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| 伯氏疟原虫静息巯基氧化酶表达阶段的确定 |
| 郑文琪1,2,冯学敏2,刘飞3,曹雅明3,韩艳秋1,王俊瑞1* |
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(1. 内蒙古医科大学附属医院检验科, 呼和浩特 010050;
2. 内蒙古医科大学附属医院外科实验室, 呼和浩特 010050;
3. 中国医科大学基础医学院免疫学教研室, 沈阳 110021
*通信作者) |
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| 摘要: |
| 目的 观察伯氏疟原虫静息巯基氧化酶（PbQSOX）的表达阶段及表达特点。方法 分别采用SignalP-5.0 Server与TMHMM Server 2.0在线软件对PbQSOX蛋白的信号肽与跨膜区进行预测分析。利用同源重组技术，将PbQSOX-HA标签型打靶质粒经限制性内切酶NotⅠ酶切使其线性化，将线性化的质粒电转染至伯氏疟原虫株内并感染小鼠。经乙胺嘧啶筛选耐药型疟原虫血症小鼠，取小鼠外周血进行PCR鉴定，获取PbQSOX-HA标签型疟原虫。培养纯化裂殖体、配子体与动合子阶段的PbQSOX-HA标签型疟原虫，采用蛋白质印迹法与间接免疫荧光实验检测PbQSOX蛋白在伯氏疟原虫中的表达阶段与表达特点。结果 生物信息学分析发现PbQSOX蛋白存在信号肽，不存在跨膜区，蛋白均在细胞膜外表达。经PCR鉴定，PbQSOX-HA标签型打靶质粒与伯氏疟原虫同源重组正确，并成功获得了PbQSOX-HA标签型疟原虫及纯化的PbQSOX-HA标签型裂殖体、配子体与动合子。蛋白质印迹法检测显示PbQSOX蛋白主要在配子体与动合子阶段表达，间接免疫荧光实验结果显示PbQSOX表达于配子体、雄配子、合子、retort与动合子表面。结论 PbQSOX是伯氏疟原虫在有性生殖阶段表达的蛋白，主要表达于配子体、雄配子、合子、retort与动合子表面。 |
| 关键词: 伯氏疟原虫 基因打靶 表达阶段 伯氏疟原虫静息巯基氧化酶 |
| DOI：10.16781/j.0258-879x.2021.07.0731 |
| 投稿时间：2019-11-15修订日期：2020-03-31 |
| 基金项目:国家自然科学基金（81760367），内蒙古自治区高等学校"青年科技英才计划"（NJYT-20-B14），内蒙古自治区自然科学基金（2020MS08126）. |
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| Expression stage analysis of Plasmodium berghei quiescin sulfhydryl oxidase |
| ZHENG Wen-qi1,2,FENG Xue-min2,LIU Fei3,CAO Ya-ming3,HAN Yan-qiu1,WANG Jun-rui1* |
(1. Department of Laboratory Medicine, Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, Inner Mongolia Autonomous Region, China;
2. Laboratory of Surgery, Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, Inner Mongolia Autonomous Region, China;
3. Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang 110021, Liaoning, China
*Corresponding author) |
| Abstract: |
| Objective To observe the expression stages and characteristics of Plasmodium berghei quiescin sulfhydryl oxidase (PbQSOX). Methods SignalP-5.0 Server and TMHMM Server 2.0 were used to predict and analyze the signal peptide and transmembrane regions of PbQSOX protein. With homologous recombination, PbQSOX-HA tag plasmids were linearized by restriction enzyme NotⅠ, and the linearized plasmids were electroporated into Plasmodium berghei strain and infected mice. The PbQSOX-HA tag Plasmodium berghei was obtained from the peripheral blood of pyrimethamine resistant mice and was identified by polymerase chain reaction (PCR). Schizonts, gametocytes and ookinetes of PbQSOX-HA tag Plasmodium berghei were purified. Western blotting and indirect immunofluorescence assay (IFA) were used to detect the expression stages and characteristics of PbQSOX protein in Plasmodium berghei. Results Bioinformatics analysis showed that PbQSOX protein had a signal peptide, but had no transmembrane region. All the proteins were expressed outside the cell membrane. PbQSOX-HA tag Plasmodium berghei was successfully obtained by homologous recombination of PbQSOX-HA tag targeting plasmid and Plasmodium berghei and confirmed by PCR. The purified schizonts, gametocytes and ookinetes of PbQSOX-HA tag Plasmodium berghei were obtained. Western blotting revealed that PbQSOX protein was mainly expressed in the gametocyte and ookinete stages, and IFA showed that PbQSOX was expressed on the surface of gametocytes, male-gametes, zygotes, retorts and ookinetes. Conclusion PbQSOX is a protein expressed at sexual reproduction stage of Plasmodium berghei, and it is mainly expressed on the surface of gametocytes, male-gametes, zygotes, retorts and ookinetes. |
| Key words: Plasmodium berghei gene targeting expression stage Plasmodium berghei quiescin sulfhydryl oxidase |
