| Since 1970s, many new vaccine strategies were developed with the advent of genetic engineering, so the safer and more efficient live vaccine using attenuated Salmonella spp. as the vector received wide attention in medical and veterinary medicine. The major advantage of the attenuated Salmonella spp. is that it can be administered by mucosal route (oral or intranasal), easily genetically manipulated and cause little side effect on animals. In addition, as an intracellular invasive bacterium, Salmonella spp. can effectively present antigen, induce specific humoral and cellular immune response against Salmonella spp. and heterologous antigen, and trigger both mucosal and systemic immune at the same time. To avoid using antibiotic as the screening marker in the construction of genetic engineering live vaccine, nutrition vicious attenuated Salmonella spp. based on balanced-lethal host-vector system was designed to deliver the expressed protein of foreign gene. Such vaccine design brings new hope to animal under the threat of contagious disease.Strain C500 of Salmonella choleraesuis is an avirulent vaccine strain attenuated by chemical method, it has good immunogenicity and safety and has been used to prevent piglet paratyphoid in China for over 40 years. Strain Salmonella choleraesuis C500 used as a delivery system for heterologous antigens based on the host-vector balanced lethal system would have great prospect in the near future. Newcastle disease (ND) is a highly contagious disease that causes substantial economic losses for poultry. Newcastle disease virus (NDV) belongs to the newly defined genus Avulavirus in the family of Paramyxoviridae. Its vaccine is frequently used for the prevention and control of ND in some countries when the disease spreads widely. The F protein, which mediates fusion of viral envelope and cellular membrane, is an important factor for viruse invasion and also a good protective immunogen. In this work, Salmonella choleraesuis C500â–³asd mutant was constructed, and used as a delivery system for F gene based on the balanced-lethal host-vector system. The immune responses of i.n. and i.m. vaccination with the recombinant Salmonella choleraesuis C500 vaccine strain expressing F antigen of NDV both in mouse and chicken were invaluated, the results showed that both of them produced specific antibody against F protein. It suggests that this balanced-lethal host-vector system of Salmonella choleraesuis could express foreign gene(s), delivery the target protein and intiate specific immunity, and could be used as vaccine vector against infectious disease.1 Construction and characterization of Salmonella choleraesuis C500â–³asd host-vector balanced lethal systemTwo sets of primers were designed and synthesized based on the sequences of asd gene of Salmonella choleraesuis in GenBank, then the upstream and downstream fragments of asd gene were amplified and cloned from Salmonella choleraesuis C500 genome DNA. The recombinant suicide plasmid was constructed in which asd gene was replaced by Cm gene. Strains with the deletion of asd gene were selected by two-step protocol and named Salmonella choleraesuis C500â–³asd mutant, which could not live without additive DAP. The mutant was confirmed by PCR method. Its phenotypes, growth properties and virulence characteristics were further verified, the results showed that the chracteristics ofâ–³asd mutant were consistent with those of its wild type, and its virulence had a drop compared with its parent strain. The dsRED plasmid was transformed into Salmonella choleraesuis C500â–³asd mutant by electroporation to generate Salmonella choleraesuis C500â–³asd (pYA3334-dsRed), the recombinant could express dsRED stably and efficiently after 100 generations in vitro. The results showed that the Salmonella choleraesuis C500â–³asd mutant was successfully constructed and it could be used as a vector based on the host-vector balanced-lethal system to express foreign gene(s) stably and efficiently, this study would pave a way for developing Salmonella choleraesuis C500 vaccine strain as a live vectored vaccine.2 Expression of F protein of Newcastle disease virus in Salmonella choleraesuis C500â–³asd mutant and analysis of its immunogenicityThe recombinant Salmonella typhimurium X4550(pYA3334-F) was indentified by PCR and SDS-PAGE, the positive plasmid pYA3334-F was extracted and electroporated into Salmonella choleraesuis C500â–³asd mutant, the recombinant was screened and designated as Salmonella choleraesuis C500â–³asd (pYA3334-F), SDS-PAGE was conducted to detect the expression of F gene in recombinant Salmonella choleraesuis C500â–³asd (pYA3334-F). It was shown that Salmonella choleraesuis C500Aasd (pYA3334-F) induced specific antibody against F protein of NDV both in mouse and chicken.In summary, the F protein expressed in attenuated Salmonella choleraesuis C500â–³asd had good immunogenity, and could initiate humoral and celluar immune responses. Salmonella choleraesuis C500â–³asd might be used as a live vaccine vector. |
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