新孢子虫GRA16和MYR2基因功能的初步研究

A preliminary study on the functions of the GRA16 and MYR2 genes in Neospora caninum

目的制备抗新孢子虫致密颗粒蛋白NcGRA16和原癌基因调控蛋白NcMYR2多克隆抗体,对NcGRA16和NcMYR2蛋白进行亚细胞定位;构建NcGRA16基因敲除虫株(△NcGRA16)和NcMYR2基因敲除虫株(△NcMYR2),探究NcGRA16和NcMYR2蛋白在虫体入侵、生长及致病中的作用。方法原核表达NcGRA16和NcMYR2蛋白,纯化后免疫小鼠,制备抗NcGRA16和NcMYR2蛋白多克隆抗体,免疫荧光法观察NcGRA16和NcMYR2蛋白亚细胞定位;运用CRISPR-cas9技术构建△NcGRA16和△NcMYR2虫株,通过噬斑试验、入侵试验、增殖试验、逸出试验和动物试验探究NcGRA16和NcMYR2蛋白在虫体入侵、生长及致病中的作用。结果成功制备抗NcGRA16和NcMYR2蛋白多克隆抗体,免疫荧光法观察NcGRA16蛋白定位于虫体致密颗粒中,NcMYR2蛋白定位于虫体高尔基体;成功构建△NcGRA16和△NcMYR2虫株,与野生虫株相比△NcGRA16虫株的噬斑面积减小(P<0.05);入侵率降低(P<0.05)。与感染野生虫株小鼠相比,感染△NcGRA16虫株的小鼠死亡时间延长3~5d;血清中IL-6和INF-γ显著下降(P<0.05);脑、心和脾荷虫量显著降低(P<0.05);脑和心脏病理变化减轻。感染△NcMYR2虫株的小鼠死亡时间延长1~2d;血清中IL-6水平显著下降(P<0.05)。结论NcGRA16为新孢子虫入侵和毒力相关基因,敲除NcMYR2基因对虫体的生长、入侵及毒力等无明显影响,这为揭示新孢子虫致病机制和新孢子虫病防控提供了理论依据。

Objectives To prepare polyclonal antibodies against Neospora caninum dense granule protein NcGRA16 and Myc-Regulation protein NcMYR2 in order to determine the subcellular localization of the NcGRA16 and NcMYR2 proteins.To generate an NcGRA16 knockout strain(△NcGRA16)and an NcMYR2 knockout strain(△NcMYR2)in order to study the functions of the NcGRA16 and NcMYR2 proteins in the invasion,growth,and pathogenesis of N.caninum.Methods Recombinant NcGRA16 and NcMYR2 proteins were expressed and purified.Mice were immunized with r-NcGRA16 and r-NcMYR2 proteins to produce polyclonal antibodies against the NcGRA16 and NcMYR2 proteins.Subcellular localization of the NcGRA16 and NcMYR2 proteins was determined using immunofluorescence.The CRISPR-cas9 genome editing system was used to generate NcGRA16 and NcMYR2 knockout strains,and the functions of the NcGRA16 and NcMYR2 proteins in the invasion,growth,and pathogenesis of N.caninum were analyzed through a plaque assay,invasion assay,proliferation assay,evasion assay,and animal experiments.Results Polyclonal antibodies against NcGRA16 and NcMYR2 proteins were successfully prepared.NcGRA16 protein was localized in dense granules of N.caninum,while NcMYR2 protein was localized in the Golgi apparatus recruited by N.caninum.NcGRA16 and NcMYR2 knockout strains were successfully generated,and the plaque area of the△NcGRA16 strain(821 pixels)and its rate of invasion(40.44%)decreased compared to the wild type strain(P<0.05).The△NcMYR2 strain did not differ significantly in terms of plaque area,invasion,proliferation,to evasion compared to the wild type strain.Compared to mice infected with the wild type strain,the time of death of mice infected with△NcGRA16 strain was prolonged by 3-5 dand the secretion of IL-6(29.2 pg/ml)and INF-γ(3505 pg/ml)in serum decreased(P<0.05).The parasite burden in the brain(106.7/200 ng DNA),heart(135.3/200 ng DNA)and spleen(4.7/200 ng DNA)decreased(P<0.05),and pathological changes in the brain and heart were alleviated.The time of death of mice infected with the△NcMYR2strain was prolonged by 1-2 d,and serum IL-6 secretion(33.5 pg/ml)decreased(P<0.05).Conclusion NcGRA16 is an invasion and virulence-related gene of N.caninum,and knockout of the NcMYR2 gene has no significant effect on the growth,invasion,or virulence of N.caninum.These findings provide the theoretical basis for revealing the pathogenesis of N.caninumand the prevention and control of neosporosis.