鼠伤寒沙门菌SL1344株分泌性蛋白K1缺失株的构建及其生物学特性的初步研究

发布时间：2018-12-13 08:14

【摘要】：为研制更加安全有效且遗传背景清晰的鼠伤寒沙门菌(S.typhimurium)疫苗弱毒株,本研究首先构建了含缺失1 011 bp sseK1基因的重组自杀性质粒PREΔsseK1,利用重组自杀性质粒介导的等位基因交换技术,两步法筛选SL1344的sseK1缺失株。PCR、双酶切及测序结果表明SL1344ΔsseK1构建成功。生物学特性初步研究表明,与亲本菌株SL1344相比,缺失株SL1344ΔsseK1保留了亲本株SL1344的血清型1,4,5,12∶i∶1,2,且能够稳定遗传缺失后的sseK1基因,生长速度及生化特性没有发生明显改变。小鼠毒力试验表明,SL1344ΔsseK1缺失株口服感染6周龄BALB/c小鼠的LD_(50)为6.63×10~8CFU,毒力较亲本株SL1344下降至0.048%。免疫保护效力试验显示,小鼠免疫后第14天血清抗体Ig G含量达到最高,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。以上研究结果表明,SL1344株sseK1基因缺失株构建成功,且遗传稳定,毒力明显降低,具有较好的免疫原性,为研究sseK1基因的致病机制及将其开发为更加安全有效的沙门菌弱毒疫苗候选菌株奠定了基础。
[Abstract]:In order to develop a more safe and effective attenuated strain of Salmonella typhimurium (S.typhimurium) vaccine with clear genetic background, a recombinant suicide plasmid PRE 螖 sseK1, containing the deletion of 1 011 bp sseK1 gene was constructed in this study. Two step method was used to screen the sseK1 deletion strain of SL1344 by using recombinant suicide plasmid mediated allele exchange technique. The results of PCR, double digestion and sequencing showed that SL1344 螖 sseK1 was successfully constructed. The preliminary study of biological characteristics showed that the missing strain SL1344 螖 sseK1 retained the serotype of the parent SL1344, and could stabilize the sseK1 gene after genetic deletion. The growth rate and biochemical characteristics of the missing sseK1 gene did not change obviously. The virulence test of mice showed that the LD_ (50) of 6-week-old BALB/c mice infected orally with SL1344 螖 sseK1 deletion strain was 6.63 脳 10 ~ (8) CFU, and the virulence of LD_ (50) was decreased to 0.048% than that of parent strain SL1344. The protective effect test showed that the content of serum Ig G reached the highest on the 14th day after immunization, and the protective rate of the wild strain of Salmonella typhimurium was 62.5% on the 17th day after immunization. The results showed that the sseK1 gene deletion strain of SL1344 strain was successfully constructed, and its genetic stability, virulence and immunogenicity were significantly decreased. The results provide a basis for studying the pathogenicity of sseK1 gene and developing it as a more safe and effective candidate for Salmonella attenuated vaccine.
【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)
【分类号】：S852.61

【相似文献】