| In recent years attenuated live bacteria vectors have elicited great concerns for they can induce humoral, cellular and local mucosal immune responses against live vectors themselves and the carried foreign antigens. Especially some latest researches have confirmed that many live bacteria vectors can act as DNA vaccine transporting vehicles through oral immunization, which can induce different immune responses against specific antigens encoded by DNA vaccine. But to date the interaction relationship and regulatory mechanisms between live bacteria vectors and host are rarely known. To understand the underlying mechanisms will provide important theoretic foundation for designing new types of genetic vaccines and give important information for the prevention and control of related diseases. To this end the recombinant attenuated Salmonella typhimurium expressing green fluorescent protein (GFP) was constructed. Its dynamic distribution in vitro and in vivo infection experiments and its interaction with antigen presenting cells (APCs) were studied. This study has laid the foundation for the development of recombinant Salmonella and related genetic vaccines vectors.1 Construction and characterization of recombinant attenuated Salmonella typhimurium expressing GFPA recombinant attenuated Salmonella typhimurium expressing GFP was constructed as follow by DNA recombinant techniques. The EGFP gene was excised from plasmid pEGFP by EcoR I and Nco I sites and then cloned into prokaryotic expression plasmid pYA3334 using the same sites. This recombinant plasmid was named pYAGFP and subsequently transformed into E. coli X6212, intermediated hostSalmonella typhimurium X3731, and final host Salmonella typhimurium X4550. The recombinants carrying pYAGFP, designated as X4550(pYAGFP), were screened, and showed green on LB plates and all bacteria cells illuminated green fluorescence under fluorescence microscopy. In in vitro stability experiment this recombinant bacteria still showed green after pass through 15 generations on LB plates or in liquid culture, which confirmed its stability. The SDS-PAGE result showed the molecule weight of GFP expressed in X4550 was about 27 kD as expected. BALB/c mice were immunized with X4550(pYAGFP) and there were no abnormal phenomenon and organ lesions 1 month. The construction of recombinant Salmonella typhimurium expressing GFP offers a good model for the studying and designing genetic vaccines based on attenuated Salmonella and offers a strong tool for studying the immunological properties of recombinant Salmonella and for preventing and controlling related diseases.2 Dynamic changes of recombinant attenuated Salmonella in murine model of infectionBALB/c mice were intravenously injected with recombinant attenuated Salmonella typhimurium X4550 expressing GFP, X4550(pYAGFP) and then the spleens and livers were aseptically removed at various time intervals postimmunization. Single-cell suspensions of the tissues were prepared and plated on LB plates, and the number of bacteria were colony-forming units (CFU) counted. At the same time serum samples were obtained from retro-orbital sinus and used for the detection of the antibodies against recombinant Salmonella. The results showed the CFU of recombinant Salmonella had two peaks during the observation period, but the tendency was to diminish. The recombinant bacteria could still be separated after 41 days, which was beneficial for host to develop effective immune responses; the antibody level in serum was rising in the same period, which showed the immunized mice could produce a relatively high humoral immune responses by intravenous immunization. The first CFU peak appeared in 11-13 days postimmunization, and for the mice produced antibodies which can cause opsonic reaction to bacteria, so the number of bacteria fell down and into the lowest point at the 17th day, and the level of antibodies rose gradually. Afterwards the bacteria number rose again and the elicited antibodies elevated rapidly.The antibod... |
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