| 羊口疮病毒129蛋白互作宿主蛋白的筛选与C1QBP基因的克隆分析 |
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| 引用本文： | 但一昕,向华,张焕容,杨璐,任玉鹏,徐颂为,何翃闳,朱江江.羊口疮病毒129蛋白互作宿主蛋白的筛选与C1QBP基因的克隆分析[J].中国畜牧兽医,2023,50(1):1-14. |
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| 作者姓名： | 但一昕 向华 张焕容 杨璐 任玉鹏 徐颂为 何翃闳 朱江江 |
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| 作者单位： | 1. 西南民族大学畜牧兽医学院, 成都 610041;2. 青藏高原动物遗传资源保护与利用四川省重点实验室, 成都 610041 |
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| 基金项目： | 西南民族大学优秀学生培养工程（2022NYXXS025）；国家自然科学基金（32072723）；动物遗传育种四川省重点实验室开放基金；四川省科技计划（2020JDJQ0010）；四川省畜禽育种攻关（2021YFYZ0003）；浙江省科技计划（2022C04017） |
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| 摘 要： | 【目的】以羊口疮病毒129（ORFV129）蛋白为研究对象，在构建山羊鼻甲骨原代细胞cDNA文库的基础上，通过酵母双杂交筛选与其相互作用的蛋白。【方法】采用SmartTM技术构建山羊鼻甲骨原代细胞cDNA文库。构建诱饵质粒pGBKT7-129，并将其转化至Y2HGold酵母感受态细胞，验证质粒pGBKT7-129是否具有自激活性。对转化了质粒pGBKT7-129的菌液进行生长曲线测定，验证该质粒是否对酵母细胞有毒性作用。以ORFV129为诱饵蛋白，利用酵母双杂交系统筛选出与ORFV129相互作用的宿主胞内蛋白并对阳性菌落进行PCR和测序鉴定。利用DAVID 6.7的GO数据库对胞内宿主蛋白进行功能分类和通路分析，并依据Cytoscape v 3.8.0软件绘制ORFV129与胞内蛋白相互作用的网络图。以山羊脾脏为组织样本，采用RT-PCR技术克隆山羊补体C1q结合蛋白（complement C1q binding protein，C1QBP）基因CDS区序列，并采用在线软件进行生物信息学分析。将C1QBP基因CDS区序列连接至pcDNA3.1（+）构建真核表达载体pcDNA3.1（+）-C1QBP，并将其转染至山羊鼻甲骨原代细胞进行亚细胞定位分析。【结果】试验成功构建山羊鼻甲骨原代细胞cDNA文库，文库容量约为6.0×106 CFU/mL。成功构建诱饵质粒pGBKT7-129，重组质粒pGBKT7-129无自激活能力且对酵母细胞无毒性。利用酵母双杂交筛选出14个与ORFV129进行互作的胞内蛋白并对阳性克隆进行PCR、测序验证。试验成功克隆山羊C1QBP基因CDS区，长度为837 bp，编码279个氨基酸。系统进化树显示，山羊和绵羊亲缘关系最近。C1QBP蛋白为不稳定的亲水性蛋白，无信号肽结构和跨膜结构域，主要包括3种磷酸化位点，分别是18个丝氨酸、4个苏氨酸和3个酪氨酸位点。C1QBP蛋白二级结构由α-螺旋（33.81%）、无规则卷曲（46.40%）、延伸链（16.91%）和β-转角（2.88%）组成，三级结构与二级结构一致。间接免疫荧光试验表明，C1QBP蛋白散在分布于细胞质中。【结论】筛选出了与ORFV129蛋白相互作用且在天然免疫应答中发挥作用的宿主胞内蛋白C1QBP，通过间接免疫荧光试验验证C1QBP定位于细胞质中，推测ORFV129与能C1QBP相互作用诱导炎症，为进一步验证ORFV129蛋白介导ORFV抑制机体免疫应答的过程奠定基础。
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| 关 键 词： | 山羊鼻甲骨原代细胞(GFTCs) 羊口疮病毒129(ORFV129) 酵母双杂交 互作蛋白 C1QBP |
| 收稿时间： | 2022-06-21 |
Screening of Host Proteins Interacting with ORFV129 Protein and Cloning and Analysis of C1QBP Gene |
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| DAN Yixin,XIANG Hua,ZHANG Huanrong,YANG Lu,REN Yupeng,XU Songwei,HE Honghong,ZHU Jiangjiang.Screening of Host Proteins Interacting with ORFV129 Protein and Cloning and Analysis of C1QBP Gene[J].China Animal Husbandry & Veterinary Medicine,2023,50(1):1-14. |
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| Authors: | DAN Yixin XIANG Hua ZHANG Huanrong YANG Lu REN Yupeng XU Songwei HE Honghong ZHU Jiangjiang |
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| Institution: | 1. College of Animal & Veterinary Sciences, Southwest Minzu University, Chengdu 610041, China;2. Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Chengdu 610041, China |
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| Abstract: | 【Objective】 The aim of the present study was to screen and validate the interacting protein of Orf virus 129(ORFV129) using yeast two-hybrid experiment with the cDNA library from goat fetal turbinate cells (GFTCs).【Method】 The SmartTM technology was used to construct the GFTCs cDNA library.A bait vector pGBKT7-129 was constructed, which was then transformed into Y2HGold yeast competent cells, to verify the activity of self-activating.The toxicity effect induced by pGBKT7-129 transfection was also evaluated by measuring the growth curve of the bacterial.Using ORFV129 as the bait vector, the host protein that interact with ORFV129 were screened and the positive colonies were identified by PCR method and sequencing.The GO databases was used for function annotation and pathway analysis, following which a Cytoscape v 3.8.0 software was used for protein-protein interaction visualization.The coding domain sequence of Complement C1q binding protein (C1QBP) gene was cloned by RT-PCR method from goat spleen tissue, which was then ligated to pcDNA3.1(+) to construct a eukaryotic expression vector pcDNA3.1-C1QBP, and it was transfected into GFTCs for subcellular localization analysis.【Result】 The GFTCs cDNA library was constructed successfully with a capacity of 6.0×106 CFU/mL.The bait plasmid pGBKT7-129 was successfully constructed with no self-activation ability and no toxicity to yeast cells.A total of 14 cellular proteins interacted with ORFV129 were selected from yeast two-hybrid experiment and the positive clones were confirmed by PCR and sequencing.The CDS region of goat C1QBP gene was successfully cloned, with a length of 837 bp, encoding 279 amino acids.The phylogenetic tree showed that there was the closest genetic relationship between Capra hircus and Ovis aries.C1QBP protein was an unstable hydrophilic protein without signal peptide structure and transmembrane domain, mainly including three kinds of phosphorylation sites, including 18 serine, 4 threonine and 3 tyrosine sites.The secondary structure of C1QBP protein was composed of alpha helix (33.81%), random coil (46.40%), extended chain (16.91%) and beta turn (2.88%), and the tertiary structure was consistent with the secondary structure.Indirect immunofluorescence test showed that C1QBP protein was scattered in the cytoplasm.【Conclusion】 The host intracellular protein C1QBP gene that interacts with ORFV129 protein and plays a role in the natural immune response was screened.The indirect immunofluorescence test verified that C1QBP was located in the cytoplasm.It was speculated that ORFV129 interacted with C1QBP to induce inflammation, laying a foundation for further verifying the process of ORFV129 protein mediated ORFV inhibiting the immune response of the body. |
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| Keywords: | goat fetal turbinate cells (GFTCs) Orf virus 129 (ORFV129) yeast two-hybrid interacting protein C1QBP |
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