Nasiri Khadijeh, Zibaee Saeed, Nassiri Mohammadreza, Tahmoorespur Mojtaba, Haghparast Alireza
Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.
Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Mashhad, Iran.
Iran J Basic Med Sci. 2016 Aug;19(8):883-889.
Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC.
FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA.
We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC.
The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans.
产肠毒素大肠杆菌（ETEC）菌株是新生犊牛、人类、猪和绵羊腹泻的主要病因之一。卵黄抗体（IgY）技术已被确定为一种有前景的替代方法，可用于大量制备用于免疫治疗和免疫诊断的特异性抗体。本研究的目的是通过卵黄抗体（IgY）产生针对ETEC重组FanC蛋白的特异性抗体。
通过特异性引物和聚合酶链反应从ETEC中扩增FanC（K99）基因。该基因分别被克隆并亚克隆到pTZ57R/T和pET32a（+）载体中。重组载体被转入大肠杆菌BL21 CodonPlus（DE3）。通过1 mM异丙基-β-D-硫代半乳糖苷（IPTG）诱导研究蛋白表达。用纯化的重组FanC蛋白免疫母鸡。通过斑点印迹、蛋白质印迹和间接酶联免疫吸附测定（ELISA）检测IgY抗体的活性和特异性。
通过用重组FanC蛋白免疫母鸡，我们获得了FanC特异性IgY。抗FanC IgY显示出与ETEC的FanC蛋白特异性结合。
结果强调，针对重组FanC蛋白的特异性IgY可被推荐作为动物和人类被动免疫预防ETEC感染的候选物。
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