摘要
为了探究弓形虫组蛋白乙酰转移酶MYST-A对弓形虫蛋白质翻译后修饰的影响,在弓形虫ToxoDB数据库获取基因全长序列,构建表达载体,大量纯化His标签重组蛋白质,免疫实验动物,制备多克隆抗体并纯化。利用间接免疫荧光实验对其进行亚细胞定位分析,用不同浓度的抗体作用于弓形虫并提取全虫蛋白,进行Western-blot验证,分析其是否对虫体蛋白质翻译后修饰产生影响。ALP2a蛋白质是组成组蛋白乙酰转移酶复合物的重要组成部分,MYST-A与ALP2a可能形成组蛋白乙酰转移酶复合物共同参与组蛋白的翻译后修饰,拟通过分子互作实验验证两者是否发生相互作用。结果表明:成功构建重组表达载体并纯化重组蛋白质His-MYST-A。His-MYST-A免疫小鼠以及兔子,并获得了特异性多克隆抗体。IFA分析结果表明:在细胞内的虫体中和游离速殖子中,弓形虫MYST-A均在虫体细胞质内广泛分布。MYST-A对弓形虫蛋白质翻译后修饰影响结果表明,随着抗体浓度的增加,泛素化、单甲基/二甲基化、丙二酰化、琥珀酰化的修饰水平均增强(尤其是55 kDa处),说明该蛋白质起负调控作用。分子互作实验验证MYST-A与ALP2a两者发生相互作用。研究进一步揭示了弓形虫组蛋白乙酰转移酶MYST-A对蛋白质翻译后修饰的重要调控作用,为深入研究其生物学功能奠定了基础。蛋白质翻译后修饰的研究对了解弓形虫的致病机制及其与宿主的相互作用、致病机制有重要意义,同时也为新型弓形虫疫苗研制及药物靶标的筛选提供一定的参考价值和理论依据。
The aim of this study was to investigate the effect of the histone lysine acetyltransferase MYST-A on the protein post-translational modifications of Toxoplasma gondii.The full-length gene sequence was obtained from ToxoDB database of Toxoplasma gondii,the expression vector was constructed,the recombinant protein of His tag was purified in large quantities,and the experimental animals were immunized,polyclonal antibodies were prepared and purified.The subcellular localization of Toxoplasma gondii was analyzed by indirect immunofluorescence assay,and different concentrations of antibodies were used to act on Toxoplasma gondii and the whole parasite protein was extracted for Western-blot verification to analyze whether it had an effect on the protein post-translational modifications of Toxoplasma gondii.ALP2a protein is an important component of histone acetyltransferase complex.MYST-A and ALP2a may form a histone acetyltransferase complex to participate in post-translational modifications of histones.This study intends to verify whether they interact with each other through molecular interaction experiments.The results showed that the recombinant expression vector was successfully constructed,and the recombinant protein His-MYST-A were purified.His-MYST-A immunized mice and rabbits and produced specific polyclonal antibodies.The results of IFA analysis showed that MYST-A was widely distributed in the cytoplasm of Toxoplasma gondii both in the vacuoles and in the tachyzoites.The effect of MYST-A on the protein post-translational modifications of Toxoplasma gondii showed that with the increase of antibody concentration,the levels of ubiquitination,monomethyl/dimethylation,malonylation and succinylation increase(especially at 55 kDa),indicating that the protein played a negative regulatory role in them.The interaction between MYST-A and ALP2a was verified by molecular interaction experiment.This study further revealed that the histone lysine acetyltransferase MYST-A of Toxoplasma gondii plays an important role in the regulation of protein post-translational modifications,which lays a foundation for further study of its biological functions.The study of protein post-translational modifications is great significance to understand the pathogenic mechanism of Toxoplasma gondii and its interaction with the host.It also provides some reference value and theoretical basis for the development of novel Toxoplasma gondii vaccines and the screening of drug targets.
作者
姜宁
程昶
尹德琦
张义伟
桑晓宇
冯颖
陈冉
JIANG Ning;CHENG Chang;YIN Deqi;ZHANG Yiwei;SANG Xiaoyu;FENG Ying;CHEN Ran(College of AnimalScience and Medicine,Shenyang Agricultural University,Shenyang 110161,China)
出处
《沈阳农业大学学报》
CAS
CSCD
北大核心
2024年第3期257-266,共10页
Journal of Shenyang Agricultural University
基金
“十四五”国家重点研发计划项目(2022YFD1800200)。
关键词
弓形虫
MYST-A
原核表达
多克隆抗体
蛋白质翻译后修饰
Toxoplasma gondii
MYST-A
prokaryotic expression
polyclonal antibody
protein post-translational modifications
作者简介
第一作者:姜宁(1969-),女,博士,教授,博士生导师,从事人兽共患寄生虫病学研究,E-mail:jiangning@syau.edu.cn。
