伯氏疟原虫红细胞膜相关蛋白1缺失突变体的构建和鉴定  

Construction and identification of plasmodium berghei erythrocyte membrane associated protein 1 deletion mutant

作　　者：魏超 史小雨 王倩 WEI Chao;SHI Xiao-yu;WANG Qian(Department of Immunology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学基础医学院免疫学系,天津300070

出　　处：《天津医科大学学报》2022年第2期135-139,共5页Journal of Tianjin Medical University

基　　金：国家自然科学基金面上项目(32070701)。

摘　　要：目的:研究伯氏疟原虫输出蛋白——红细胞膜相关蛋白1(EMAP1)在疟原虫生长发育和侵染中的作用,构建EMAP1缺失的伯氏疟原虫突变体。方法:以pL0035质粒为载体,PCR扩增EMAP1编码区上游和下游序列作为同源臂,用酶切连接和无缝克隆的方法将两条同源臂分别插入pL0035,获得重组质粒pL0035-ΔPbEMAP1;利用疟原虫转染技术在伯氏疟原虫中敲除EMAP1,有限稀释法筛选EMAP1基因敲除的单克隆虫株,PCR和Southern印迹鉴定基因型;定制抗PbEMAP1多克隆抗体,利用Western印迹鉴定EMAP1缺失疟原虫。结果:(1)成功构建用于敲除PbEMAP1的重组质粒pL0035-ΔPbEMAP1。(2)获得能有效识别PbEMAP1蛋白的抗体。(3)基因型和蛋白水平鉴定重组疟原虫为PbEMAP1缺失的伯氏疟原虫单克隆虫株。结论:本研究构建的PbEMAP1缺失伯氏疟原虫突变体,为研究PbEMAP1在疟原虫发育和侵染中的作用提供了必要的工具。Objective:To investigate the role of Plasmodium berghei exported protein PbEMAP1 in the development and invasion of malaria parasites,to construct PbEMAP1-deleted parasite strain.Methods:The upstream and downstream non-coding sequences of PbEMAP1 coding region were amplified as the homologous recombination arms using pL0035 plasmid as vector.The two homologous arms were inserted into pL0035 vector by T4 DNA ligase and HiFi DNA Assembly kit in order to obtain the recombinant plasmid pL0035-ΔPbEMAP1.The plasmid pL0035-ΔPbEMAP1 was transfected into Plasmodium berghei,by electro-transfection,and the single clone ofΔPbEMAP1 parasite strain were screened by limiting dilution.The genotype ofΔPbEMAP1 parasite was determined by PCR and Southern blotting,and the protein expression level was detected by Western blotting using the customized polyclonal antibody against PbEMAP1.Results:(1)The recombinant plasmid pL0035-ΔPbEMAP1 for knocking out PbEMAP1 was successfully constructed.(2)Antibodies that could effectively recognize PbEMAP1 were obtained.(3)The recombinant Plasmodium was identified as a single clone ofΔPbEMAP1 deletion parasite of Plasmodium berghei by genotype and protein level.Conclusion:The PbEMAP1 deletion mutant of Plasmodium berghei constructed in this study provides a necessary tool for studying the role of PbEMAP1 in the development and infection of Plasmodium.

关 键 词：伯氏疟原虫 输出蛋白 红细胞膜相关蛋白1 

分 类 号：R382.31[医药卫生—医学寄生虫学]