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

以抗乙胺嘧啶的DHFR-TS和绿色荧光蛋白（GFP）基因为双重选择标记，建立了一种改进的表达外源基因的重组弓形虫的构建方法。这种改进的方法使得表达外源基因的重组弓形虫的构建具有极大的情况和速度。由于外源基因位于DHFR-TS和GFP基因之间，所有的耐药和荧光克隆都被证实是表达的外源基因。利用该方法构建了表达犬新孢子虫主要抗原SRS 2基因（Tg/NcSRS 2）和微小隐孢子虫主要抗原P23基因（Tg/CpP 23）的重组弓形虫。NcSRS 2和CpP 23在重组体Tg/NcSRS 2和Tg/CpP 23中的表达水平分别与犬诺氏菌和小隐孢子虫中的表达水平相似。重组蛋白NcSRS 2和CpP 23的分子量和抗原性分别与天然蛋白NaSRS 2和CpP 23相似。用重组Tg/NcSRS 2免疫的小鼠被部分保护免于犬奈瑟氏菌攻击感染。另一方面，用重组Tg/CpP 23免疫的小鼠产生针对小隐孢子虫的特异性中和抗体。此外，一种生物活性分子，如小鼠干扰素γ，成功地表达弓形虫载体。这些结果表明，弓形虫载体不仅可以为生产抗动物原生动物寄生虫感染的重组疫苗，而且还可以为生产生物活性分子提供新的工具。

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.