猪霍乱沙门氏菌ΔasdC500株的生物学特性及作为活疫苗表达载体的应用

Characterization of attenuated Salmonella C500 strain with a Δasd mutant and use as an Asd^+ balanced-lethal host-vector system

为了评价猪霍乱沙门氏菌ΔasdC500株作为沙门氏菌Asd+平衡表达系统的可行性,对ΔasdC500株和其亲本菌株C500的生物表型、生长特性、毒力、生物安全性、表达特性等进行比较研究。结果表明:ΔasdC500缺失株的生化特性和血清型与亲本菌株C500一致,符合猪霍乱沙门氏菌的表型特征;携带平衡表达质粒pYA3493的重组菌株ΔasdC500(pYA3493)与C500的生长速度没有明显差别;根据Reed-Muench法,测定ΔasdC500(pYA3493)腹腔感染BALB/c小鼠的LD50为1.1×107CFU,毒力稍低于C500;口服接种ΔasdC500(pYA3493)和C500的所有仔猪未见任何发病症状,两者没有显著差别;携带重组质粒pYA-F1P2(含有支气管败血波氏杆菌抗原基因fhaB的TypeI区域和prn的R2区域)的重组菌株ΔasdC500(pYA-F1P2)能够稳定遗传重组质粒及其外源基因片段,并能稳定、高效、分泌性表达外源保护性抗原。由此表明,ΔasdC500保留了亲本菌株C500的一系列生物学特征,并可高效表达外源抗原,可作为沙门氏菌Asd+平衡表达系统开发基因工程重组疫苗。

Salmonella enterica serovar Choleraesuis strain C500 is a live, attenuated vaccine that has been used in China for over 40 years to prevent piglet paratyphoid. The objective of this study was to evaluate the potential of attenuated Salmonella enterica serovar Choleraesuis C500 strain with a Aasd mutant as an effective live vaccine vector by the Asd^＋ balanced-lethal host-vector system. Here, we compared the characteristics of S. enterica serovar Choleraesuis △asdC500 strain with the parent C500 strain, including phenotype, growth rate, virulence, safety, and expression for heterologous antigen. The mean generation times of △asdC500 mutant, the vector control △asdC500 （pYA3493）, and the parent avirulent C500 vaccine strain in Luria broth were 30.7, 28.1, and 27.9 min, respectively. The fermentation patterns of theses three strains on different carbohydrates, and the levels of production of H2S, were similar. The O and H antigens of △asdC500 mutant, △asdC500 （pYA3493） and AasdC500 （pYA-F1P2） were 6,7：C：1,5, identical to the parent strain C500. By the method of Reed and Muench, groups of mice were challenged by the intraperitoneal route with different amounts of △asdC500 （pYA3493） or the parent C500 strain, and the virulence of △asdC500 （pYA3493） with LDs0 of 1.lxl07 CFU was a little lower than C500 with LDs0 of 4.4×106 CFU. All piglets inoculated with AasdC500 （pYA3493） or C500 survived, and no signs of disease were observed during the entire experimental period. No major differences were found in these two groups. In addition, the recombinant pYA-F1P2 plasmid was very stable in the recombinant △asdC500 （pYA-F1P2） strain, which expressed secretorily a large amount of the recombinant filamentous hemagglutinin type I domain and pertactin region 2 domain antigen （rF1P2） of Bordetella bronchiseptica. In this study, we have shown that the AasdC500 mutant had a series of biological characteristics silimar to the parent vaccine strain C500. Furthermore, the strain could express secretorily a large amount of heterologous antigen. It is likely that this Salmonella expression and delivery system could be easily adapted to develop multivalent recombinant Salmonella vaccines against infectious agents using the Asd^＋ balanced-lethal host-vector system.