疟原虫纯化新方法的研究

A new method for Plasmodium purification

目的:拟应用去白细胞输血器,建立一种快速简便纯化疟原虫的新方法,以去除宿主白细胞DNA干扰。方法:建立Pb ANKA株鼠疟模型;39只小鼠分为A组(新鲜虫血组,n=18)、B组(冻存虫血组,-80℃保存,n=16)及C组(正常对照组,n=5);应用去白细胞输血器对血样进行过滤纯化,观察比较过滤速度及血样处理(新鲜、冻存)对去除白细胞效果的影响;血细胞计数板和血涂片吉姆萨染色法计数各样本过滤前后白细胞、红细胞、原虫率并观察疟原虫形态和疟原虫密度。滤过后的血液提取DNA,PCR验证白细胞滤过效果。结果:经血细胞计数板计数,3组白细胞平均去除率比较,差异无统计学意义(P>0.05);C组红细胞的回收率高于A组,差异具有统计学意义(P<0.05);B组则仅见原虫。白细胞过滤后疟原虫形态及密度无明显改变。PCR鉴定PbGAPDH结果 A、B组均为阳性,而小鼠GAPDH均为阴性。结论:建立了白细胞输血器一步法纯化疟原虫的新方法。该方法简便、快速、廉价,是进行疟原虫分子生物学、免疫学等方面的研究不可或缺的实用技术。

Objective. The primal problem for existing methods of plasmodium purification and concentration is complicated or expensive. In order to overcome the defects, it is necessary to develop a simple and effective method to suppress interference of host DNA in the malaria parasites research. Methods. Plasmodium berghei （Pb） ANKA strain mouse model has been set up for the experiment. Total of 39 Kunming mice were divided into three groups, fresh parasitemia blood samples （group A, n= 18）, frozen parasitemia blood samples （group B, n= 16） and normal blood control （group C, n= 5）. Parasitemia blood samples were collected from Pb infected mice 6d to 9d after inoculating and were treated with 0.1 heparin sodium. Each sample （〈1 mL） was diluted with RPMI 1640 to 5 mL and filtered with leukocyte removing filters made with non-woven fabric for blood transfusion. Observing and comparing the influence of filtration velocity and blood processing （fresh and frozen） for the plasmodium purification. Leukocyte removal rate, erythrocyte recovery rate,Pb infected red blood cell rate, Pb density and morphology were examined and analyzed by hemocytometer and/or Giemisa-stained blood smear method. DNAs were extracted from filtered blood samples. PCR was conducted for verifying effects of filtration. Results： No significant difference in WBC removal rate was found among group A, B and C. The RBC recovery rate of group C （93.71±1.74） % was higher than that of group A （78.48±3.87） % （P〈0.05）. RBC could not be seen in frozen parasitemia blood samples （group B）. The density and morphology of Pb between A and B groups had no noticeable difference after filtration. PCR results for Pb GAPDH gene amplification were positive, while negative for mouse GAPDH. Conclusion： One-step method for Plasmodium purification and concentration by using leukocyte removal filters for blood transfusion has been developed. It is a simple, fast, cheap, efficient technique for the molecular and immune study of malaria parasite.