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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。
OBJECTIVE: To construct a lethal system of attenuated Salmonella typhimurium secreted protein K1 deletion strain and to develop a live vaccine vector capable of stably carrying exogenous genes. Methods: By using the allele exchange technique mediated by recombinant suicide plasmid (pREΔasd) and SL1344Δsse K1 attenuated Salmonella typhimurium strain as the parent strain, the asd gene deletion strain SL1344Δsse K1Δasd was screened by two-step method. The non-resistant p YA3493 plasmid carrying the asd gene was electroporated to the above-described deletion strain SL1344Δsse K1Δasd to construct a recombinant strain of SL1344Δsse K1Δasd (p YA3493). Results: PCR and sequencing showed that SL1344Δsse K1Δasd (pYA3493) was successfully constructed. Further studies showed that the serotype of the recombinant strain SL1344Δsse K1Δasd (p YA3493) was the same as the virulent strain SL1344 and the parent strain SL1344Δsse K1, and could stably inherit the asd gene after genetic deletion; its biochemical characteristics and growth rate were similar to those of the virulent strain SL1344 and the parent strain SL1344Δsse K1 compared to no significant difference. The virulence of mice showed that the LD50 of BALB / c mice orally infected with SL1344Δsse K1Δasd (p YA3493) at 6 weeks was 5.24 × 108CFU, and the virulent strain SL1344 decreased to about 0.048%. The protective efficacy of immunization showed that, The S. Typhimurium wild strain had a 62.5% protection against challenge with the 17th day post-immunization mice, essentially identical to the parent strain SL1344Δsse K1. Conclusion: The above results show that the host-carrier lethal system of Salmonella typhimurium secreted protein K1 strain was constructed successfully, and its genetic stability, virulence was significantly reduced, with good immunogenicity, for further study of Salmonella typhimurium As a carrier of oral polyvalent vaccine to lay the foundation.