Division of Microbiology, Defence Food Research Laboratory, Mysore, Karnataka 570011, India.
Lett Appl Microbiol. 2010 Feb;50(2):131-7. doi: 10.1111/j.1472-765X.2009.02755.x. Epub 2009 Oct 12.
Rapid detection of pathogenic Yersinia enterocolitica isolates by using antisera raised against recombinant attachment-invasion locus (Ail) protein.
The complete gene (471 bp) encoding for the Ail protein was amplified by PCR and cloned in pQE 30 UA vector. The recombinant clones were selected by polymerase chain reaction (PCR). Recombinant protein was expressed using induction with 1 mmol l(-1) final concentration of isopropylthiogalactoside (IPTG). Polyclonal antibodies were raised in mice against this purified recombinant protein. An indirect plate ELISA was standardized based on rAil protein for the detection of Y. enterocolitica. Western blot analysis with the sera raised against recombinant Ail protein exhibited reaction at 17 kDa region of the native Ail protein present in pathogenic Y. enterocolitica standard strains and strains isolated from pork samples suggesting that the antigenicity of recombinant Ail protein was similar to that of native Ail protein. Nonpathogenic Y. enterocolitica and the other species of Yersinia, namely, Y. pseudotuberculosis, Y. intermedia, Y. kristenseni, Y. fredrickseni and also the Enterobacteriaceae organisms tested were not found reacting to polyclonal antisera against this recombinant Ail protein.
The antibodies raised against recombinant Ail protein could specifically identify pathogenic Y. enterocolitica strains both by indirect plate ELISA and Western blot immunoassay.
The method developed in this study may find application in the detection of pathogenic Y. enterocolitica not only from food and environmental samples but also from clinical samples.
利用针对重组黏附侵袭位点（Ail）蛋白的抗血清快速检测致病性耶尔森氏菌。
通过 PCR 扩增并克隆了编码 Ail 蛋白的完整基因（471bp）到 pQE 30UA 载体中。通过聚合酶链反应（PCR）选择重组克隆。使用 1mmol/L 终浓度异丙基硫代半乳糖苷（IPTG）诱导表达重组蛋白。用纯化的重组蛋白免疫小鼠制备多克隆抗体。基于 rAil 蛋白建立了间接平板 ELISA 检测肠侵袭性大肠杆菌的方法。用针对重组 Ail 蛋白的血清进行 Western blot 分析，在致病性肠侵袭性大肠杆菌标准株和从猪肉样品中分离的菌株的天然 Ail 蛋白 17kDa 区域显示出反应，表明重组 Ail 蛋白的抗原性与天然 Ail 蛋白相似。非致病性肠侵袭性大肠杆菌和其他耶尔森氏菌，即假结核耶尔森氏菌、中间耶尔森氏菌、克氏耶尔森氏菌、弗雷德里克森氏菌以及肠杆菌科的其他生物体均未与针对该重组 Ail 蛋白的多克隆抗血清发生反应。
针对重组 Ail 蛋白的抗体可通过间接平板 ELISA 和 Western blot 免疫测定特异性识别致病性肠侵袭性大肠杆菌菌株。
本研究中开发的方法不仅可用于从食品和环境样本中，还可用于从临床样本中检测致病性肠侵袭性大肠杆菌。
