Tian Lu, Li Wenyu, Huang Xinmei, Tian Di, Liu Jianhua, Yang Xinchao, Liu Lianrui, Yan Ruofeng, Xu Lixin, Li Xiangrui, Song Xiaokai
College of Veterinary Medicine, Nanjing Agricultural UniversityNanjing, China.
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural SciencesNanjing, China.
Front Microbiol. 2017 Jul 18;8:1245. doi: 10.3389/fmicb.2017.01245. eCollection 2017.
Coccidiosis is an intestinal disorder of poultry and often caused by simultaneous infections of several species. GAPDH is one of the immunogenic common antigens among , , and identified in our previous study. The present study was performed to further evaluate its immunogenicity and protective efficacy. The genes of cloned from and were named as and , respectively. The immunogenicity of recombinant proteins of EaGAPDH and EmGAPDH were analyzed by Western blot. The transcription and expression of pVAX-EaGAPDH and pVAX-EmGAPDH in the injected muscles were detected by reverse transcription PCR (RT-PCR) and Western blot, respectively. GAPDH-induced changes of T lymphocytes subpopulation, cytokines production, and antibody were determined using flow cytometry, quantitative real-time PCR (qPCR), and ELISA, respectively. Finally, the protective efficacies of pVAX-EaGAPDH and pVAX-EmGAPDH were evaluated by vaccination and challenge experiments. The results revealed that the recombinant GAPDH proteins reacted with the corresponding chicken antisera. The genes were successfully transcribed and expressed in the injected muscles. Vaccination with pVAX-EaGAPDH and pVAX-EmGAPDH significantly increased the proportion of CD4 and CD8 T lymphocytes, the cytokines productions of IFN-γ, IL-2, IL-4 et al., and IgG antibody levels compared to controls. The vaccination increased the weight gains, decreased the oocyst outputs, alleviate the enteric lesions compared to controls, and induced moderate anti-coccidial index (ACI). In conclusion, the coccidial common antigen of GAPDH induced significant humoral and cellular immune response and effective protection against , , , and mixed infection of the three species.
球虫病是家禽的一种肠道疾病，通常由几种球虫同时感染引起。甘油醛-3-磷酸脱氢酶（GAPDH）是我们之前研究中在柔嫩艾美耳球虫、巨型艾美耳球虫和堆型艾美耳球虫中鉴定出的免疫原性共同抗原之一。本研究旨在进一步评估其免疫原性和保护效果。从柔嫩艾美耳球虫和巨型艾美耳球虫中克隆的GAPDH基因分别命名为EaGAPDH和EmGAPDH。通过蛋白质免疫印迹法分析EaGAPDH和EmGAPDH重组蛋白的免疫原性。分别通过逆转录PCR（RT-PCR）和蛋白质免疫印迹法检测pVAX-EaGAPDH和pVAX-EmGAPDH在注射肌肉中的转录和表达。分别使用流式细胞术、定量实时PCR（qPCR）和酶联免疫吸附测定（ELISA）来测定GAPDH诱导的T淋巴细胞亚群变化、细胞因子产生和抗体。最后，通过疫苗接种和攻毒实验评估pVAX-EaGAPDH和pVAX-EmGAPDH的保护效果。结果显示，重组GAPDH蛋白与相应的鸡抗血清发生反应。EaGAPDH和EmGAPDH基因在注射肌肉中成功转录和表达。与对照组相比，用pVAX-EaGAPDH和pVAX-EmGAPDH进行疫苗接种显著增加了CD4和CD8 T淋巴细胞的比例、IFN-γ、IL-2、IL-4等细胞因子的产生以及IgG抗体水平。与对照组相比，疫苗接种增加了体重增加，减少了卵囊产量，减轻了肠道病变，并诱导了中等的抗球虫指数（ACI）。总之，球虫的共同抗原GAPDH诱导了显著的体液和细胞免疫反应，并对柔嫩艾美耳球虫、巨型艾美耳球虫、堆型艾美耳球虫以及这三种球虫的混合感染产生了有效的保护作用。
