胞嘧啶鸟嘌呤二核苷酸-脱氧寡核苷酸抑制疟原虫红前期发育

Inhibition of CpG Oligodeoxynucleotide on the Development of Pre-erythrocytic Stage of Plasmodium

目的观察胞嘧啶鸟嘌呤二核苷酸-脱氧寡核苷酸(cytidine-phosphate-guanosine oligodeoxynucleotide,CpG ODN)对疟原虫红前期发育的影响。方法通过构建约氏疟原虫BY265株18S rRNA外标准品质粒,与小鼠三磷酸甘油醛脱氢酶(GAPDH)内标准品共同组成TaqMan RT-PCR分析模型。用不同剂量(0.05×105、0.1×105、0.5×105、1×105和2×105个)约氏疟原虫唾液腺子孢子感染BALB/c小鼠,42h后处死小鼠取其肝脏,制备约氏疟原虫cDNA进行TaqMan RT-PCR反应,以小鼠肝脏虫荷指标检验模型的有效性。将12只BALB/c小鼠随机均分为CpG组、CpG对照组和PBS对照组,CpG组和CpG对照组小鼠分别尾静脉注射脱氧寡核苷酸1826(ODN1826)及其对照(ODN1826 control)各30μg,PBS对照组注射0.01mol/LPBS溶液200μl。24h后各组每鼠感染100个子孢子,于感染后42h处死小鼠取肝脏,制备约氏疟原虫cDNA进行TaqMan RT-PCR,定量分析感染疟原虫24h前CpGODN预处理小鼠肝脏虫荷的变化。结果构建的约氏疟原虫外标准品质粒所插入的BY265株18Sr RNA基因与17XNL株18Sr RNA基因相似性为98%,它与小鼠GAPDH内标准品共同组成的TaqMan RT-PCR分析模型能够正确反映小鼠肝脏虫荷与唾液腺子孢子感染量的正比关系。感染疟原虫24h前给予CpG ODN处理,CpG组小鼠肝脏虫荷为CPG对照组的1/5(0.28/1.33),两者差异有统计学意义(P<0.05)。结论本实验建立的TaqMan RT-PCR分析模型适用于红前期疟原虫(肝脏)虫荷指标的研究。CpG ODN能显著抑制红前期疟原虫的发育。

Objective To study the role of cytidine-phosphate-guanosine oligodeoxynucleotide （CpG ODN） on the development of Plasmodium liver stage. Methods Plasmodium yoelii BY265 18S rRNA was cloned, and the TaqMan real-time PCR was established on P. yoelii BY265 18S rRNA and mouse GAPDH as quantitative analysis model. The model was tested by the level of liver Plasmodium load with the liver eDNA in BALB/c mice infected by salivary gland sporozoites for 42 hours. Twelve BALB/c mice were randomly divided into CpG group, CpG control group and PBS control group which were injected respectively by ODN1826 30 μg, ODN1826 control 30 μg and 0.01 mol/L PBS 200 μl via vena caudalis. Twenty-four hours later, each mouse was inoculated with 100 sporozoites. Mice were sacrificed in 42 hours after infection, and the liver load of Plosmodium was analyzed by TaqMan real-time PCR. Results The cloned Py BY265 18S rRNA gene showed 98% similarity to Py 17XNL. The quantitative analysis model consisted by 18S rRNA and GAPDH showed positive correlation between the level of liver Plasmodium load and the sporozoite inoculation dose to mice. The Plasmodium load in CpG ODN pre-treated mice was reduced to one fifth of the control group （0.28/1.33） （P〈0.05）. Conclusion The quantitative analysis model of TaqMan RT-PCR can detect the liver load of Plasmodium parasites, and CpG ODN can inhibit the development of its pre-erythrocytic stage.