摘要
目的构建动合子阶段表达绿色荧光蛋白的基因工程疟原虫,为该阶段相关的研究提供工具虫株。方法构建含有GFP基因序列和pb25基因位点重组同源臂的打靶载体,线性化后电转染至伯氏疟原虫P.berghei。经药物筛选后使用有限稀释法进行克隆,利用PCR进行基因型鉴定。通过观察记录感染小鼠的发病情况、疟疾血症、红内期各阶段和合子、动合子的形态发育,分析基因工程疟原虫的生物学特性。结果基因工程伯氏疟原虫P.bzoGFP,经PCR基因型鉴定与预期一致,且能在动合子阶段表达绿色荧光蛋白。P.bzoGFP与野生型疟原虫在红细胞内的形态和体外培养合子、动合子的形态均无差异,感染小鼠的生存时间无统计学差异(χ^(2)=0.309,P>0.05),体外培养动合子的转化情况无显著差别(t=0.341,P>0.05)。结论成功构建基因工程疟原虫P.bzoGFP,其生物学特性与野生型疟原虫无明显不同,能够在合子形成后有效表达GFP,无需抗体标记,可直接用于疟疾传播阻断效果的评估等此阶段相关研究。
Objective To construct a genetically engineered Plasmodium berghei strain that expresses green fluorescent protein at the ookinete stage,providing a tool for studies related to this stage.Methods A targeting vector comprising the GFP gene sequence and the recombinant homology arms of the pb25 gene locus was constructed,linearised and then electro-transfected into Plasmodium berghei.Following drug screening,cloning was carried out using limited dilution,and genotype of parasites was detected using PCR.The biological characteristics of the genetically engineered Plasmodium berghei were analyzed through observation of the morphological characteristics of the intraerythrocytic phase,zygote and ookinete phase and by comparing the parasitemia and survival of mice infected with WT and genetically engineered parasites.Results Genetically engineered Plasmodium berghei P.bzoGFP,genotyped by PCR,exhibited consistent results with the predicted outcomes and demonstrated the capacity to express green fluorescent protein at the ookinete stage.P.bzoGFP did not display any significant deviations from the wild strain with respect to the morphology of the intra-erythrocytic phase,the zygote and ookinete phase.The survival curves of the two groups did not differ significantly(χ^(2)=0.309,P>0.05).The number of ookinetes obtained from in vitro culture was not statistically significant(t=0.341,P>0.05).Conclusion The genetically engineered Plasmodium berghei strain P.bzoGFP has been successfully constructed,exhibiting biological characteristics that are not significantly different from those of the wild-type parasite.This strain can effectively express GFP after zygote formation,enabling its direct use in studies related to this phase,such as evaluating the efficacy of malaria transmission blockade,without the need for anti-body labeling.
作者
范长一
巩小惠
魏延钊
张雨琦
王姗姗
杜峰
FAN Changyi;GONG Xiaohui;WEI Yanzhao;ZHANG Yuqi;WANG Shanshan;DU Feng(Affiliated Hospital of Jining Medical University(School of Clinical Medicine);School of Stomatology,Jining Medical University,Jining 272000,China;Department of Pathology,Institute of Precise on Medicine,Jining Medical University,Jining 272000,China;School of Basic Medicine,Jining Medical University,Jining 272000,China)
出处
《济宁医学院学报》
2025年第1期12-18,共7页
Journal of Jining Medical University
基金
国家自然科学基金项目(82101867)
济宁医学院大学生创新训练计划项目(cx2023077z)。
关键词
疟疾
GFP
基因工程
动合子
Malaria
GFP
Genetic engineering
Ookinete
作者简介
通信作者:杜峰,Email:hurryman@126.com。
