鸡白痢沙门菌ipaJ基因的克隆与鉴定

Cloning and identification of ipaJ gene in Salmonella pullorum

摘要:

摘要：【目的】从鸡白痢沙门菌C79-13中克隆ipaJ基因，体外表达该蛋白后进行免疫原性分析。【方法】鸡白痢沙门菌C79-13与肠炎沙门菌50041进行抑制差减杂交后获得的片段PEA2、PE31和PE44与猪霍乱沙门菌C500疫苗株pSFD10质粒上ipaJ基因高度同源，拼接后获得了鸡白痢沙门菌完整的ipaJ基因序列。从鸡白痢沙门菌中克隆出ipaJ基因并将其构建到原核表达载体pET-30a(+)上，Western-blot检测体外表达蛋白的免疫原性，同时检测了该基因在鸡白痢沙门菌分离株中的分布。【结果】从鸡白痢沙门菌中克隆了大小为840 bp的ipaJ基因序列，并获得了体外原核表达的大小为37 kDa融合蛋白。该蛋白可与鸡白痢沙门菌阳性血清反应。PCR结果显示该基因广泛存在于鸡白痢沙门菌菌株中。 【结论】本文首次报道和克隆了鸡白痢沙门菌ipaJ基因，并证明了IpaJ蛋白具有免疫原性。

Abstract:

Abstract: [Objective] Cloning of ipaJ gene from Salmonella pullorum C79-13, and identification of expressed IpaJ protein as an immunogen of the pathogen. [Methods] With suppression subtractive hybridization (SSH) between Salmonella pullorum strain C79-13 (tester) and Salmonella enteritidis strain 50041 (driver), three subtracted fragments PEA3, PE31 and PE44 showed high homology with ipaJ in plasmid pSFD10 of Salmonella choleraesuis C500. The three subtracted sequences were spliced together into the whole sequence of ipaJ in Salmonella pullorum. Then the ipaJ gene was amplified from Salmonella pullorum by polymerase chain reaction (PCR) and cloned into prokaryotic expressive vector pET-30a(+). Western-blot was used to identify it as an immunogen. The distribution of the gene was also detected in Salmonella pullorum isolates. [Results] The ipaJ gene cloned from Salmonella pullorum was 840 bp, and the expressed fusion protein was 37 kDa. Specific reaction was found between Salmonella pullorum positive serum and expressed protein by Western-blot assay, confirming its identification as an immunogen of Salmonella pullorum. The PCR results showed that the gene exists in all Salmonella pullorum strains. [Conclusion] The ipaJ gene from Salmonella pullorum was first reported and cloned, and the expressed IpaJ protein was confirmed as an immunogen of Salmonella pullorum.