Zhang Zhenchao, Li Yuhua, Li Haoran, Song Xiaoxiao, Ma Zhongshan, Lu Haoran, Liu Shuyue, Zhao Yi, Tan Mengyao, Wang Shuai, Li Xiangrui
Xinxiang Key Laboratory of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang 453003, China.
MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Vaccines (Basel). 2020 Apr 1;8(2):158. doi: 10.3390/vaccines8020158.
The neurotropic parasite () infection can change the behavior of rodents and cause neuropsychological symptoms in humans, which may be related to the change in neurotransmitter dopamine in the host brain caused by infection. tyrosine hydroxylase (TgTH) is an important factor in increasing the neurotransmitter dopamine in the host brain. In this study, the enzyme activity of TgTH catalytic substrate for dopamine production and the molecular characteristics of TgTH were identified. In order to amplify the open reading frame (ORF), the designing of the specific primers for polymerase chain reaction (PCR) was on the basis of the TgTH sequence (GenBank Accession No. EU481510.1), which was inserted into pET-32a (+) for the expression of recombined TgTH (rTgTH). The sequence analysis indicated that the gene of TgTH directed the encoding of a 62.4-kDa protein consisting of 565 amino acid residues, which was predicted to have a high antigen index. The enzyme activity test showed that rTgTH and the soluble proteins extracted separately from RH strain and PRU strain could catalyze the substrate to produce dopamine in a dose-dependent manner, and the optimum catalytic temperature was 37 °C. The result of the Western Blotting assay revealed that the rTgTH and the native TgTH extracted from somatic of RH tachyzoite were successfully detected by the sera of mice infected with and the rat serum after rTgTH immune, respectively. Immunofluorescence analysis using antibody against rTgTH demonstrated that the protein was expressed and located on the surface of RH tachyzoite. Freund's adjuvant was used to emulsify the rTgTH, which was subsequently applied to BALB/c mouse immune thrice on week 0, week 2, and week 4, respectively. The result of the animal challenge experiments showed an integral increase in IgG, IgG2a, IgG1, and IFN-γ, IL-4, and IL17 were as well significantly increased, and that the rTgTH vaccinated animals apparently had a prolonged survival time (14.30 ± 2.41) after infection with the RH strain of compared with that of the non-vaccinated control animals, which died within 11 days. Additionally, in the rTgTH vaccination group, the number of brain cysts (1275 ± 224) significantly decreased ( < 0.05) compared to the blank control group (2375 ± 883), and the size of the brain cysts in the animals immunized with rTgTH vaccination was remarkably smaller than that of the control mice. All the findings prove that TgTH played an important role in increasing the neurotransmitter dopamine in the host brain and could be used as a vaccine candidate antigen to mediate cell-mediated and humoral immunity.
嗜神经寄生虫（）感染可改变啮齿动物的行为，并导致人类出现神经心理症状，这可能与感染引起宿主大脑中神经递质多巴胺的变化有关。酪氨酸羟化酶（TgTH）是增加宿主大脑中神经递质多巴胺的一个重要因素。在本研究中，鉴定了TgTH催化底物生成多巴胺的酶活性以及TgTH的分子特征。为扩增开放阅读框（ORF），基于TgTH序列（GenBank登录号EU481510.1）设计聚合酶链反应（PCR）特异性引物，并将其插入pET-32a（+）中用于重组TgTH（rTgTH）的表达。序列分析表明，TgTH基因指导编码一个由565个氨基酸残基组成的62.4 kDa蛋白，预测其具有较高的抗原指数。酶活性测试表明，rTgTH以及分别从RH株和PRU株中提取的可溶性蛋白能够以剂量依赖的方式催化底物生成多巴胺，最佳催化温度为37℃。蛋白质印迹分析结果显示，感染的小鼠血清和rTgTH免疫后的大鼠血清分别成功检测到rTgTH和从RH速殖子体细胞中提取的天然TgTH。使用抗rTgTH抗体的免疫荧光分析表明，该蛋白在RH速殖子表面表达并定位。用弗氏佐剂乳化rTgTH，随后分别在第0周、第2周和第4周对BALB/c小鼠进行三次免疫。动物攻毒实验结果显示，IgG、IgG2a、IgG1以及IFN-γ、IL-4和IL17均整体增加，且rTgTH疫苗接种动物在感染RH株后明显比未接种疫苗的对照动物存活时间延长（14.30±2.41），对照动物在11天内死亡。此外，在rTgTH疫苗接种组中，脑包囊数量（1275±224）与空白对照组（2375±883）相比显著减少（<0.05），且用rTgTH疫苗免疫的动物脑包囊大小明显小于对照小鼠。所有这些发现证明，TgTH在增加宿主大脑中神经递质多巴胺方面发挥了重要作用，并且可作为候选疫苗抗原来介导细胞免疫和体液免疫。
