【摘 要】
：
目的 构建并验证一种可用作生物免疫治疗载体的非复制尿嘧啶营养缺陷型弓形虫(NRTUA),并鉴定其表型.方法 通过经典的基于同源重组的基因敲除技术构建稳定的NRTUA虫株;构建p-OM1-HXGPRT-OM2、p-OM1-OM2和p-UP1-HXGPRT-UP2质粒用于敲除RHΔku80Δhxgprt弓形虫中的乳清苷-5\'-磷酸脱羧酶(OMPDC)基因和尿苷磷酸化酶(UP)基因,并通过电转染的方法转染到RHΔku80Δ hxgprt弓形虫中,最后经过药物筛选和极限稀释得到RHΔku80ΔompdcΔ
【机 构】
：
中山大学中山医学院,广东广州510080;中山大学附属第一医院,广东广州510080;中山大学中山医学院,广东广州510080;中山大学热带病防治研究教育部重点实验室,广东广州510080
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目的 构建并验证一种可用作生物免疫治疗载体的非复制尿嘧啶营养缺陷型弓形虫(NRTUA),并鉴定其表型.方法 通过经典的基于同源重组的基因敲除技术构建稳定的NRTUA虫株;构建p-OM1-HXGPRT-OM2、p-OM1-OM2和p-UP1-HXGPRT-UP2质粒用于敲除RHΔku80Δhxgprt弓形虫中的乳清苷-5\'-磷酸脱羧酶(OMPDC)基因和尿苷磷酸化酶(UP)基因,并通过电转染的方法转染到RHΔku80Δ hxgprt弓形虫中,最后经过药物筛选和极限稀释得到RHΔku80ΔompdcΔ up∷HXGPRT弓形虫(NRTUA虫株).利用PCR实验和全基因组测序对其进行基因结构验证,然后通过噬斑实验鉴定NRTUA虫株的细胞表型,并通过NRTUA虫株感染小鼠的毒力实验检测其在正常小鼠体内的毒性.结果 p-OM1-HXGPRT-OM2、p-OM1-OM2和p-UP1-HXGPRT-UP2质粒进行酶切鉴定,经NdeI酶切后,三种质粒酶切后的骨架DNA片段均为3.0 kb左右.RHΔku80Δompdc∷HXGPRT弓形虫和RHΔ ku80△ompdc△hxgprt弓形虫中OMPDC基因被敲除;而RHΔku80ΔompdcΔup∷HXGPRT弓形虫中OMPDC基因和UP基因均被敲除.其中敲除的OMPDC基因序列为3.3 kb左右,敲除的UP基因序列为3.1 kb左右,与理论值相吻合.成功构建并通过全基因组测序验证了弓形虫NRTUA株,噬斑实验显示NRTUA虫株在未添加尿嘧啶的培养基中没有噬斑形成,在添加尿嘧啶的培养基中能产生噬斑.小鼠毒力实验中,活的NRTUA虫株感染小鼠后直到实验周期结束无小鼠死亡,对照原株弓形虫感染小鼠后从第7～9天所有小鼠均死亡.结论 NRTUA虫株在体外尿嘧啶缺乏的条件下无法在宿主细胞内增殖且在正常小鼠体内不具有致死毒性,是一种减毒甚至基本无毒的弓形虫活疫苗,具有作为生物免疫治疗载体的应用潜力.
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