Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str., 32, 119991 Moscow, Russia.
Bach Institute of Biochemistry, Federal Research Centre "Fundamentals of Biotechnology" of the Russian Academy of Sciences, 119071 Moscow, Russia.
Int J Mol Sci. 2021 Oct 28;22(21):11657. doi: 10.3390/ijms222111657.
Approximately 1/6 of humanity is at high risk of experiencing cholera epidemics. The development of effective and safe vaccines against the primary cause of cholera, is part of the public health measures to prevent cholera epidemics. Natural nontoxigenic isolates represent a source of new genetically improved and relatively safe vaccine strains. However, the genomic engineering of wild-type strains is difficult, and these strains are genetically unstable due to their high homologous recombination activity. We comprehensively characterized two isolates using genome sequencing, bioinformatic analysis, and microscopic, physiological, and biochemical tests. Genetic constructs were Gibson assembled and electrotransformed into . Bacterial colonies were assessed using standard microbiological and immunological techniques. As a result, we created a synthetic chromoprotein-expressing reporter operon. This operon was used to improve the genome engineering approach and monitor the stability of the genetic constructs. Finally, we created a stable candidate vaccine strain bearing a deletion and expressing the β-subunit of cholera toxin. Thus, we developed a strategy for the rapid creation of genetically stable and relatively safe candidate vaccine strains. This strategy can be applied not only to but also to other important human bacterial pathogens.
约六分之一的人类面临霍乱流行的高风险。针对霍乱的主要病原体开发有效和安全的疫苗是预防霍乱流行的公共卫生措施的一部分。天然无毒力分离株代表了新型遗传改良和相对安全的疫苗株的来源。然而，野生型菌株的基因组工程较为困难，并且由于其同源重组活性高，这些菌株的遗传稳定性较差。我们使用基因组测序、生物信息学分析以及微观、生理和生化测试对两种分离株进行了全面表征。通过 Gibson 组装和电转化将遗传构建体导入到。通过标准微生物学和免疫学技术评估细菌集落。结果，我们创建了一个表达合成色蛋白的报告基因操纵子。该操纵子用于改进基因组工程方法并监测遗传构建体的稳定性。最后，我们创建了一种携带缺失并表达霍乱毒素β亚单位的稳定候选疫苗株。因此，我们开发了一种快速创建遗传稳定和相对安全的候选疫苗株的策略。该策略不仅可应用于，还可应用于其他重要的人类细菌病原体。
