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

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...

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

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 of ΔPbEMAP1 parasite strain were screened by limiting dilution. The genotype of ΔPbEMAP1 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 of ΔPbEMAP1 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.