| 范长一,巩小惠,魏延钊,张雨琦,王姗姗,杜峰.基因工程疟原虫P.bzoGFP的构建及其生物特性[J].济宁医学院学报,2025,48(1):12-18 |
| 基因工程疟原虫P.bzoGFP的构建及其生物特性 |
| Construction and biological characterisation of a genetically engineered Plasmodium berghei P.bzoGFP |
| 投稿时间：2024-11-07 |
| DOI：10.3969/j.issn.1000-9760.2025.01.003 |
| 中文关键词: 疟疾;GFP;基因工程;动合子 |
| 英文关键词: Malaria;GFP;Genetic engineering;Ookinete |
| 基金项目: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) |
| 作者 | 单位 | E-mail | | 范长一 | 济宁医学院附属医院(临床医学院), 济宁 272000 | | | 巩小惠 | 济宁医学院附属医院(临床医学院), 济宁 272000 | | | 魏延钊 | 济宁医学院附属医院(临床医学院), 济宁 272000 | | | 张雨琦 | 济宁医学院口腔医学院, 济宁 272000 | | | 王姗姗 | 济宁医学院精准医学研究院, 济宁 272000 | | | 杜峰 | 济宁医学院基础医学院, 济宁 272000 | hurryman@126.com |
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| 中文摘要: |
| 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(χ2=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。 |
| 英文摘要: |
| 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 intra-erythrocytic 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 (χ2=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 antibody labeling. |
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