Chen Wenjun, Wang Xiaoyun, Lv Xiaoli, Tian Yanli, Xu Yanquan, Mao Qiang, Shang Mei, Li Xuerong, Huang Yan, Yu Xinbing
Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, 510080, China.
Parasitol Res. 2014 Sep;113(9):3409-18. doi: 10.1007/s00436-014-4006-6. Epub 2014 Jul 3.
Clonorchis sinensis excretory/secretory products (ESP) have gained high attentions because of their potential to be vaccine candidates and drug targets in C. sinensis prevention. In this study, we extensively profiled the characteristics of four C. sinensis cathepsin B cysteine proteases (CsCB1, CsCB2, CsCB3, and CsCB4). Bioinformatics analysis showed all CsCBs contained signal peptides at the N-terminal. Functional domains and residues were found in CsCB sequences. We expressed four CsCBs and profiled immune responses followed by vaccine trials. Recombinant CsCBs could induce high IgG titers, indicating high immunogenicity of CsCB family. Additionally, ELISA results showed that both IgG1 and IgG2a levels apparently increased post-immunization with all four CsCBs, showing that combined Th1/Th2 immune responses were triggered by CsCB family. Both Real-time polymerase chain reaction (RT-PCR) and Western blotting confirmed that four CsCBs have distinct expression patterns in C. sinensis life stages. More importantly, we validated our hypothesis that CsCBs were C. sinensis excretory/secretory products. CsCBs could be recognized by C. sinensis-infected sera throughout the infection period, indicating that secreted CsCBs are immune triggers during C. sinensis infection. The protective effect was assessed by comparing the worm burden and egg per gram (EPG) between CsCB group and control group, showing that worm burden (P < 0.01) and EPG (P < 0.01) in CsCB2 and CsCB3 groups were significantly lower than in control group. In conclusion, we profiled secreted cathepsin B cysteine proteases family for the first time and demonstrated that all CsCB family were C. sinensis excretory/secretory products that may regulate host immune responses.
华支睾吸虫排泄/分泌产物（ESP）因其在华支睾吸虫预防中作为疫苗候选物和药物靶点的潜力而备受关注。在本研究中，我们广泛分析了四种华支睾吸虫组织蛋白酶B半胱氨酸蛋白酶（CsCB1、CsCB2、CsCB3和CsCB4）的特性。生物信息学分析表明，所有CsCBs在N端均含有信号肽。在CsCB序列中发现了功能域和残基。我们表达了四种CsCBs并分析了免疫反应，随后进行了疫苗试验。重组CsCBs可诱导高IgG滴度，表明CsCB家族具有高免疫原性。此外，ELISA结果显示，用所有四种CsCBs免疫后，IgG1和IgG2a水平均明显升高，表明CsCB家族触发了Th1/Th2联合免疫反应。实时聚合酶链反应（RT-PCR）和蛋白质印迹法均证实，四种CsCBs在华支睾吸虫生活史各阶段具有不同的表达模式。更重要的是，我们验证了我们的假设，即CsCBs是华支睾吸虫的排泄/分泌产物。在整个感染期，CsCBs可被华支睾吸虫感染血清识别，表明分泌的CsCBs是华支睾吸虫感染期间的免疫触发因素。通过比较CsCB组和对照组之间的虫体负荷和每克虫卵数（EPG）来评估保护效果，结果显示，CsCB2和CsCB3组的虫体负荷（P<0.01）和EPG（P<0.01）均显著低于对照组。总之，我们首次分析了分泌型组织蛋白酶B半胱氨酸蛋白酶家族，并证明所有CsCB家族都是华支睾吸虫的排泄/分泌产物，可能调节宿主免疫反应。
