鼠伤寒沙门菌SL1344株环化腺苷酸合成酶缺失株平衡致死系统的构建及其雏鸡免疫保护试验

Construction of host-vector balanced lethal system of Salmonella typhimurium SL1344Δcya mutant and immune protection test of chickling

摘要:

摘要:【目的】为构建鼠伤寒沙门菌环化腺苷酸合成酶缺失株平衡致死系统，并对其生物学特性进行检测。【方法】以鼠伤寒沙门菌SL1344Δcya株为亲本株，利用重组自杀性质粒(pREΔasd)介导的同源重组技术，经两步法缺失并筛选asd基因缺失株(SL1344Δcya Δasd)。将含asd基因且无抗性的pYA3493质粒电转化至缺失株SL1344ΔcyaΔasd，构建重组菌株SL1344ΔcyaΔasd (pYA3493)。【结果】试验结果表明，重组菌株SL1344ΔcyaΔasd(pYA3493)生化特性和生长速度与参考株SL1344相比差异较大，与亲本株SL1344Δcya基本一致，SL1344ΔcyaΔasd(pYA3493)失去了利用麦芽糖、乳糖、山梨醇等碳源的能力，也不能分解H2 S、半乳糖和鼠李糖，但仍保留了利用葡萄糖的能力。对1日龄雏鸡攻毒试验表明，SL1344ΔcyaΔasd(pYA3493)毒力较参考株SL1344降低了约104倍。免疫保护试验显示，1日龄雏鸡免疫SL1344ΔcyaΔasd(pYA3493)后第17天，利用鼠伤寒沙门菌参考株攻毒，保护率为62.5%。【结论】鼠伤寒沙门菌SL1344株环化腺苷酸合成酶缺失株平衡致死系统构建成功，且具有较好的免疫保护性，为深入研究以鼠伤寒沙门菌为载体的口服疫苗奠定基础。

Abstract:

Abstract:［Objective］ To develop a host-vector balanced lethal system of Salmonella typhimurium adenylate cyclase mutant，and detect its biological characteristics.［Methods］ We constructed SL1344ΔcyaΔasd mutant strain by recombinant suicide plasmid ( pＲEΔasd)，and screened by two-step method，transformed pYA3493 plasmid containing the asd gene without resistance electric into the lack of SL1344 ΔcyaΔasd，then the recombinant strains SL1344 ΔcyaΔasd (pYA3493) was constructed successfully.［Results］The biochemical characteristics and growth rate of the mutant were different from that of the wild strain SL1344，but almost the same as that of the parent strain SL1344Δcya. The mutant strain could neither ferment maltose，lactose，and sorbitol，nor decompose H2 S，galactose and rat lee sugar，but still retained the ability to use glucose． The one-day chicken lethal test showed that SL1344ΔcyaΔasd (pYA3493) mutant was at least 104 times lower than SL1344 strain． The protection rate induced by the SL1344ΔcyaΔasd ( pYA3493) mutant was 62.5%.［Conclusion］The SL1344ΔcyaΔasd (pYA3493) mutant was successfully constructed，and had good immune protection，it laid a foundation for developing potential oral vaccines.