Evaluation of the immunogenicity of auxotrophic Lactobacillus with CRISPR-Cas9D10A system-mediated chromosomal editing to express porcine rotavirus capsid protein VP4
Fengsai Li,
Zhuyuan Mei,
Ning Ju,
Ling Sui,
Xiaolong Fan,
Zi Wang,
Jiaxuan Li,
Yanping Jiang,
Wen Cui,
Zhifu Shan,
Han Zhou,
Li Wang,
Xinyuan Qiao,
Lijie Tang,
Xiaona Wang,
Yijing Li
Affiliations
Fengsai Li
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Zhuyuan Mei
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Ning Ju
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Ling Sui
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Xiaolong Fan
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Zi Wang
Tongliao Institute of agriculture and animal husbandry, Tongliao City, China
Jiaxuan Li
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Yanping Jiang
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Wen Cui
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Zhifu Shan
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Han Zhou
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Li Wang
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Xinyuan Qiao
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Lijie Tang
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Xiaona Wang
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
Yijing Li
College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China
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.
