Jawale Chetan V, Lee John Hwa
College of Veterinary Medicine, Chonbuk National University, Jeonju, Republic of Korea.
PLoS One. 2013 Oct 18;8(10):e78193. doi: 10.1371/journal.pone.0078193. eCollection 2013.
In the development of genetically inactivated bacterial vaccines, plasmid retention often requires the antibiotic resistance gene markers, the presence of which can cause the potential biosafety hazards such as the horizontal spread of resistance genes. The new lysis plasmid was constructed by utilizing the approach of balanced-lethal systems based on auxotrophic gene Aspartate semialdehyde dehydrogenase (asd). The PhiX174 lysis gene E and λPR37-cI857 temperature-sensitive regulatory system was cloned in the asd gene positive plasmid and this novel approach allowed the production of antibiotic resistance marker free Salmonella Enteritidis (S. Enteritidis) ghost. The immunogenic potential of the biosafety enhanced antibiotic resistance gene free S. Enteritidis ghost was evaluated in chickens by employing the prime-boost vaccination strategy using a combination of oral and intramuscular routes. A total of 75 two-week-old chickens were equally divided into five groups: group A (non-immunized control), group B (intramuscularly primed and boosted), group C (primed intramuscularly and boosted orally), group D (primed and boosted orally), and group E (primed orally and boosted intramuscularly). Chickens from all immunized groups demonstrated significant increases in plasma IgG, intestinal secretory IgA levels, and antigen-specific lymphocyte proliferative response. After a virulent S. Enteritidis challenge, all immunized groups showed fewer gross lesions and decreased bacterial recovery from organs in comparison with the non-immunized control group. Among the immunized chickens, groups B and D chickens showed optimized protection, indicating that the prime-booster immunization with the ghost via intramuscular or oral route is efficient. Taken together, our results demonstrate that an antibiotic resistance gene free lysis plasmid was successfully constructed and utilized for production of safety enhanced S. Enteritidis ghost, which can be used as a safe and effective vaccine against virulent S. Enteritidis infections.
在基因灭活细菌疫苗的研发过程中，质粒保留通常需要抗生素抗性基因标记，而这些标记的存在可能会导致潜在的生物安全隐患，如抗性基因的水平传播。通过利用基于营养缺陷型基因天冬氨酸半醛脱氢酶（asd）的平衡致死系统构建了新的裂解质粒。将PhiX174裂解基因E和λPR37-cI857温度敏感调节系统克隆到asd基因阳性质粒中，这种新方法使得无抗生素抗性标记的肠炎沙门氏菌（S. Enteritidis）菌蜕得以产生。通过采用口服和肌肉注射相结合的初免-加强免疫策略，在鸡中评估了生物安全性增强的无抗生素抗性基因肠炎沙门氏菌菌蜕的免疫原性潜力。总共75只两周龄的鸡被平均分为五组：A组（未免疫对照）、B组（肌肉注射初免和加强免疫）、C组（肌肉注射初免，口服加强免疫）、D组（口服初免和加强免疫）和E组（口服初免，肌肉注射加强免疫）。所有免疫组的鸡血浆IgG、肠道分泌型IgA水平以及抗原特异性淋巴细胞增殖反应均显著增加。在用强毒肠炎沙门氏菌攻击后，与未免疫对照组相比，所有免疫组的大体病变更少，器官中的细菌回收量减少。在免疫鸡中，B组和D组的鸡表现出最佳的保护效果，表明通过肌肉注射或口服途径用菌蜕进行初免-加强免疫是有效的。综上所述，我们的结果表明成功构建了无抗生素抗性基因的裂解质粒，并用于生产生物安全性增强的肠炎沙门氏菌菌蜕，其可作为一种安全有效的疫苗用于预防强毒肠炎沙门氏菌感染。
