| Salmonella is a kind of very important zoonotic bacteria, mostly causing fever, enteritis, septicemia in human and animal in the worldwide. Salmonella as an intracellular invasive bacterium, it can effectively present antigen, stimulate specific humoral and cellular immune response against both Salmonella and heterologous antigen, stimulate mucosal and systemic immune at the same time. In recent years, Salmonella spp. were being developed as novel vaccine vectors based on host-vector balanced lethal system.In our previous studies, attenuated S. gallinarum vaccine strain 97A was obtained by gene transposition and coarse mutation, its safety, immunogenicity, pathogenesis had been evaluted, the results showed that it had good safety and immunogenicity. This study was aimed to develop a safer attenuated S. gallinarum vaccine strain and exploit 97A as a live vaccine vector. The asd (aspartate beta-semialdehyde) gene was deleted from 97A genome to construct host-vector balanced lethal system for stably expressing heterologous genes. This system was used to express dsRED gene and F gene of Newcastle disease virus (NDV). It provided essential base for developing novel vectored vaccines. 1. Construction and Characterization of the 97A△asd Mutant of an Attenuated S. gallinarum StrainIn order to develop a safer vaccine strain and exploit S. gallinarum vaccine strain as a live vaccine vector, a△asd mutant of S. gallinarum 97A strain was constructed and its host-vector balanced lethal system was developed. Two pair of primers were designed and synthesized based on the asd sequences of S. gallinarum in GenBank, and then the upstream and downstream fragments of asd gene from S. gallinarum genome DNA were amplified and cloned. The recombinant suicide plasmid was constructed in which asd gene was replaced by Cm gene. The asd deleted strain with Cm resistance was selected by two–step protocol and named 97A△asd mutant, and its phenotypes, growth properties and biochemical characteristics were further identified, the results showed that the properties of△asd mutant were consistent with those of its wildtype. The△asd mutant could express dsRED stably based on the plasmid (including asd gene) complementation experiment. All the results showed that a△asd mutant of S. gallinarum 97A strain was successfully constructed and it could be used as a carrier for the host-vector balanced lethal system to express heterologous genes stably and efficiently, this study would pave a way for developing S. gallinarum vaccine strain as a live vectored vaccine.2. Expression of NDV-F gene in S. gallinarum 97A△a sd mutantThe 594bp fragment of NDV-F gene was amplified from recombinant plasmid pVAX1-F and subcloned into pYA3334, and transformed into E.coli X6212, the positive clone was named X6212 (pYA3334-F). And then pYA3334-F plasmid was electroporated into S. typhimurium X3730 for methylating, the methylated plasmid pYA3334-F was electroporated into attenuated S. typhimurium X4550. The result of SDS-PAGE and immunological experiment showed that NDV-F gene was expressed in X4550. Furthermore, the methylated plasmid pYA3334-F was electroporated into 97A △asd mutant. SDS-PAGE was conducted to detect the expression of F gene in recombinant 97A (pYA3334). Chicken were immunized by intramuscular injection with 97A (pYA3334-F) at a dose of 1×109 CFU, and 97A (pYA3334) at a dose of 1×109 CFU as control, the result showed that this recombinant bacteria was safe. The immunized groups were induced specific antibodies (Ab) against F protein of NDV. The level of Ab reached 1:200 at the second week after the second immunization; the level of Ab were up to 1:400 at the second week after the third immunization. These results provided base for developing new NDV vaccine. |
