摘要
目的利用规律间隔成簇短回文重复序列关联蛋白9(CRISPR/Cas9)技术构建刚地弓形虫(Toxoplasma gondii)类枯草杆菌蛋白酶基因sub3敲除株并进行体外表型分析,探究TgSUB3对弓形虫黏附、入侵和生长繁殖的作用。方法利用定点突变方法将pSAG1::CAS9⁃U6::sgUPRT中的sgUPRT突变为Tgsub3的单向导RNA(sgRNA),构建sub3基因敲除质粒pSAG1::CAS9⁃U6::sgSUB3。PCR扩增带有sub3上下游40 bp同源臂的二氢叶酸还原酶抗性供体片段,将质粒和供体片段通过电转化的方式导入弓形虫RHΔku80速殖子,经过乙胺嘧啶抗性选择和单克隆筛选,PCR鉴定敲除株RHΔku80Δsub3(Δsub3)。鉴定正确的敲除株Δsub3进行噬斑试验、入侵试验和增殖试验,分析其体外表型,以RHΔku80为对照,使用软件GraphPad Prism 9进行统计学分析。结果经PCR鉴定,在Δsub3中扩增到的条带大小符合预期,Δsub3敲除株构建成功。噬斑试验结果显示,RHΔku80和Δsub3形成的噬斑面积分别为(60.42±23.20)任意单位和(2.21±1.89)任意单位,二者差异有统计学意义(t=17.79,P<0.01);入侵实验结果显示,RHΔku80和Δsub3的入侵效率分别为(37.94±18.18)%和(22.97±15.36)%,差异无统计学意义(t=0.89,P>0.05)。增殖试验结果显示,RHΔku80和Δsub3纳虫泡中含有8个及以上速殖子的纳虫泡数量分别为(56.33±8.58)%和(39.67±11.84)%;含有4个及以下速殖子的纳虫泡数量分别为(43.67±8.58)%和(60.33±11.84)%,与RHΔku80虫株相比,纳虫泡内速殖子个数显著减少(F=17.93,P<0.01)。结论成功构建弓形虫Δsub3敲除株,TgSUB3的缺失影响弓形虫速殖子的生长繁殖。
Objective To generate the Toxoplasma gondii sub3(Tgsub3)gene knockout strain using the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR⁃associated protein 9(Cas9)system,investigate the in⁃vitro phenotypes of the Tgsub3 gene knockout strain,and examine the effect of Tgsub3 gene on adhesion,invasion and proliferation of T.gondii.Methods SgUPRT on the pSAG1::CAS9U6::SgUPRT plasmid was mutated to single guide RNA(sgRNA)using site mutation,and the pSAG1::CAS9⁃U6::sgSUB3 plasmid with the sub3 gene knockout was generated.The DHFR resistant donor fragments containing 40 bp upstream and downstream homology arms of the sub3 gene were amplified,and the sub3 gene knockout plasmid and donor fragments were co⁃transfected into T.gondii by electroporation.Following resistance selection by pyrimethamine and monoclonal screening,the sub3 gene knockout strain RHΔku80Δsub3(Δsub3)was identified using PCR assay.The in⁃vitro phenotypes of theΔsub3 strain were analyzed with plaque,invasion,and proliferation assays.Using RHΔku80 strain as a control,all statistical analyses were conducted using the software GraphPad Prism 9.Results PCR assay identified bands with expected sizes in theΔsub3 strain,indicating successful generation of theΔsub3 strain.Plaque assay showed that the sizes of plaques formed by RHΔku80 andΔsub3 strains were(60.42±23.20)au and(2.21±1.89)arbitrary unit,respectively(t=17.79,P<0.01),and invasion assay showed that the invasion efficiencies of RHΔku80 andΔsub3 strains were(37.94±18.18)%and(22.97±15.36)%,respectively(t=0.89,P>0.05).Proliferation assay showed that the proportions of parasitophorous vacuoles containing 8 and more tachyzoites of RHΔku80 andΔsub3 strainswere(56.33±8.58)%and(39.67±11.84)%,respectively,and the proportions of parasitophorous vacuoles containing 4 and fewer tachyzoites of RHΔku80 andΔsub3 strains were(43.67±8.58)%and(60.33±11.84)%,respectively.Compared with control strain,the number of tachyzoites within the parasitophorous vacuole was significantly decreased(F=17.93,P<0.01).Conclusion TheΔsub3 gene knockout strain is successfully generatedand absence of the Tgsub3 gene affects the growth and reproduction of T.gondii tachyzoites.
作者
王龙江
吴燕
李瑾
谢金晶
张欣
孙慧
WANG Longjiang;WU Yan;LI Jin;XIE Jinjing;ZHANG Xin;SUN Hui(Shandong Institute of Parasitic Diseases,Shandong First Medical University(Shandong Academy of Medical Sciences),Jining 272033,Shandong,China)
出处
《中国寄生虫学与寄生虫病杂志》
北大核心
2025年第3期324-328,共5页
Chinese Journal of Parasitology and Parasitic Diseases
基金
山东省自然科学基金(ZR2022MH271)
山东省医药卫生科技项目(202301011242、202101050153)。
作者简介
王龙江(ORCID:0000⁃0003⁃0439⁃1945),男,硕士,助理研究员,从事寄生虫病防治研究。E⁃mail:ljwang880108@163.com;通信作者:孙慧(ORCID:0009⁃0002⁃3967⁃6393),女,博士,副研究员,从事寄生虫病防治。E⁃mail:sunhui123aq@126.com。
