Hu Yuanqing, Shang Yuwei, Huang Jinlin, Wang Yan, Ren Fangzhe, Jiao Yang, Pan Zhiming, Jiao Xin-An
Jiangsu Key Laboratory of Zoonosis, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, 88 South Daxue Road, Yangzhou, Jiangsu 225009, PR China.
Biochim Biophys Acta. 2013 Nov;1830(11):5229-35. doi: 10.1016/j.bbagen.2013.06.042. Epub 2013 Jul 11.
Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.
In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.
Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI-TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.
We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.
This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.
空肠弯曲菌是一种重要的食源性病原体和人畜共患病原体，在全球范围内广泛分布。人类和鸡是这种病原体的宿主。目前，尚无理想的疫苗来控制人类弯曲菌病或鸡体内空肠弯曲菌的携带。细菌体内诱导抗原可用作潜在的疫苗候选物和毒力生物标志物。
在本研究中，我们开发了一种新颖的系统免疫蛋白质组学方法，使用来自空肠弯曲菌感染患者的预吸附血清，从空肠弯曲菌的总细胞蛋白中鉴定体内诱导抗原。
总体而言，使用针对空肠弯曲菌的预吸附人血清在聚偏二氟乙烯（PVDF）膜上检测到14个免疫反应斑点。然后，我们从重复的凝胶中切下这些蛋白斑点，并使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行鉴定。通过肽质量指纹图谱（PMF）和BLAST分析，总共鉴定出14种体内诱导抗原。鉴定出的蛋白质包括CadF（CadF-1和CadF-2）、CheW、TufB、DnaK、MetK、LpxB、HslU、DmsA、PorA、ProS、CJBH_0976、CSU_0396和假设蛋白cje135_05017。对9个基因进行实时逆转录聚合酶链反应（RT-PCR），以比较它们在体内和体外的表达水平。数据显示，9个分析基因中的8个在体内相对于体外显著上调。
我们成功开发了一种新颖的免疫蛋白质组学方法，通过使用来自感染患者的预吸附血清来鉴定空肠弯曲菌的体内诱导抗原。
这种新的分析方法可能被证明对鉴定任何细菌感染宿主内的体内诱导抗原有用，并将有助于新型亚单位疫苗的开发。
