弓形虫pcDNA3.1(+)-Flag-ROP18质粒构建及对RAW264.7细胞凋亡影响研究  

Construction of recombinant plasmid pcDNA3.1(+)-Flag-ROP18 of Toxoplasma gondii and study the effect on apoptosis of RAW264.7 cells

作　　者：刘俊[1] 孙涛 LIU Jun;SUN Tao(Department of Laboratory Medicine,the First Affiliated Hospital of Shaoyang University,Shaoyang,Hunan 422000;Hematologic Oncology Lab,Shaoyang Central Hospital,Shaoyang,Hunan 422000,China)

机构地区：[1]邵阳学院附属第一医院检验科,湖南邵阳422000 [2]邵阳市中心医院血液肿瘤实验室,湖南邵阳422000

出　　处：《热带医学杂志》2021年第9期1125-1128,F0003,共5页Journal of Tropical Medicine

基　　金：湖南省教育厅科学研究项目(17C1419)。

摘　　要：目的构建pcDNA3.1(+)-Flag-ROP18真核表达载体,体外研究弓形虫ROP18蛋白是否具有抑制H2O2诱导RAW264.7细胞凋亡的作用。方法通过PCR扩增ROP18基因,构建pcDNA3.1(+)-Flag-ROP18真核表达载体,转染RAW264.7细胞,为重组质粒组;并设置未含POP18基因的空白质粒为对照组。分别检测重组质粒组与对照组线粒体及细胞质中细胞色素c含量。结果成功构建pcDNA3.1(+)-Flag-ROP18重组质粒,通过双酶切鉴定,得到2个符合预期大小的条带。使用H2O2分别诱导重组质粒组与对照组对RAW264.7细胞的凋亡作用,分别检测线粒体及细胞质中细胞色素c含量,发现两组细胞色素c含量水平无区别。结论ROP18没有抑制H2O2经线粒体途径诱导RAW264.7细胞凋亡,为进一步探索ROP18蛋白功能及弓形虫病的发病机制提供了理论依据。Objective The pcDNA3.1(+)-Flag-ROP18 eukaryotic expression vector was constructed to study whether Toxoplasma gondii ROP18 protein can inhibit H2O2 induced apoptosis of RAW264.7 cells in vitro.Methods The ROP18 gene was amplified by PCR,pc DNA3.1(+)-Flag-ROP18 eukaryotic expression vector was constructed and transfected into RAW264.7 cells.The recombinant plasmid group and control group were set up to detect the content of cytochrome C in mitochondria and cytoplasm of recombinant plasmid group and control group respectively.Results The recombinant plasmid pc DNA3.1(+)-Flag-ROP18 was successfully constructed and identified by double enzyme digestion,and two bands of expected size were obtained.H2O2 was used to induce apoptosis of RAW264.7 cells in the recombinant plasmid group and the control group,and the cytochrome C content was detected separately,and there was no difference in the cytochrome C content level between the two groups.Conclusions ROP18 did not inhibit the apoptosis of RAW264.7 cells induced by H2O2 via the mitochondrial pathway.This study provided a theoretical basis for further exploring the function of ROP18 protein and the pathogenesis of toxoplasmosis.

关 键 词：弓形虫 ROP18蛋白 凋亡 

分 类 号：R382.5[医药卫生—医学寄生虫学]