Generation of a temperature-sensitive Edwardsiella tarda mutant and its potential as a prophylactic vaccine in olive flounder (Paralichthys olivaceus)
Generation of a temperature-sensitive Edwardsiella tarda mutant and its potential as a prophylactic vaccine in olive flounder (Paralichthys olivaceus)
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DOI：
10.1111/j.1365-2672.2012.05328.x
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发表时间：
2012-08-01
影响因子：
4
通讯作者：
Kim, K. H.
中科院分区：
文献类型：
--
作者：
Choi, S. H.;Kwon, S. R.;Kim, K. H.
目的：本研究的目的是产生温度敏感的迟缓爱德华氏菌突变体，并评估突变体作为橄榄比目鱼（Paralichthys olivaceus）疫苗的潜力。方法和结果：将alr基因启动子替换为cI 857-？PR启动子系统加上另一个CI 857表达盒，该表达盒由与tarda（EtPR C28-1）类似的E.在39 ℃的培养条件下，突变株的生长与野生型E.类似于迟缓菌的生长没有差异。然而，在25或20 ℃不加d-丙氨酸的条件下培养，突变株的生长受到阻碍。为了进一步抑制泄漏？PR启动子，突变菌株用携带EtPR C28-1驱动的cI 857盒（pEtPR-cI 857）的载体转化，与没有质粒的突变体相比，其导致更有限的生长。alr基因在类似tarda的突变体和携带pEtPR-cI 857的突变体中的转录水平与细菌生长的结果一致。用类似迟缓型肠杆菌突变体免疫的橄榄比目鱼鱼种死亡率降低，加强免疫诱导了100%的抗类似迟缓型肠杆菌感染保护。鱼的保护率与其血清对迟缓型肠杆菌的凝集效价成正比。结论：首次获得了一株低温诱导的类似迟缓突变株的减毒大肠杆菌。用该突变体免疫鱼导致对毒性E.的保护，类似于迟缓攻击。本研究的意义和影响：本研究结果表明，目前的温度敏感的E.类似tarda突变体可以作为一个很好的候选疫苗，用于预防爱德华氏菌病。此外，由于本发明的类似迟缓肠杆菌突变体可以在不补充特定营养物的情况下培养，并且可以仅通过将温度降低到30 ℃以下来减毒，因此基于本发明的类似迟缓肠杆菌突变体的疫苗在经济方面是有利的。
Aims: The aim of this study was to generate temperature-sensitive Edwardsiella tarda mutant and to evaluate potential of the mutant as a vaccine in olive flounder (Paralichthys olivaceus). Methods and Results: A temperature-sensitive E.similar to tarda mutant was generated by replacement of alr gene promoter with cI857-?PR promoter system plus another CI857 expression cassette that was driven by a constitutive promoter of E.similar to tarda (EtPR C28-1). Growth of the mutant strain was not different to that of wild-type E.similar to tarda under conditions of culture at 39 degrees C. However, growth of the mutant strain was retarded by culturing at 25 or 20 degrees C without d-alanine. To further inhibit leakage of ?PR promoter, the mutant strain was transformed with a vector harbouring an EtPR C28-1-driven cI857 cassette (pEtPR-cI857), which resulted in more limited growth compared to the mutant without the plasmids. The level of alr gene transcription in the mutant E.similar to tarda and the mutant harbouring pEtPR-cI857 was well coincided with the result of bacterial growth. Olive flounder fingerlings immunized with the E.similar to tarda mutant showed decreased mortality, and a boost immunization induced 100% protection against E.similar to tarda infection. The protection rate of fish was proportional to the serum agglutination titre against E.similar to tarda. Conclusions: An attenuated E.similar to tarda mutant induced by shifting down temperature below 30 degrees C was firstly generated. Immunization of fish with the mutant led to protection against virulent E.similar to tarda challenge. Significance and Impact of the Study: The results suggest that the present temperature-sensitive E.similar to tarda mutant can be a good candidate for effective vaccines for prophylaxis of edwardsiellosis. Moreover, as the present E.similar to tarda mutant can be cultured without supplementation of the specific nutrient and can be attenuated just by decreasing temperatures below 30 degrees C, vaccines based on the present E.similar to tarda mutant would be advantageous in an economical aspect.