【作者】 李敏；

【导师】 姜宁；

【作者基本信息】 沈阳农业大学 ， 预防兽医学， 2024， 硕士

【摘要】 刚地弓形虫(Toxoplasma gondii)是一种属于顶复门的专性细胞内寄生原虫,可以感染各种温血动物,弓形虫引起的人畜共患病带来了广泛影响。目前,参与调节弓形虫基因表达的核因子中,只有少数被研究。弓形虫核因子(Nuclear factor,TgNF)3是一种动态染色质相关成分,可调节核仁结构和寄生虫毒力,密切参与虫体的代谢、转录及翻译等调控过程。弓形虫核因子TgNF7具有Nudix水解酶活性,在虫体代谢中发挥一定作用,并与虫体入侵相关。其他核因子家族的蛋白质尚未被深入研究。Pinin结构域是一种桥粒相关白,可能参与调节蛋白质-蛋白质相互作用。本课题选择了弓形虫核因子家族中一个具有Pinin结构域的蛋白质TgNF2(TGME49＿286790)进行研究。首先,对TgNF2进行生物信息学分析和抗原表位分析。然后通过CRISPR/Cas9技术对弓形虫TgNF2进行基因内源性标记,成功构建了TgNF2-HA虫株。利用TgNF2-HA虫株,通过间接免疫荧光(Indirect immunofluorescent assay,IFA)对TgNF2进行了亚细胞定位分析。结果发现TgNF2蛋白质广泛分布在弓形虫细胞质中。接下来,利用pGEX-4T-1和p ET-28a表达载体对TgNF2进行原核表达,诱导并纯化出GST标签和His标签两种重组蛋白质,用于后续研究TgNF2与虫体内其他蛋白质间的相互作用。根据抗原表位结果设计并合成多肽肽段,免疫大鼠后成功制备了抗TgNF2的多克隆抗体。纯化Ig G后进行免疫沉淀(Immunoprecipitation,IP)和免疫共沉淀(Co-immunoprecipitation,Co-IP)实验以验证TgNF2与虫体内其他蛋白质间的相互作用。结果显示,TgNF2与弓形虫疑似热休克蛋白90(Heat shock protein 90,Hsp90)、假定钙离子结合蛋白(Putative calcium binding protein,pCaBP)、棒状体蛋白9(Rhoptry protein 9,ROP9)和TgNF7均发生相互作用。为进一步确定与该蛋白相互作用的虫体蛋白,使用质谱方法进行鉴定,共检测到包含ROP9、TgNF7、热休克蛋白、EF-Hand结构域蛋白等500多个蛋白质。后续又利用重组蛋白质进行了分子互作实验,证实TgNF2确实能够与弓形虫疑似Hsp90、pCaBP、ROP9和TgNF7发生结合,结合亲和力常数分别是5.73E-07、2.92E-06、1.19E-05、8.01E-07。综上,TgNF2可与虫体内多种蛋白质互相作用,在虫体入侵、生长发育等方面具有潜在作用。更多还原

【Abstract】 Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of warmblooded animals,and the zoonosis caused by Toxoplasma gondii has a wide impact.Currently,only a few of the nuclear factors involved in regulating Toxoplasma gene expression have been studied.Toxoplasma gondii nuclear factor 3(TgNF3)is a dynamic chromatin related component of Toxoplasma gondii,which regulates nucleolar structure and parasite virulence,and is closely involved in metabolism,transcription,translation and other regulatory processes.Toxoplasma gondii nuclear factor 7(TgNF7)has Nudix hydrolase activity,plays a role in the metabolism of the parasite,and is related to the invasion of the parasite.Other nuclear factor families of proteins have not been studied in depth.The Pinin domain is a desmosome associated white that may be involved in regulating protein-protein interactions.In this study,a protein with Pinin domain,nuclear factor 2(TGME49＿286790),was selected from the Toxoplasma gondii family of nuclear factors.Firstly,bioinformatic analysis and epitope analysis of Toxoplasma gondii Nuclear factor 2(TgNF2)were performed.Then the TgNF2-HA strain of T.gondii TgNF2 was successfully constructed by using CRISPR/Cas9 technique for endogenous gene labeling.Subcellular localization of TgNF2 was analyzed by Indirect immunofluorescent assay(IFA)using TgNF2-HA strains.The results showed that TgNF2 protein was widely distributed in Toxoplasma.Next,pGEX-4T-1 and p ET-28 a expression vectors were used for prokaryotic expression of TgNF2,and two recombinant proteins,GST tag and His tag,were induced and purified for subsequent study of the interaction between TgNF2 and other proteins in worms.Polyclonal antibody against TgNF2 was successfully prepared by immunizing rats with polypeptide segments designed and synthesized according to the results of epitope.After purification of the Ig G,Immunoprecipitation(IP)and Co-immunoprecipitation(Co-IP)tests were conducted to verify the interaction between TgNF2 and other proteins in the parasite.The results showed that TgNF2 interacts with T.gondii putative heat shock protein 90(Hsp90),putative calcium ion binding protein(pCaBP),rod-like protein 9(ROP9),and TgNF7.In order to further identify the insect body proteins interacting with this protein,more than 500 proteins including ROP9,TgNF7,heat shock protein,and EF-Hand domain proteins were detected by mass spectrometry.Subsequent molecular interaction experiments with recombinant proteins confirmed that TgNF2 could indeed bind to T.gondii hypothesized Hsp90,pCaBP,ROP9 and TgNF7,with binding affinity constants of 5.73E-07,2.92E-06,1.19E-05 and 8.01E-07,respectively.In summary,TgNF2 can interact with various proteins in insect body,and has potential role in invasion,growth and development of insect body.更多还原