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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

25卷

期数:

2019年04期

页码:

324-328

栏目:

基础医学

出版日期:

2019-07-20

文章信息/Info

Title:

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

文章编号:

1006-8147(2019)04-0324-05

作者:

刘 影¹; 史小雨¹; 王 倩¹

（1.天津医科大学基础医学院免疫学系，天津300070）

Author(s):

LIU Ying¹;  SHI Xiao-yu¹;  WANG Qian¹

(1. Department of Immunology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China)

关键词:

伯式疟原虫; PbSEY1; pL0034; 疟原虫转染

Keywords:

Plasmodium;  PbSEY1;  pL0034;  Plasmodium transfection

分类号:

R784

DOI:

-

文献标志码:

A

摘要:

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T4 DNA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。

Abstract:

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.

参考文献/References:

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备注/Memo

备注/Memo:

基金项目 天津市教委科研计划项目（11601501/2016KJ0138）
作者简介 刘影（1993-），女，硕士在读，研究方向：免疫学；通信作者：王倩，E-mail: wangq@tmu.edu.cn。

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更新日期/Last Update: 2019-08-28