Dong Zhiling, Xu Lei, Yang Jing, He Yonglin, She Qian, Wang Jingxian, Zhang Zhuangmiao, Feng Xin, Li Dairong, Yang Chun
Department of Pathogenic Organisms, Chongqing Medical University, Chongqing, China; Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing, China.
Department of Respiratory Disease, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Hum Immunol. 2014 May;75(5):428-32. doi: 10.1016/j.humimm.2014.02.017. Epub 2014 Feb 12.
PPE68 protein is absent from BCG and the attenuated strains of Mycobacterium tuberculosis (MTB). In this study, the shuttle plasmid pBudCE4.1/PPE68/OriM was constructed and transformed into BCG to obtain PPE68 recombination BCG (PPE68-rBCG), and BALB/c mice were immunized with PPE68-rBCG to evaluate the immunological characterization of PPE68-rBCG. The level of lgG2a, IFN-γ, IL-12 and IL-4 in serum of immunized mice were detected, the proliferation response of spleen lymphocyte were measured, the frequency of CD4(+), CD8(+) and CD4(+)/CD8(+) were determined, and the spleen and lung tissue were prepared for pathological analysis. PPE68-rBCG was constructed successfully and could induce powerful Th1 immune response in mice. Besides, we took the purified recombination PPE68 (rPPE68) protein as diagnostic antigen to detect pulmonary tuberculosis patients (n=252) and extrapulmonary tuberculosis patients (n=66). We also used anti-PPE68 polyclonal antibody as coating antibody to detect specific antigen in the same serum samples. Our data provide an experimental basis for potential application of rPPE68 in the diagnosis of tuberculosis, especially for extrapulmonary tuberculosis.
卡介苗（BCG）和结核分枝杆菌（MTB）减毒株中不存在PPE68蛋白。在本研究中，构建了穿梭质粒pBudCE4.1/PPE68/OriM并将其转化到卡介苗中以获得PPE68重组卡介苗（PPE68-rBCG），然后用PPE68-rBCG免疫BALB/c小鼠以评估PPE68-rBCG的免疫学特性。检测免疫小鼠血清中lgG2a、IFN-γ、IL-12和IL-4的水平，测量脾淋巴细胞的增殖反应，测定CD4(+)、CD8(+)和CD4(+)/CD8(+)的频率，并制备脾和肺组织用于病理分析。成功构建了PPE68-rBCG，其可在小鼠中诱导强烈的Th1免疫反应。此外，我们将纯化的重组PPE68（rPPE68）蛋白作为诊断抗原检测肺结核患者（n=252）和肺外结核患者（n=66）。我们还用抗PPE68多克隆抗体作为包被抗体检测相同血清样本中的特异性抗原。我们的数据为rPPE68在结核病诊断中的潜在应用，特别是肺外结核的诊断，提供了实验依据。
