Evaluation of the immunogenicity of auxotrophic Lactobacillus with CRISPR-Cas9D10A system-mediated chromosomal editing to express porcine rotavirus capsid protein VP4.
Evaluation of the immunogenicity of auxotrophic Lactobacillus with CRISPR-Cas9D10A system-mediated chromosomal editing to express porcine rotavirus capsid protein VP4.
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DOI：
10.1080/21505594.2022.2107646
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发表时间：
2022-12
期刊：
影响因子：
5.2
通讯作者：
中科院分区：
文献类型：
--
作者：
猪轮状病毒（Porcine Rotavirus，PoRV）是引起猪病毒性腹泻的重要病原。由于感染具有明显的肠嗜性，肠粘膜免疫是抵抗病原体入侵的重要防线。此外，由于乳酸菌（LAB）具有耐酸性、耐胆盐性和免疫调节性，因此开发口服疫苗具有重要意义。大多数传统的质粒递送载体使用抗生素基因作为选择性标记，容易导致抗生素积累。因此，在基因工程食品级微生物中选择食品级标记是至关重要的。基于CRISPR-Cas 9D 10A系统，我们构建了一株稳定的营养缺陷型副干酪乳杆菌HLJ-27（Lactobacillus △Alr HLJ-27）菌株。此外，许多质粒在宿主细菌中复制，导致内部基因缺失。在本研究中，我们使用温度敏感的基因编辑质粒将VP 4基因插入到基因组中，产生插入突变株VP 4/△Alr HLJ-27、VP 4/△Alr W56和VP 4/W56。该重组细菌有效地诱导基于分泌型免疫球蛋白A（SIgA）的粘膜和基于免疫球蛋白G（IgG）的体液免疫应答。这些口腔粘膜疫苗有可能在未来作为抗生素应用的替代品，并诱导针对PEDV感染的有效免疫应答。
Porcine rotavirus (PoRV) is an important pathogen, leading to the occurrence of viral diarrhoea . As the infection displays obvious enterotropism, intestinal mucosal immunity is the significant line of defence against pathogen invasion. Moreover, as lactic acid bacteria (LAB) show acid resistance, bile salt resistance and immune regulation, it is of great significance to develop an oral vaccine. Most traditional plasmid delivery vectors use antibiotic genes as selective markers, easily leading to antibiotic accumulation. Therefore, to select a food-grade marker in genetically engineering food-grade microorganisms is vital. Based on the CRISPR-Cas9D10A system, we constructed a stable auxotrophic Lactobacillus paracasei HLJ-27 (Lactobacillus △Alr HLJ-27) strain. In addition, as many plasmids replicate in the host bacteria, resulting in internal gene deletions. In this study,we used a temperature-sensitive gene editing plasmidto insert the VP4 gene into the genome, yielding the insertion mutant strains VP4/△Alr HLJ-27, VP4/△Alr W56, and VP4/W56. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses. These oral mucosal vaccines have the potential to act as an alternative to the application of antibiotics in the future and induce efficient immune responses against PEDV infection.
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