弓形虫鸡尾酒DNA疫苗的免疫保护作用研究

doi:10.19640/j.cnki.jtau.2023.03.008

天津农学院学报 ›› 2023, Vol. 30 ›› Issue (3): 40-46.doi: 10.19640/j.cnki.jtau.2023.03.008

弓形虫鸡尾酒DNA疫苗的免疫保护作用研究

何金灵¹, 杜荣起¹, 郑淇月¹, 宋淇淇¹, 张东超^{1,通信作者}, 金天明^{2,通信作者}

1.天津农学院动物科学与动物医学学院天津市农业动物繁育与健康养殖重点实验室,天津 300392;
2.天津市农业科学院,天津 300192

收稿日期:2022-04-26 出版日期:2023-06-30 发布日期:2023-09-06
通讯作者: 张东超（1991—）,男,讲师,博士,主要从事人兽共患病分子病原学与免疫学研究。E-mail：zdc2991@163.com。金天明（1968—）,男,教授,博士,主要从事分子免疫病理学研究。E-mail：jtm680@163.com。
作者简介:何金灵（1999—）,女,硕士在读,主要从事人兽共患病分子病原学与免疫学研究。E-mail：hjl101999@163.com。
基金资助:
天津市教委科技计划项目（2019KJ032）

Study on immune protection of Toxoplasma gondii cocktail DNA vaccine

He Jinling¹, Du Rongqi¹, Zheng Qiyue¹, Song Qiqi¹, Zhang Dongchao^{1,Corresponding Author}, Jin Tianming^{2,Corresponding Author}

1. Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China;
2. Tianjin Academy of Agricultural Sciences, Tianjin 300192, China

Received:2022-04-26 Online:2023-06-30 Published:2023-09-06

摘要/Abstract

摘要： 研究旨在构建弓形虫鸡尾酒DNA疫苗并评价其对小鼠的免疫保护效果。研究将编码弓形虫棒状体蛋白ROP5、ROP9和ROP17的基因分别构建至真核表达载体pVAX1上,获得重组真核表达质粒pVAX1-ROP5、pVAX1-ROP9和pVAX1-ROP17,将上述质粒进行PCR和双酶切验证,并通过Western blot检测重组质粒在真核细胞中表达。将三种重组质粒按1∶1∶1的比例混合后,取100 μg通过腿部肌肉注射免疫小鼠,加强免疫后,检测小鼠血清抗体、脾淋巴细胞增殖及细胞因子水平,并接种弓形虫RH强毒株进行攻毒,评价免疫后小鼠的保护效果。真核表达质粒pVAX1-ROP5、pVAX1-ROP9和pVAX1-ROP17经PCR和双酶切验证的结果显示：分别在1 686 bp、1 128 bp和1 836 bp处出现特异性目的条带;Western blot检测结果发现,在约为62、41和68 kDa处出现目的条带,分别与预期目的蛋白ROP5、ROP9和ROP17的分子量大小相符。与空载体组和生理盐水组小鼠相比,疫苗免疫组小鼠产生的血清中抗体IgG和淋巴细胞产生的细胞因子IFN-γ和IL-6水平、脾脏淋巴细胞增殖的刺激指数（SI）均极显著升高（P<0.01）,且接种弓形虫RH强毒株后,免疫组小鼠的存活时间（16.1±2.07）比对照组小鼠存活时间（9.2±0.74或9.0±0.77）极显著延长（P<0.01）。研究结果表明,成功构建的弓形虫鸡尾酒DNA疫苗pAX1-ROP5/ROP9/ROP17能激发小鼠的体液免疫反应和细胞免疫反应,并增强小鼠对弓形虫感染的抵抗能力。研究结果为弓形虫新型疫苗研制提供借鉴和参考。

关键词: 弓形虫, DNA疫苗, ROP5, ROP9, ROP17

Abstract: The aim of this study is to construct a DNA cocktail vaccine against Toxoplasma gondii（T. gondii）and evaluate its immunoprotective effect on mice. The genes encoding T. gondii ROP5, ROP9, and ROP17 were constructed into the eukaryotic expression vector pVAX1, and the recombinant eukaryotic expression plasmids were named pVAX1-ROP5, PVAX1-ROP9, and PVAX1-ROP17, respectively. The target genes of the recombinant plasmids were verified by PCR and double enzyme digestion, and the expression of the plasmids in eukaryotic cells was detected by Western blot. The three recombinant plasmids were mixed at the ratio of 1∶1∶1, subsequently, the 100 μg plasmid mixture was used to immunize mice by intramuscular injection of leg. After the booster immunization, serum antibody, lymphocyte proliferation, and cytokine levels of mice were detected, and T. gondii RH virulent strain was inoculated to evaluate the protection effect of the immunized mice. The results showed that when the eukaryotic expression plasmids pVAX1-ROP5, pVAX1-ROP9, and pVAX1-ROP17 were verified by PCR and double-digestion, the specific target bands were found at 1 686 bp, 1 128 bp, and 1 836 bp, respectively. Western blot analysis showed that the target bands were detected at 62, 41, and 68 kDa, which were consistent with the molecular weight of the expected target proteins ROP5, ROP9, and ROP17, respectively. Compared with pVAX1 and normal saline group, all the levels of antibody IgG in serum, cytokines IFN-γ and IL-6 produced by splenic lymphocytes, and stimulation index（SI）of splenic lymphocyte proliferation produced by mice immunized with cocktail vaccine were extremely significantly increased（P<0.01）. After inoculation with T. gondii RH virulent strain, the survival time of the immunized mice（16.1±2.07）was extremely significantly prolonged（P<0.01）than that of control mice（9.2±0.74 or 9.0±0.77）. The results indicated that the constructed T. gondii cocktail DNA vaccine pVAX1-ROP5/ROP9/ROP17 could stimulate the humoral and cellular immune responses in mice and enhance the resistance of mice to the parasite infection. The results provide reference for the development of the novel T. gondii vaccine.

Key words: Toxoplasma gondii, DNA vaccine, ROP5, ROP9, ROP17

中图分类号:

S855.9

何金灵, 杜荣起, 郑淇月, 宋淇淇, 张东超, 金天明. 弓形虫鸡尾酒DNA疫苗的免疫保护作用研究[J]. 天津农学院学报, 2023, 30(3): 40-46.

He Jinling, Du Rongqi, Zheng Qiyue, Song Qiqi, Zhang Dongchao, Jin Tianming. Study on immune protection of Toxoplasma gondii cocktail DNA vaccine[J]. Journal of Tianjin Agricultural University, 2023, 30(3): 40-46.

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http://xuebao.tjau.edu.cn/CN/Y2023/V30/I3/40

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弓形虫鸡尾酒DNA疫苗的免疫保护作用研究

Study on immune protection of Toxoplasma gondii cocktail DNA vaccine

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