Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, South Korea.
Fish Shellfish Immunol. 2011 Jul;31(1):58-65. doi: 10.1016/j.fsi.2011.03.006. Epub 2011 Mar 17.
Two auxotrophic genes that play essential roles in bacterial cell wall biosynthesis--alanine racemase (alr) gene and aspartate semialdehyde dehydrogenase (asd) gene--knock-out Edwardsiella tarda (Δalr Δasd E. tarda) was generated by the allelic exchange method to develop a combined vaccine system. Green fluorescent protein (GFP) was used as a model foreign protein, and was expressed by transformation of the mutant E. tarda with antibiotic resistant gene-free plasmids harboring cassettes for GFP and asd expression (pG02-ASD-EtPR-GFP). In vitro growth of the mutant E. tarda was similar to wild-type E. tarda when D-alanine and diaminopimelic acid (DAP) were supplemented to growth medium. However, without d-alanine and/or DAP supplementation, the mutant showed very limited growth. The Δalr Δasd E. tarda transformed with pG02-ASD-EtPR-GFP showed a similar growth pattern of wild-type E. tarda when D-alanine was supplemented in the medium, and the expression of GFP could be observed even with naked eyes. The virulence of the auxotrophic mutant E. tarda was decreased, which was demonstrated by approximately 10⁶ fold increase of LD₅₀ dose compared to wild-type E. tarda. To assess vaccine potential of the present combined vaccine system, olive flounder (Paralichthys olivaceus) were immunized with the GFP expressing mutant E. tarda, and analyzed protection efficacy against E. tarda challenge and antibody titers against E. tarda and GFP. Groups of fish immunized with 10⁷ CFU of the Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP showed no mortality, which was irrespective to boost immunization. The cumulative mortality rates of fish immunized with 10⁶ or 10⁵ CFU of the mutant bacteria were lowered by a boost immunization. Fish immunized with the mutant E. tarda at doses of 10⁶-10⁷ CFU/fish showed significantly higher serum agglutination activities against formalin-killed E. tarda than PBS-injected control fish. Furthermore, fish immunized with 10⁶-10⁷ CFU/fish of the mutant E. tarda showed significantly higher ELISA titer against GFP antigen than fish in other groups. These results indicate that the present double auxotrophic genes knock-out E. tarda coupled with a heterologous antigen expression has a great strategic potential to be used as combined vaccines against various fish diseases.
通过等位基因交换方法生成了两种在细菌细胞壁生物合成中起关键作用的营养缺陷型基因——丙氨酸消旋酶（alr）基因和天门冬氨酸半醛脱氢酶（asd）基因敲除的迟钝爱德华氏菌（Δalr Δasd E. tarda），以开发联合疫苗系统。绿色荧光蛋白（GFP）被用作模型外源蛋白，并通过转化携带 GFP 和 asd 表达盒的抗生素抗性基因缺失质粒的突变型 E. tarda 来表达（pG02-ASD-EtPR-GFP）。当在培养基中补充 D-丙氨酸和二氨基庚二酸（DAP）时，突变型 E. tarda 的体外生长与野生型 E. tarda 相似。然而，没有 D-丙氨酸和/或 DAP 的补充，突变型的生长非常有限。当培养基中补充 D-丙氨酸时，转化了 pG02-ASD-EtPR-GFP 的Δalr Δasd E. tarda 表现出与野生型 E. tarda 相似的生长模式，甚至可以用肉眼观察到 GFP 的表达。营养缺陷型突变型 E. tarda 的毒力降低，与野生型 E. tarda 相比，LD₅₀ 剂量增加了约 10⁶ 倍。为了评估本联合疫苗系统的疫苗潜力，用表达 GFP 的突变型迟钝爱德华氏菌免疫橄榄鲷（Paralichthys olivaceus），并分析对迟钝爱德华氏菌攻毒的保护效力和对迟钝爱德华氏菌和 GFP 的抗体滴度。用 10⁷ CFU 的携带 pG02-ASD-EtPR-GFP 的Δalr Δasd E. tarda 免疫的鱼群没有死亡，这与加强免疫无关。用 10⁶ 或 10⁵ CFU 的突变细菌免疫的鱼的累积死亡率通过加强免疫降低。用 10⁶-10⁷ CFU/鱼剂量的突变迟钝爱德华氏菌免疫的鱼对福尔马林杀死的迟钝爱德华氏菌的血清凝集活性明显高于 PBS 注射对照鱼。此外，用 10⁶-10⁷ CFU/鱼的突变迟钝爱德华氏菌免疫的鱼对 GFP 抗原的 ELISA 滴度明显高于其他组的鱼。这些结果表明，本研究中双重营养缺陷型基因敲除迟钝爱德华氏菌与异源抗原表达相结合，具有很大的战略潜力，可作为防治各种鱼类疾病的联合疫苗。
