伯氏疟内质网塑形蛋白SEY1敲除质粒的构建及转染

Construction of Plasmodium berghei ER-shaping protein SEY1 knockout plasmid and Plasmodium transfection

目的:为了研究重要内质网塑形蛋白SEY1在疟原虫致病性方面的作用,本研究构建敲除伯氏疟原虫PbSEY1的质粒,结合疟原虫转染技术筛选PbSEY1缺失疟原虫突变体。方法:选取PbSEY1编码区中相邻的两个片段作为同源臂进行PCR扩增,并引入特定酶切位点,借助T4DNA连接酶和In-Fusion无缝克隆的方法将其插入载体pL0034中;利用疟原虫同步化培养及电转染技术将该质粒转入疟原虫,基于基因同源重组原理在疟原虫中敲除PbSEY1并进行药物筛选。结果:(1)获得可用于基因敲除及回复实验的重组质粒pL0034-△PbSey1;(2)在伯式疟原虫中敲除PbSEY1。结论:利用重组质粒pL0034-△PbSey1和疟原虫转染技术初步获得PbSEY1缺失疟原虫突变体,为进一步研究PbSEY1在疟原虫致病性中的作用提供了必要工具。

Objective: To investigate the role of endoplasmic reticulum-shaping protein SEY1 in Plasmodium virulence, and to construct pL0034-△PbSey1 plasmid to transfect it to Plasmodium berghei to obtain PbSEY1-deleted parasite strain. Methods:Two adjacent segments within PbSEY1 coding sequence were amplified by PCR as the homologous recombination arms and inserted into the pL0034 vector by T4 DNA ligase and In-Fusion cloning kit. The recombinant plasmid was transfected into Plasmodium berghei by synchronized culture and electro-transfection in order to delete PbSEY1 based on homologous recombination. Results:(1)The plasmid pL0034-△PbSey1 aiming at knockout and rescue experiments was obtained.(2) PbSey1 was deleted in Plasmodium berghei. Conclusion: Utilizing the recombinant plasmid pL0034-△PbSey1 and Plasmodium transfection, PbSey1-deleted parasites could be acquired, and may be a necessary tool for further study of PbSey1 in Plasmodium virulence.