巨噬细胞鸟苷酸结合蛋白5在新孢子虫感染中的作用

Role of macrophage guanylate-binding protein 5 in Neospora caninum infection

探究新孢子虫感染对小鼠腹腔巨噬细胞鸟苷酸结合蛋白(mGBPs)表达的影响,以及mGBP5在新孢子虫感染中的作用。将新孢子虫速殖子感染小鼠腹腔巨噬细胞后,通过RT-PCR和Western blot检测,筛选出上调表达的mGBPs,构建pcDNA3.1-mGBP5真核表达载体并转染293T细胞,再通过Western blot验证mGBP5蛋白的过表达,最后感染新孢子虫并采用荧光定量PCR检测虫体数量。结果显示,新孢子虫感染引起mGBP1-mGBP11 mRNA表达水平升高,且mGBP5蛋白表达水平显著升高,并可诱导细胞质内散在分布的mGBP5向新孢子虫的纳虫泡膜聚集;成功构建pcDNA3.1-mGBP5真核表达载体并能够在293T细胞中表达;与空载体组相比,细胞中过表达mGBP5可显著减少胞内新孢子虫的数量。结果表明,新孢子虫感染小鼠腹腔巨噬细胞会导致mGBP5表达上调,并靶向新孢子虫的纳虫泡膜,从而起到抑制新孢子虫增殖的作用。

The purpose of this study is to investigate the effect of Neospora caninum(N. caninum) infection on the expression of guanylate-binding proteins(mGBPs) in mouse peritoneal macrophages and the role of mGBP5 in N. caninum infection.Peritoneal macrophages of mouse were infected with N. caninum tachyzoites.The expression of mGBPs were detected by RT-PCR and Western blot, then pcDNA3.1-mGBP5 eukaryotic expression vector was constructed and transfected into 293 T cells, the overexpression of mGBP5 was confirmed by Western blot.Finally, 293 T cells transfected with pcDNA3.1-mGBP5 or pcDNA3.1 vector were infected with N.caninum and the number of parasites was detected by RT-PCR.The results showed that the expression level of mGBP1-mGBP11 mRNAs increased after N. caninum infection, the expression level of mGBP5 protein was significantly increased, and the dispersed mGBP5 in the cytoplasm could be induced to aggregate into the vesicle of the parasitoid of N. caninum.The eukaryotic expression vector pcDNA3.1-mGBP5 was successfully constructed and expressed in 293 T cells.Compared with the empty vector group, the number of N.caninum in the overexpressed mGBP5 group was significantly reduced.Thus, it indicated that the up-regulated expression of mGBP5 in peritoneal macrophages of mice infected with N. caninum inhibits the proliferation of N. caninum by targeting the parasitoid vesicle.