Development and applications of a protozoan parasite vector using Toxoplasma gondii to express heterologous genes

弓形虫表达异源基因的原生动物寄生虫载体的研制及应用

• 批准号: 14360172
• 负责人: XUAN Xuenan
• 金额: $ 9.34万
• 依托单位: Obihiro University of Agriculture and Veterinary Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14360172/
• 关键词: T.gondii; N.canunim; C.parvum; NcSRS2; CpP23; Recombinant vaccine; Vector vaccine; Protection; mIFNγ; トキソプラズマ; ネオスポラ; クリプトスポリジウム; TgSAG1; TgSAG2; NcSAG1; T.gondii; cDNAライブラリー; 組換えトキソプラズマ原虫; 遺伝子KO原虫

An improved method for the construction of recombinant Toxoplasma gondii expressing heterologous genes was developed using pyrimethamine-resistant DHFR-TS and green fluorescent protein (GFP) genes as double selection markers. This improved method allows the construction of recombinant T.gondii expressing foreign genes with great case and speed. Because the foreign genes were placed between the DHFR-TS and GFP genes, all of the drug-resistant and fluorescent clones were confirmed to be expressed foreign genes. This method was employed to construct recombinant T.gondii expressing the Neospora caninum major antigen SRS2 gene (Tg/NcSRS2) and the Cryptosporidium parvum major antigen P23 gene (Tg/CpP23). The expression levels of NcSRS2 and CpP23 in recombinant Tg/NcSRS2 and Tg/CpP23 were similar to those in N.caninum and C.parvum, respectively. The molecular weight and antigenic property of recombinant NcSRS2 and CpP23 were similar to those of native NaSRS2 and CpP23, respectively. Mice immunized with recombinant Tg/NcSRS2 were partially protected from N.caninum challenge infection. On the other hand, the mice immunized with recombinant Tg/CpP23 produced specific neutralizing antibodies against C.parvum. In addition, a bioactive molecule, such as mouse interferon gamma was successfully expressed by T.gondii vector. These results indicate that the T.gondii vector may provide a new tool not only for the production of recombinant vaccines against protozoan parasite infections in animals, but also for the production of bioactive molecules.

以抗乙胺药DHFR-TS和绿色荧光蛋白（GFP）基因为双选择标记，构建了表达异源基因的重组刚地弓形虫。这种改进的方法使表达外源基因的刚地弓形虫重组体的构建更加快捷。由于外源基因被置于DHFR-TS和GFP基因之间，所有耐药克隆和荧光克隆均被证实表达了外源基因。利用该方法构建了表达犬新孢子虫主抗原SRS2基因（Tg/NcSRS2）和细小隐孢子虫主抗原P23基因（Tg/CpP23）的重组弓形虫。重组Tg/NcSRS2和Tg/CpP23中NcSRS2和CpP23的表达水平分别与犬链球菌和细小链球菌相似。重组NcSRS2和CpP23的分子量和抗原性分别与天然的NaSRS2和CpP23相似。用重组Tg/NcSRS2免疫小鼠，可部分保护小鼠免受犬瘟病毒攻击感染。另一方面，用重组Tg/CpP23免疫的小鼠产生了针对小孢子虫的特异性中和抗体。此外，还成功地在弓形虫载体上表达了小鼠干扰素γ等生物活性分子。这些结果表明，弓形虫载体不仅可以为生产抗动物原生寄生虫感染的重组疫苗提供新的工具，而且可以为生产具有生物活性的分子提供新的工具。

项目成果

Tanaka, T., et al.: "The detection of bovine lactoferrin binding protein on Toxoplasma gondii"J.Vet.Med.Sci.. 65・12. 1377-1380 (2003)

Tanaka, T., et al.：“弓形虫上牛乳铁蛋白结合蛋白的检测”J.Vet.Med.Sci. 65・12（2003）。

Neospora caninum NcSRS2 is a transmembrance protein that contains a glycosylphosphatidylinositol anchor in insect cells.

犬新孢子虫 NcSRS2 是一种跨膜蛋白，在昆虫细胞中含有糖基磷脂酰肌醇锚。

• 发表时间: 2002
• 期刊: Vet.Parasitol. 109
• 作者: Nishikawa;Y.;Tragoolpua;K.;Makala;L.;Xuan;X.;Nagasawa;H.
• 通讯作者: H.

Koyama, T., et al.: "A 14-3-3 protein homologue is expressed in feline enteroepithelia-stages of Toxoplasma gondii"Vet.Parasitol.. 96.1. 65-74 (2001)

Koyama, T. 等人：“14-3-3 蛋白同源物在弓形虫的猫肠上皮阶段表达”Vet.Parasitol.. 96.1。

獣医微生物学

兽医微生物学

• 发表时间: 2012
• 作者: 見上彪;関崎勉;高井伸二;堀本泰介;望月雅美
• 通讯作者: 望月雅美

Huang, X,. et al.: "Development and evaluation of an ELISA with recombinant SAG2 for diagnosis of Toxoplasma gondii infection in cats"J.Parasitol.. 88・4. 804-807 (2002)

Huang, X, 等：“用于诊断猫弓形虫感染的重组 SAG2 ELISA 的开发和评估”J.Parasitol.. 88・4 (2002)。