减毒鼠伤寒沙门菌SL1344ΔsseK1Δasd宿主-载体平衡致死系统的构建及其生物学特性研究

发布时间：2019-06-18 11:03

【摘要】：目的:为构建减毒鼠伤寒沙门菌分泌性蛋白K1缺失株平衡致死系统,并将其开发为能够稳定携带外源基因的活疫苗载体。方法:利用重组自杀性质粒(pREΔasd)介导的等位基因交换技术,以减毒鼠伤寒沙门菌株SL1344Δsse K1为亲本株,运用二步法筛选asd基因缺失株SL1344Δsse K1Δasd。将携带有asd基因的无抗性p YA3493质粒电转至上述缺失菌株SL1344Δsse K1Δasd,构建SL1344Δsse K1Δasd(p YA3493)重组菌株。结果:PCR及测序结果表明SL1344Δsse K1Δasd(p YA3493)构建成功。进一步研究表明重组菌株SL1344Δsse K1Δasd(p YA3493)血清型和强毒株SL1344及亲本株SL1344Δsse K1相同,且能稳定遗传缺失后的asd基因;其生化特性和生长速度与强毒株SL1344及亲本株SL1344Δsse K1相比均无明显差异。小鼠毒力试验表明,SL1344Δsse K1Δasd(p YA3493)口服感染6周龄BALB/c小鼠的LD50为5.24×108CFU,毒力较强毒株SL1344约下降至0.048%;免疫保护效力试验显示,运用鼠伤寒沙门菌野生毒株对免疫后第17天的小鼠攻毒有62.5%的保护率,与亲本株SL1344Δsse K1基本一致。结论:以上结果表明,鼠伤寒沙门菌分泌性蛋白K1缺失株宿主-载体平衡致死系统构建成功,且遗传稳定,毒力明显降低,具有较好的免疫原性,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。
[Abstract]:Aim: to construct a balanced lethal system of attenuated Salmonella typhimurium secretory protein K1 deletion strain and develop it into a live vaccine vector which can stably carry foreign genes. Methods: SL1344 螖 sse K1 螖 asd. strain SL1344 螖 sse K1 螖 asd. was screened by two-step method using attenuated Salmonella typhimurium strain SL1344 螖 sse K1 as parent strain mediated by recombinant suicide plasmid (PRE 螖 ASD). The non-resistant p YA3493 plasmid carrying asd gene was transferred to the deleted strain SL1344 螖 sse K1 螖 asd, to construct the recombinant strain SL1344 螖 sse K1 螖 ASD (p YA3493). Results: the results of PCR and sequencing showed that SL1344 螖 sse K1 螖 ASD (p YA3493) was successfully constructed. Further studies showed that the serotype of recombinant strain SL1344 螖 sse K1 螖 ASD (p YA3493) was the same as that of virulent strain SL1344 and its parent strain SL1344 螖 sse K1, and it could stabilize the asd gene after genetic deletion, and its biochemical characteristics and growth rate were not significantly different from those of virulent strain SL1344 and parent strain SL1344 螖 sse K1. The virulence test of mice showed that the LD50 of 6-week-old BALB/c mice infected with SL1344 螖 sse K1 螖 ASD (p YA3493) was 5.24 脳 108 CFU, and the virulence strain SL1344 decreased to 0.048%, and the protective effect of Salmonella typhimurium wild strain on mice on the 17th day after immunization was 62.5%, which was basically consistent with that of its parent strain SL1344 螖 sse K1. Conclusion: the above results show that the host-vector balanced lethal system of Salmonella typhimurium secretory protein K1 deletion strain has been successfully constructed, and its genetic stability is stable, its toxicity is obviously reduced, and it has good immunogenicity, which lays a foundation for further study of oral multivalent vaccine with Salmonella typhimurium as carrier.
【作者单位】：河南科技大学动物疫病与公共卫生重点实验室/洛阳市活载体生物材料与动物疫病防控重点实验室;
【基金】：河南省科技攻关项目(112102110019)资助
【分类号】：R392

【相似文献】