Zhao Li, Guo Zhihou, Liu Jiali, Wang Zi, Wang Ruichong, Li Yijing, Wang Li, Xu Yigang, Tang Lijie, Qiao Xinyuan
Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Department of Preventive Veterinary, College of Veterinary, Northeast Agricultural University, Harbin, China.
Department of Radiological and Protection, Heilongjiang Province Center for Disease Control and Prevention, Harbin, China.
Vaccine. 2017 Jul 13;35(32):4010-4021. doi: 10.1016/j.vaccine.2017.05.076.
The present study used Lactobacillus casei ATCC 393 as antigen delivery system to express C. perfringens toxoids α-β2-ε-β1 to construct the recombination Lactobacillus casei pPG-2-α-β2-ε-β1/L. casei 393. After being induced by 1% xylose, the specificity and integrity of recombinant strain were determined by Western-blotting. Rabbits as native animal model were immunized orally with pPG-2-α-β2-ε-β1/L. casei 393 and the titers of specific IgG and sIgA were determined by ELISA. The result showed that oral administration with the recombinants could elicit both local mucosal and systemic immune responses. The proliferation of spleen lymphocytes in rabbits immunized with pPG-2-α-β2-ε-β1/L. casei 393 was observed. Levels of IL-4 and IFN-γ produced were significantly higher in lymphocytes isolated from the vaccine group than those from the control groups. Flow cytometry assay showed that both the percentages of CD4+T cells and CD8+T cells from the vaccine group were significantly increased than the control groups. All these results showed that immunizing with recombinants can elicit both humoral immunity and cellular immunity. Besides, in order to determine the effectiveness of oral immunization with pPG-2-α-β2-ε-β1/L. casei 393, rabbits of vaccine group and control groups were challenged with 1×LD unit of culture filtrate of C. perfringens type C and type D toxins respectively. After challenge, 100% of the immunized rabbits survived, while the rabbits of the control group were killed within 48h. Observation on histopathology showed that histopathological changes were obviously found in heart, liver, spleen, lung, kidney, intestine and brain of rabbits from the control groups, while no apparent histopathological change was observed in the vaccine group. All the results show that pPG-2-α-β2-ε-β1/L. casei 393 can eliciteffective immunoprotection against C. perfringens. All of these suggest that the use of pPG-2-α-β2-ε-β1/L. casei 393 can be regarded as candidate for the development of a vaccine against C. perfringens.
本研究以干酪乳杆菌ATCC 393作为抗原递送系统，表达产气荚膜梭菌类毒素α-β2-ε-β1，构建重组干酪乳杆菌pPG-2-α-β2-ε-β1/L. casei 393。经1%木糖诱导后，通过蛋白质免疫印迹法检测重组菌株的特异性和完整性。以家兔作为天然动物模型，口服pPG-2-α-β2-ε-β1/L. casei 393进行免疫，通过酶联免疫吸附测定法检测特异性IgG和sIgA的效价。结果表明，口服重组菌可引发局部黏膜免疫反应和全身免疫反应。观察了用pPG-2-α-β2-ε-β1/L. casei 393免疫的家兔脾脏淋巴细胞的增殖情况。疫苗组分离的淋巴细胞产生的IL-4和IFN-γ水平显著高于对照组。流式细胞术检测表明，疫苗组CD4+T细胞和CD8+T细胞的百分比均显著高于对照组。所有这些结果表明，用重组菌免疫可引发体液免疫和细胞免疫。此外，为了确定口服pPG-2-α-β2-ε-β1/L. casei 393免疫的有效性，分别用1×LD单位的C型和D型产气荚膜梭菌毒素培养滤液对疫苗组和对照组家兔进行攻毒。攻毒后，100%的免疫家兔存活，而对照组家兔在48小时内死亡。组织病理学观察表明，对照组家兔的心、肝、脾、肺、肾、肠和脑出现明显的组织病理学变化，而疫苗组未观察到明显的组织病理学变化。所有结果表明，pPG-2-α-β2-ε-β1/L. casei 393可引发针对产气荚膜梭菌的有效免疫保护。所有这些表明，使用pPG-2-α-β2-ε-β1/L. casei 393可被视为开发产气荚膜梭菌疫苗的候选物。
