摘要
为克隆亚洲璃眼蜱(Hyalomma asiaticum)的防御素基因(defensin),并分析defensin蛋白分子结构,通过提取亚洲璃眼蜱总RNA,构建cDNA文库,然后利用PCR技术成功鉴定并克隆了defensin基因,其开放阅读框大小为330 bp。序列同源性比对发现,该基因与NCBI公布的抗菌肽(XP_065292496)序列相似性最高,为93.27%。随后,对defensin蛋白进行了理化性质测定、信号肽剪切位点预测、二硫键预测以及二级结构和三级结构分析,并构建了系统进化树。结果显示:defensin蛋白的相对分子质量约为11.9 kDa,理论等电点为6.33,为亲水性蛋白,其N端含有一段长度为19个氨基酸的信号肽;该蛋白的成熟形式主要由α-螺旋结构组成,具有保守的由6个半胱氨酸残基形成的3对分子内二硫键,这种结构与抗菌肽的抗菌活性有关;系统进化分析显示,亚洲璃眼蜱defensin基因与革蜱属和璃眼蜱属的抗菌肽基因聚为一支,具有较近的亲缘关系。本研究成功克隆了亚洲璃眼蜱defensin基因,明确了其编码蛋白的分子结构特征,为进一步研究该基因的生物学功能奠定了基础。
In order to clone the defensin gene of Hyalomma asiaticum and analyze the molecular structure of defensin protein,total RNA was extracted from the Hyalomma asiaticum to construct a cDNA library,the defensin gene with the open reading frame of 330 bp was successfully identified and cloned using PCR technology.Sequence homology comparison revealed that the gene shared the highest similarity with the antimicrobial peptide(XP_065292496)published in NCBI,that was 93.27%.Subsequently,physicochemical property determination,signal peptide cleavage site prediction,disulfide bond prediction,and secondary and tertiary structure analysis were conducted for defensin protein,and a phylogenetic tree was constructed.The results revealed that the defensin protein,with a relative molecular mass of about 11.9 kDa and a theoretical isoelectric point of 6.33,was a hydrophilic protein,and a signal peptide with a length of 19 amino acids was contained in its N-terminal;the mature form of the protein mainly consisted ofα-helical structures with 3 pairs of conserved intramolecular disulfide bond formed by 6 cysteine residues,which was related to the antimicrobial activity of the antimicrobial peptide;and phylogenetic analysis showed that the defensin gene of Hyalomma asiaticum clustered with the antimicrobial peptide genes of the genus Dermacentor and Hyalomma,indicating a close genetic relationship.In conclusion,the defensin gene of Hyalomma asiaticum was successfully cloned,and the molecular structure characteristics of its encoded protein were defined,providing a basis for further research into the biological functions of the gene.
作者
罗亭祥
队森·木拉力别克
杨德鹏
余权威
妥小丽
高胜红
马祯
杨森
呼尔查
胡政香
Luo Tingxiang;Duisen Mulalibieke;Yang Depeng;Yu Quanwei;Tuo Xiaoli;Gao Shenghong;Ma Zhen;Yang Sen;Hu'ercha;Hu Zhengxiang(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,Xinjiang,China;Xinjiang Key Laboratory ofNew Drug Research and Development for Herbivorous Animals,Unumqi 830052,Xinjiang,China;Veterinary Medicine Post-doctoral Station,Xinjiang Agricultural University,Unmqi 830052,Xinjiang,China;College of Grassland Science,Xinjiang Agricultural University,Unmqi 830052,Xinjiang,China)
出处
《中国动物检疫》
2025年第3期127-132,共6页
China Animal Health Inspection
基金
新疆维吾尔自治区自然科学基金面上项目(2022D01A65)
新疆草食动物新药研究与创制重点实验室2023年度开放课题项目(XJCDVM-HDRC-T202303)
天山英才创新团队项目(2023TSYCTD0008)
国家重点研发计划项目(2021YFD1600702-3)
新疆重要马病诊断与预防关键技术的集成创新与示范项目(ZYYD2023C03)
新疆维吾尔自治区重大科技专项(2022A02013-2-7)
新疆农业大学大学生创新项目(dxscx2024448)。
关键词
亚洲璃眼蜱
防御素基因
抗微生物肽
分子克隆
免疫防御
Hyalomma asiaticum
defensin gene
antimicrobial peptide
molecular cloning
immune defense
作者简介
罗亭祥,同等贡献作者;队森·木拉力别克,同等贡献作者;通信作者:胡政香,E-mail:huzhengxiang300@163.com;通信作者:呼尔查,E-mail:huercha300@sina.com。
