Lv Qiang, Xing Shenyang, Gong Pengtao, Chang Le, Bian Zhengzheng, Wang Lidong, Zhang Xichen, Li Jianhua
College of Veterinary Medicine of Jilin University, 5333 Xian Road, Changchun 130062, China; Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China.
College of Animal Science of Jilin University, 5333 Xian Road, Changchun 130062, China.
Exp Parasitol. 2015 Jan;148:56-65. doi: 10.1016/j.exppara.2014.10.006. Epub 2014 Nov 4.
Neosporosis is an intracellular protozoan disease caused by Neospora caninum. Until now, there is no effective vaccine to prevent neosporosis. The host cell binding protein has the potential as neosporosis vaccine. In the present study, a T7 phage display library was constructed and screened using Vero cells to obtain host cell binding protein of N. caninum. Two host cell binding proteins, a hypothetical protein of 78 kDa (named as NcP78) homologous to the acylglycerol lipase of Toxoplasma gondii ME49 (XP_002370319.1) and NcGRA7 (known as a dense granules protein that is involved in the invasion of N. caninum to the host cells), were identified. Immune responses induced by recombinant NcP78 and NcGRA7 proteins and their protective efficacies against homologous challenge in BALB/c mice were evaluated respectively. Results showed that recombinant NcP78 and NcGRA7 could elicit both Th1 and Th2 immune responses (with the elevated levels of IgG1 and IgG2a antibody), but predominately a Th2 immune response with a high level of IgG1. The ani-NcP78 and anti-NcGRA7 serum also had inhibitory effects on N. caninum invasion to Vero cells in vitro, which indicated that both NcP78 and NcGRA7 proteins were involved in host cell invasion. Recombinant NcP78 and NcGRA7 could not prolong the survival times and improve the survival rates of dams, but could prolong the survival times and improve the survival rates of offspring significantly. Moreover, the recombinant NcP78 and NcGRA7 could reduce the brain parasite load of dams and offspring. Though these protein vaccines could not effectively alleviate the symptom of abortion, they could increase the number of born offspring significantly, indicating that Nc78 and NcGRA7 recombinant proteins could provide a partial protection against N. caninum infection in mice.
新孢子虫病是由犬新孢子虫引起的一种细胞内原生动物疾病。到目前为止，尚无有效的疫苗来预防新孢子虫病。宿主细胞结合蛋白有作为新孢子虫病疫苗的潜力。在本研究中，构建了一个T7噬菌体展示文库，并使用Vero细胞进行筛选，以获得犬新孢子虫的宿主细胞结合蛋白。鉴定出了两种宿主细胞结合蛋白，一种是与刚地弓形虫ME49的酰基甘油脂肪酶（XP_002370319.1）同源的78 kDa假设蛋白（命名为NcP78）和NcGRA7（一种已知的致密颗粒蛋白，参与犬新孢子虫对宿主细胞的侵袭）。分别评估了重组NcP78和NcGRA7蛋白诱导的免疫反应及其对BALB/c小鼠同源攻击的保护效果。结果表明，重组NcP78和NcGRA7均可引发Th1和Th2免疫反应（IgG1和IgG2a抗体水平升高），但主要是高水平IgG1的Th2免疫反应。抗NcP78和抗NcGRA7血清在体外对犬新孢子虫侵袭Vero细胞也有抑制作用，这表明NcP78和NcGRA7蛋白均参与宿主细胞侵袭。重组NcP78和NcGRA7不能延长母鼠的存活时间和提高其存活率，但可显著延长后代的存活时间并提高其存活率。此外，重组NcP78和NcGRA7可降低母鼠和后代大脑中的寄生虫负荷。虽然这些蛋白疫苗不能有效缓解流产症状，但可显著增加出生后代的数量，表明Nc78和NcGRA7重组蛋白可为小鼠抵抗犬新孢子虫感染提供部分保护。
