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目的:为研究用于能够稳定携带外源基因的口服活疫苗载体。方法:本研究以减毒鼠伤寒沙门菌SL1344ΔsipB为研究对象,利用自杀性质粒pREΔasd介导的细菌同源重组技术,构建SL1344ΔsipBΔasd宿主-载体平衡致死系统,并进一步研究其生物学特性。结果:PCR及测序结果表明SL1344ΔsipBΔasd构建成功。生物学特性结果表明SL1344ΔsipBΔasd的血清型与SL1344ΔsipB相同,并能够稳定遗传缺失后的asd基因;其生长速度和生化特性与亲本株SL1344基本相同,并且该缺失株仍然具有运动性的能力,小鼠攻毒试验表明SL1344ΔsipBΔasd电转了携带asd基因的互补质粒pYA3493后,其毒力较亲本菌株SL1344约降低至1.4%。结论:以上结果证明,减毒鼠伤寒沙门菌SL1344ΔsipBΔasd菌株构建成功,为深入研究以鼠伤寒沙门菌为载体的口服多价疫苗奠定基础。
OBJECTIVE: To study oral live vaccine vectors for use in stable delivery of foreign genes. Methods: In this study, the attenuated Salmonella typhimurium SL1344ΔsipB was used as the study object. The suicide plasmid pREΔasd mediated bacterial homologous recombination technique was used to construct SL1344ΔsipBΔasd host-vector balance lethal system and to further study its biological characteristics. Results: PCR and sequencing results showed that SL1344ΔsipBΔasd was successfully constructed. The biological characteristics of SL1344ΔsipBΔasd showed that the serotype of SL1344ΔsipBΔasd was the same as that of SL1344ΔsipB, and was able to stably inherit the gene deleted asd gene. The growth rate and biochemical characteristics of SL1344ΔsipBΔasd were basically the same as that of the parental strain SL1344, and the mutant strain still possessed the ability of motility. Toxicity test showed that SL1344ΔsipBΔasd reduced the virulence to about 1.4% compared with the parental strain SL1344 by electroporation of the complementary plasmid pYA3493 carrying asd gene. Conclusion: The above results demonstrate that the construction of the attenuated Salmonella typhimurium SL1344ΔsipBΔasd strain is successful, which lays the foundation for the further study of an oral multivalent vaccine with Salmonella typhimurium as carrier.