| Live bacteria vector vaccine is a novel kind of vaccine by inserting heterogenous antigen genes into chromosome or plasmid of bacteria vectors, which irritate efficient and protective immune responses to heterogenous antigens, so as to prevent one or several diseases. The use of the attenuated Salmonella strain as a vaccine carrier for delivery of foreign antigens has been studied extensively, and shows a good prospect. Experiments in a mouse model have proved that using attenuated Salmonella strains, a variety of antigens including H. pylori urease, can be delivered to the immune system. Considerable progress has been made in humans with attenuated Salmonella enterica serovar Typhi strains, which can be used both as a more effective typhoid vaccine and for delivery of heterologous antigens. Among the most extensively evaluated vaccine candidates are serovar Typhi strains CVD908 (Ty2 aroC aroD) and CVD908-htrA (Ty2 aroC aroD htrA).In this work, aλRed in vivo recombinant system was used to knock out the asd gene of CVD908 (Ty2 aroC aroD) in order to constructed the host-vector balance lethal system. In the first chapter, the upstream and downstream homologous arm of asd gene was PCR amplified, with the genome of original strain CVD908 as a template. Through digestion and connecting, the upstream and downstream homologous arms are attached to both sides of the kanamycin resistance genes in targeting vector. Then the vector was used as template for the PCR amplification of linear targeting fragments with a high concentration.Plasmid pKD46 which expressed Red recombination proteins was transferred into CVD908 strain, and then L-arabinose was added to induce the expression of Red recombination proteins. Targeting fragments with a high concentration were transferred into CVD908/pKD46, and positive clone were selected by kanamycin agar plate. Subsequently, plasmid pCP20 was transferred into positive clone to delete the kanamycin resistant gene. The asd knocked out strains were identified by whole cell PCR assay and RT-PCR assay.Another target gene htrA, which encoded a periplasmic protease involving in degrading aberrant proteins was disrupted by the method ofλRed in vivo recombination too, and the final product CVD908-htrA (Ty2 aroC aroD asd htrA) mutant strains were also identified by whole cell PCR assay and RT-PCR assay. In the second chapter, we constructed a plasmid which could display a foreign antigen on the surface of bacterial outer membrane. Original plasmid pBAD-△ssdsbc carried a cat gene (chloramphenicol acetyltransferase gene). In this work, cat gene was replaced by asd gene of Escherichia coli, in order to construct a host-plasmid balanced lethal system. lpp-ompA sequence followed by a multiple clone site (MCS) was inserted just downstream of the arabinose promoter, so as to carry any foreign antigens to the surface of host cell. Hecilobacter pylori ureB gene was selected to check the efficiency of the surface display tool. The vector was transferred into attenuated strain CVD908-htrA (Ty2 aroC aroD asd htrA) and resulted strain was named CVD908-htrA-LOU. Outer membrane extraction combining with Western blot assay, immunofluorescence microscopy assay, as well as flow cytometry assay showed UreB was stably expressed on the cell surface of CVD908-htrA-LOU.In the third chapter of the work, the safety of live vaccine CVD908-htrA-LOU was evaluated by invasiveness of cells and infection of mice. Tests of invasion and intracellular yield showed CVD908-htrA-LOU decreased invasiveness of macrophages (mouse macrophages RAW264.7) and humen epithelial cell (Hela cell line), and also decreased the survival ability in these cells. When Mice were infected either by wild strain Ty2, CVD908 or CVD908-htrA-LOU via intraperitoneal injection routes, the LD50 of CVD908-htrA-LOU strain raised at least 10 times or 10 times compared with wild strain Ty2 or CVD908, respectively. It was verified that CVD908-htrA-LOU strain attenuated dramatically and may be a safer live vaccine strain.In the fourth chapter, we evaluated the immunogenicity of CVD908-htrA-LOU. Mouse were immunized orally with physiological saline, CVD908-htrA-LO (containing surface display vector but having no UreB), CVD908-htrA-LOU, as well as CVD908-htrA-U(UreB expressed in cytoplasm). CVD908-htrA-LOU would elicit higher sera IgG responses than CVD908-htrA-U although the latter had a higher expressed level of UreB antigen. It showed that live vaccine which surface displaying foreign antigen could elicit stronger immune response than those carried antigen in their cytoplasm. |
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