| 表达猪圆环病毒2型cap蛋白的重组罗伊氏乳酸杆菌在小鼠诱导的免疫应答分析 |
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| 引用本文： | 王书博,徐义刚,陈秋艳,梅朱园,崔文,姜艳平,周晗,王丽,乔薪瑗,李一经,唐丽杰.表达猪圆环病毒2型cap蛋白的重组罗伊氏乳酸杆菌在小鼠诱导的免疫应答分析[J].畜牧兽医学报,2020,51(9):2238-2249. |
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| 作者姓名： | 王书博 徐义刚 陈秋艳 梅朱园 崔文 姜艳平 周晗 王丽 乔薪瑗 李一经 唐丽杰 |
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| 作者单位： | 1. 东北农业大学 动物医学学院, 哈尔滨 150030;2. 农业部动物疫病病原生物学重点实验室东北科学观测实验站, 哈尔滨 150030 |
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| 基金项目： | “十三五”国家重点研发计划（2017YFD0501100） |
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| 摘 要： | 旨在构建表达猪圆环病毒2型（PCV2）cap蛋白的重组罗伊氏乳酸杆菌（Lactobacillus reuteri，L.reuteri），并评价其在小鼠体内诱导的免疫应答效果。利用PCR扩增实验室分离保存的PCV2b型毒株的cap蛋白基因，以猪源L.reuteri为宿主菌，构建表达cap蛋白的重组菌株pPG-T7 g10-PPT-cap/L.reuteri，通过口服免疫BALB/c小鼠。采用间接ELISA方法测定免疫后小鼠血清中抗原特异性IgG抗体水平，粪便、鼻腔洗液、生殖道洗液、肠黏液中抗原特异性sIgA抗体水平，小鼠血清中各细胞因子水平；MTT法检测小鼠脾淋巴细胞增殖水平；流式细胞技术检测小鼠脾淋巴细胞中CD4+T细胞、CD8+T细胞的水平；荧光定量PCR检测免疫后攻毒的小鼠体内器官的病毒载量。结果显示，口服免疫重组乳酸菌组小鼠血清IgG抗体水平显著高于对照组（P<0.01）；小鼠粪便、鼻腔洗液、生殖道洗液、肠黏液中sIgA抗体水平显著高于对照组（P<0.01）；小鼠血清中细胞因子水平和对照组相比，IFN-γ、IL-2、IL-4、IL-12水平升高，IL-10水平降低，IFN-α无显著变化；体外孵育PCV2和小鼠脾淋巴细胞结果表明，重组乳酸菌组小鼠脾淋巴细胞增殖刺激指数显著高于对照组（P<0.01）；流式细胞技术检测结果显示，口服免疫重组乳酸菌组小鼠脾细胞中CD4+T细胞、CD8+T细胞含量高于对照组；荧光定量PCR结果显示，相比于对照组，口服免疫重组乳酸菌组小鼠体内的病毒载量明显低于对照组。综上所述，本研究成功构建了表达PCV2 cap蛋白的重组罗伊氏乳酸杆菌，经口服途径免疫动物，构建的重组乳酸杆菌能够刺激小鼠产生体液免疫和细胞免疫应答，且具有一定的免疫保护效果。
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| 关 键 词： | 猪圆环病毒2型 cap 蛋白 重组罗伊氏乳酸杆菌 免疫应答 |
| 收稿时间： | 2020-03-06 |
Analysis of Immune Response Induced by Recombinant Lactobacillus reuteri Expressing Cap Protein of Porcine Circovirus Type 2 in Mice |
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| WANG Shubo,XU Yigang,CHEN Qiuyan,MEI Zhuyuan,CUI Wen,JIANG Yanping,ZHOU Han,WANG Li,QIAO Xinyuan,LI Yijing,TANG Lijie.Analysis of Immune Response Induced by Recombinant Lactobacillus reuteri Expressing Cap Protein of Porcine Circovirus Type 2 in Mice[J].Acta Veterinaria et Zootechnica Sinica,2020,51(9):2238-2249. |
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| Authors: | WANG Shubo XU Yigang CHEN Qiuyan MEI Zhuyuan CUI Wen JIANG Yanping ZHOU Han WANG Li QIAO Xinyuan LI Yijing TANG Lijie |
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| Institution: | 1. College of Veterinary Medicines, Northeast Agricultural University, Harbin 150030, China;2. Northeast Scientific Inspection Observation Station, Key Laboratory of Animal Pathogen Biology of Ministry of Agriculture, Harbin 150030, China |
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| Abstract: | The purpose of this study was to construct a recombinant Lactobacillus reuteri (L. reuteri) expressing the cap protein of porcine circovirus type 2 (PCV2) and evaluate its effect on immune response in mice. The cap protein gene of PCV2b strain isolated and stored in the laboratory was amplified by PCR. A recombinant strain pPG-T7 g10-PPT-cap / L. reuteri expressing the cap protein was constructed using L. reuteri of pig origin as the host strain and explored the immune effect of BALB/c mice with recombinant bacteria orogastrically. Indirect ELISA was used to determine the level of antigen-specific IgG antibodies in the serum of mice after immunization, the levels of antigen-specific sIgA antibodies in stool, nasal wash, reproductive tract wash, and intestinal mucus, and the levels of various cytokines in mouse serum; MTT method was used to detect mouse spleen lymphocyte proliferation levels; flow cytometry (FCM) was used to detect the levels of CD4+ T cells and CD8+ T cells in mouse spleen lymphocytes; fluorescence quantitative PCR was used to detect the viral load of organs in challenged mice after immunization. The results showed that the serum levels of IgG antibodies in the mice of the oral immune recombinant strain group (OIG) were significantly higher than those in the control group (P<0.01); the levels of sIgA antibodies in the stool, nasal wash, reproductive tract wash, and intestinal mucus of the mice in OIG were significantly higher than those in the control group (P<0.01); Compared with the control group, the levels of cytokines in the serum of OIG were as follows: The levels of IFN-γ, IL-2, IL-4, IL-12 increased, the levels of IL-10 decreased, and the levels of IFN-α did not change significantly; Incubation of PCV2 and mouse spleen lymphocytes in vitro showed that the proliferation stimulating index of spleen lymphocytes in OIG was significantly higher than that of the control group (P<0.01); FCM results showed that CD4+ T cells and CD8+ T cells were higher than those of the control group; the results of fluorescent quantitative PCR showed that compared with the control group, the viral load in the OIG was significantly lower than that of the control group. In summary, the recombinant L. reuteri expressing the PCV2 cap protein were successfully constructed, and the constructed recombinant L. reuteri can stimulate mice to produce humoral and cellular immune responses after oragastrical immunization, and can exert a certain immune protection effect. |
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| Keywords: | porcine circovirus cap protein recombinant Lactobacillus reuteri immune response |
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