Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, People's Republic of China.
Appl Microbiol Biotechnol. 2012 Feb;93(3):1197-205. doi: 10.1007/s00253-011-3635-2. Epub 2011 Oct 27.
Riemerella anatipestifer (RA) infections cause major economic losses in the duck industry. In this study, an immunogenic protein, chaperonin GroEL (GroEL), was identified from the outer membrane of RA strain WJ4 by immunoproteomic assay based on matrix-assisted laser desorption/ionization time of flight mass spectrometry. The complete sequence of the encoding gene, chaperonin groEL (groEL) was amplified and determined to be 1,629 base pairs in length. groEL was then cloned into expression vector pGEX-6P-1, and the expression of the recombinant GroEL (rGroEL) in Escherichia coli strain BL21 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting analysis. Immunization assay showed that ducklings or rabbits immunized with purified rGroEL generated 53- or 160-fold more anti-GroEL antibodies than those with no immunization. Importantly, bactericidal assay showed that rabbit anti-GroEL serum killed 30.0-57.3% of bacteria representing different serotypes, while rabbit anti-bacterin serum killing activity exhibits large serotype-dependent variations between 0.2% and 63.6%. Animal challenge experiment showed that ducklings immunized with rGroEL were 50%, 37.5%, and 37.5% protected from the challenge with RA strains WJ4 (serotype 1), Th4 (serotype 2), and YXb-2 (serotype 10), respectively. In addition, groEL from 34 additional RA strains was amplified by polymerase chain reaction (PCR), and products from nine were sequenced. groEL is highly conserved among RA strains, as the DNA sequence identity was over 97.5% between WJ4 and the nine additional strains. Our results suggest that GroEL may be a good candidate for new RA vaccine development.
鸭传染性浆膜炎（RA）感染给养鸭业造成了重大经济损失。本研究采用基于基质辅助激光解吸/电离飞行时间质谱的免疫蛋白质组学方法，从 RA 菌株 WJ4 的外膜中鉴定出一种免疫原性蛋白伴侣 GroEL（GroEL）。编码基因 chaperonin groEL（groEL）的全长序列被扩增并确定为 1629 个碱基对。groEL 随后被克隆到表达载体 pGEX-6P-1 中，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和 Western blot 分析确认了大肠杆菌菌株 BL21 中重组 GroEL（rGroEL）的表达。免疫试验表明，用纯化的 rGroEL 免疫的鸭或兔产生的抗-GroEL 抗体比未免疫的鸭或兔多 53-160 倍。重要的是，杀菌试验表明，兔抗-GroEL 血清可杀死代表不同血清型的 30.0-57.3%的细菌，而兔抗菌血清的杀菌活性在 0.2%至 63.6%之间存在很大的血清型依赖性变化。动物攻毒试验表明，用 rGroEL 免疫的鸭对 RA 菌株 WJ4（血清型 1）、Th4（血清型 2）和 YXb-2（血清型 10）的攻毒分别有 50%、37.5%和 37.5%的保护率。此外，通过聚合酶链反应（PCR）扩增了来自 34 株额外 RA 株的 groEL，其中 9 株进行了测序。RA 株之间 groEL 高度保守，WJ4 与 9 株额外株之间的 DNA 序列同一性超过 97.5%。我们的结果表明，GroEL 可能是新型 RA 疫苗开发的良好候选物。
