better, to develop a safe and effective vaccine is the focus of plague prevention and control. This experiment has been trying to optimize of purification of two proteins F1 and V based on the laboratory work,and to find the law of monkeys in the protection process of plague subunit vaccine and the feasibility to be an animal model the of plague vaccine.Methods: In this work, a new purification strategy that produced high-purity F1 antigen from Y. pestis EV76 was developed by the substitution of physical disruption for organic solvent one, followed by a combination of ammonium sulfate fractionation and Sephacryl S-200HR column filtration chromatography. To reduce immunosuppressive properties of LcrV antigen and retain its immuno-protection activities, rV270, a variant lacking amino acids 271 to 326 of LcrV, was cloned into pET32a vector, expressed as a His-tag fusion protein in Escherichia coli BL21, digested with Enterokinase to cut the peptide of His-tag and purified by a combination of Co2+ affinity chromatography and Sephacryl S-200HR gel filtration. Groups of monkey were immunized with F1 antigen and rV270 antigen adsorbed to 25% (v/v) Alhydrogel in phosphate-buffered saline (PBS). After primary immunization, animals were boosted with the similar dose at the same injection site on day 21. The animals from the immunized and control groups were challenged s.c. with 106 CFU of Y. pestis strain 141 at ten weeks after the primary immunization, and then closely observed for 14 days.Results: In this experiment the natural F1 antigen was purified from the Yersinia pestis EV76, and a label-free and no-immunosuppressive activity rV270 antigen was obtain with enterokinase digestion,N-terminal sequencing, mass spectrometry peptide map is used to identify the nature and purity of the two protein. Prepared subunit vaccine with the two kinds of antigen, the immuned monkeys were found to result in higher antibody titers and have a good immune protection.Conclusions: The experiment obtained a simplified purification of F1 antigen from the Yersinia pestis EV76, and non-immunosuppressive activity rV270 antigen obtained by enterokinase digestion completely, subunit vaccines was prepared and immunize monkeys. The experimental observed the sensitivity of animals infected with Yersinia pestis, evaluated the feasibility of monkeys as animal models of plague, and also give people valuable information adout the subunit vaccine immune in the protection of monkey.Construction and preliminary evaluation of live attenuated Salmonella typhimurium vaccinePurpose: The attenuated Salmonella vaccine is a harmLess micro-organism in which the antigen genes were cloned into the body of this vector to induced immune response. It has two advantages of the powerful immunogenicity of the live attenuated vaccine and the accuracy of subunit vaccine. This live vector vaccine is a can induce final clearance cellular immunity effective, and this live vaccine plague vaccine vector has a very bright prospect. This study was designed to build a stable plague vaccine presence in the body to produce good immune protection and high security with a carrier of Salmonella typhimurium.Methods: V antigen fragment was cloned into pYA248 plasmid and transformed into the final plasmid host bacteria step by step through chemical transformation and electricity transformation. SDS-PAGE gel electrophoresis and WB is used on purpose of identifing the antigen identification and secretion. Mice were used to observe the safety of the vaccine. Activation of macrophages was observed by infecting RAW264.7 cells with the bacteria constructedResults: An attenuated Salmonella typhimurium as a carrier of the plague vaccine was constructed, it is found that the expression of V antigen through secretion, and it was safety and can be exist in vitro stability, after infect RAW264.7 macrophages; we found it can activate and stimulate TNF-αsecretion.Conclusions: The subunit vaccine can only stimulate humoral immune, In order to overcome this weakness,we use Salmonella typhimurium to be the carrier of plague vaccine, and successfully obtained a stable secreted V antigen vaccine strain, immunization of mice and found a good safety profile, and it also can stimulate macrophages to secrete a large number of TNF-αwhich indicate that strains were good immunogenicity, but it also need follow-up experiments to observe the result in vivo to stimulate antibody and immune protection.