恶性疟原虫基因稳定转染方法的研究进展  

Research progress on stable gene transfection methods of Plasmodium falciparum

作　　者：李晓松 潘茂华 黄亚铭 杨照青[1] LI Xiao-song;PAN Mao-hua;HUANG Ya-ming;YANG Zhao-qing(Department of Pathogen Biology and Immunology,Kunming Medical University,Kunming,Yunnan 650500,China;Shanglin County People's Hospital,Nanning,Guangxi 530599,China)

机构地区：[1]昆明医科大学病原生物学与免疫学系,云南昆明650500 [2]广西壮族自治区上林县人民医院,广西南宁530599

出　　处：《中国热带医学》2023年第2期186-190,共5页China Tropical Medicine

基　　金：国家自然科学基金项目(No.U1802286,No.31860604);国际科技合作专项(No.202003AE140004)。

摘　　要：恶性疟原虫(Plasmodium falciparum)的转染有助于研究其基因的功能,例如抗药性。但转基因操作一直都非常具有挑战性,主要是由于疟原虫基因组的高A/T碱基序列结构(A+T含量约为82%)以及低转染效率。基于电穿孔的恶性疟原虫转染方法已成功应用于某些基因的研究中,而预载法的电穿孔是目前将外源DNA引入恶性疟原虫的首选方法。疟原虫基因的定点编辑多采用双质粒转染的方法,通常认为对疟原虫的成功转染需要大量高纯度质粒DNA以及精确的转染体系。本文除对目前常用的电转方法进行了评价外,还介绍了一种新的转染方法,即裂解-再密封红细胞转染(lyse-reseal erythrocytes for transfection),并对质粒DNA浓度、转染试剂的使用、转染参数的设置、新鲜红细胞的添加以及转染成功的标记等因素在提高疟原虫转染成功率和效率方面的作用等进行综述,为该领域研究提供参考。Transfection of Plasmodium falciparum is helpful to study the function of its genes, such as drug resistance.However, transgenic manipulation has been very challenging, mainly due to the high A/T base sequence structure(A+T content of about 82%) and low transfection efficiency of the Plasmodium genome. Electroporation-based transfection of Plasmodium falciparum has been successfully applied in the study of certain genes, and electroporation by preloading is currently the preferred method for introducing foreign DNA into Plasmodium falciparum. The site-directed editing of Plasmodium genes mostly adopts the method of two-plasmid transfection. It is generally believed that successful transfection of Plasmodium requires a large amount of high-purity plasmid DNA and an accurate transfection system. In addition to the evaluation of the current commonly used electrotransfection methods, this paper also introduces a new transfection method, namely lyse-reseal erythrocytes for transfection(LyRET). This paper also review the role of factors such as plasmid DNA concentration, the use of transfection reagents, the setting of transfection parameters, the addition of fresh red blood cells, and the markers of successful transfection in improving the success rate and efficiency of Plasmodium transfection, in the hope of providing a reference for study in this field.

关 键 词：恶性疟原虫 稳定转染 效率 质粒DNA 裂解-再密封红细胞转染 

分 类 号：R382.31[医药卫生—医学寄生虫学]