摘要
为了对山羊痘病毒(Goatpox virus,GTPV)的截短融合结构蛋白P32-L1R进行生物信息学分析、原核表达及制备多克隆抗体,使用多种生物信息学工具对P32-L1R进行预测,利用SDS-PAGE与Western blot对P32-L1R重组蛋白进行鉴定,以纯化后的P32-L1R免疫BALB/c小鼠进行多克隆抗体制备,通过间接ELISA方法及Western blot技术检测制备抗体的效价和免疫反应性。结果显示,P32-L1R由458个氨基酸组成,相对分子质量为51 ku;偏碱性,原子组成为C _(2283) H_( 3614) N _(594 )O _(707 )S_( 17);是稳定亲水蛋白;无跨膜结构域无信号肽,包含16个抗原决定簇,共有57个磷酸化位点和3个N-糖基化位点;二级结构主要由α-螺旋、β-转角、延伸链及无规则卷曲组成。将pCold I-P32-L1R转化到大肠埃希氏菌中,诱导表达重组蛋白P32-L1R,该重组蛋白主要以包涵体的形式存在;免疫Balb/c小鼠后产生的多克隆抗体效价为1∶819200。研究结果为建立GTPV检测方法及开发GTPV亚单位疫苗提供了参考。
The aim was to perform bioinformatics analysis and prokaryotic expression of the truncated fusion structural protein P32-L1R of the goatpox virus(GTPV)and to prepare polyclonal antibodies.P32-L1R gene was analysed using multiple bioinformatics tools,identified by SDS-PAGE and Western blot.The polyclonal antibodies were prepared by immunizing Balb/c mice with purified P32-L1R protein.The serum titer and immunoreactivity of the polyclonal antibodies were detected by indirect ELISA and Western blot.The results showed that P32-L1R encoded 458 amino acids,and the protein size was about 51 ku.P32-L1R is a basic protein,and its molecular formula is C_(2283)H_(3614)N_(594)O_(707)S_(17),which belongs to hydrophilic protein with good stability.P32-L1R has no signal peptide region or transmembrane domain,with 16 antigenic determinants,57 phosphorylation sites,and 3 N-glycosylation sites.The secondary structure of the P32-L1R protein contained α-helix,β-turn,extended chain,and random coil.pCold I-P32-L1R was transformed into E.coli and induced to express the recombinant protein P32-L1R,which mainly existed in the form of inclusion bodies.Immunization of Balb/c mice produced polyclonal antibodies with a potency of 1∶819200.This study can be used as a reference for establishing GTPV detection methods and for the development of GTPV subunit vaccines.
作者
王永璇
胡茜
胡鹏飞
瞿正文
朱二鹏
王乃秀
文明
肖琨
程振涛
WANG Yong-xuan;HU Qian;HU Peng-fei;QU Zheng-wen;ZHU Er-peng;WANG Nai-xiu;WEN Ming;XIAO Kun;CHENG Zhen-tao(Guizhou Key Laboratory of Animal Disease and Veterinary Public Health,College of Animal Science,Guizhou University,Guiyang,Guizhou,Guangdong 550025,China;Panzhou Center of Animal Disease Control and Prevention,Liupanshui,Guizhou,Guangdong 553000,China;Liupanshui Center of Animal Disease Control and Prevention,Liupanshui,Guizhou,Guangdong 553029,China)
出处
《动物医学进展》
北大核心
2025年第10期49-55,共7页
Progress In Veterinary Medicine
基金
贵州省科技计划项目(黔科合成果[2023]一般077)
贵州省科技计划项目(黔科合平台人才[2021]5646号)。
