论文部分内容阅读
目的:为了研制更加安全的鼠伤寒沙门菌弱毒株,本研究构建了鼠伤寒沙门菌SL1344Δsip B基因缺失突变株。方法:首先构建含缺失585 bp sipB基因的重组自杀性质粒PREΔsipB,利用重组自杀性质粒介导的等位基因交换技术,两步法筛选出SL1344的sip B缺失株。结果:PCR及测序结果表明SL1344ΔsipB构建成功。进一步生物学特性研究表明,缺失株SL1344ΔsipB保留了亲本菌株SL1344的血清型1,4,5,12:i:1,2,且能够稳定遗传缺失585 bp的sipB基因,生长速度没有发生明显改变;但是,与亲本株SL1344相比,其毒力发生明显改变,缺失株SL1344ΔsipB口服感染6周龄BALB/c小鼠的LD50为1.70×108CFU,毒力较亲本株SL1344降低至1.4%,免疫保护效力实验显示,对6周龄BALB/c小鼠免疫后第17天,鼠伤寒沙门菌野生株攻毒有50%的保护率。在给小鼠免疫后第14天血清抗体IgG达到最高。结论:结果表明,SL1344ΔsipB基因缺失株构建成功,遗传稳定,毒力明显降低,且具有较好的免疫原性,为研究鼠伤寒沙门菌sipB基因与其致病力之间的关系提供了方法,并为研制安全有效的沙门菌疫苗弱毒株奠定了基础。
OBJECTIVE: To develop a safer attenuated Salmonella typhimurium strain, we constructed a mutant strain of Salmonella typhimurium SL1344Δsip B gene deletion. Methods: A recombinant suicide plasmid PREΔsipB containing 585 bp sipB gene was constructed. The sip B deletion mutant of SL1344 was screened by a two-step method using recombinant suicide plasmid-mediated allelic exchange. Results: PCR and sequencing results showed that SL1344ΔsipB was successfully constructed. Further studies on the biological characteristics showed that the deletion strain SL1344ΔsipB retained the serotypes 1, 4, 5, 12: i: 1,2 of the parental strain SL1344 and could stably inherit the sipB gene lacking 585 bp. The growth rate did not change significantly. However, its virulence significantly changed compared with that of the parental strain SL1344. The LD50 of the 6-week-old BALB / c mice orally infected with the deletion strain SL1344ΔsipB was 1.70 × 108CFU and the virulence was reduced to 1.4% compared with the parental strain SL1344. The immunoprotective efficacy Experiments show that, on the 17th day after immunization of 6-week-old BALB / c mice, the S. typhimurium wild-type strain has a 50% protection rate. Serum IgG was highest on day 14 after immunization of mice. CONCLUSION: The results showed that the constructed SL1344ΔsipB gene deletion mutant was successfully constructed, its genetic stability was significantly reduced, its virulence was significantly reduced and its immunogenicity was good. It provided a method for studying the relationship between the sipB gene of S. Typhimurium and its virulence It lays the foundation for developing a safe and effective attenuated Salmonella vaccine strain.