摘要
【目的】分析非洲猪瘟病毒(Africa swine fever virus, ASFV)D250R蛋白潜在的生物学功能,制备其多克隆抗体,为ASFV D250R蛋白功能研究及相关诊断试剂的研发提供材料。【方法】应用生物信息学软件分析D250R蛋白的理化性质、信号肽、跨膜结构、磷酸化位点、生物功能及蛋白结构等。通过大肠杆菌表达系统表达重组D250R蛋白,采用镍亲和层析柱和分子筛纯化重组D250R蛋白,Western blotting检测纯化蛋白的反应原性,将纯化的D250R蛋白免疫BALB/c小鼠制备多克隆抗体,用间接ELISA方法测定多克隆抗体效价,用间接免疫荧光试验(IFA)检测多克隆抗体特异性。【结果】生物信息学分析结果显示,D250R蛋白共由250个氨基酸组成,理论分子质量为29 822.54 u,理论等电点为8.96,为亲水性蛋白,无信号肽及跨膜区。修饰位点预测结果显示,D250R蛋白可能存在15个磷酸化修饰位点,其中丝氨酸(Ser)6个、酪氨酸(Tyr)6个、苏氨酸(Thr)3个;2个N-糖基化修饰位点,分别位于第61和197位氨基酸处。生物学功能预测结果显示,D250R蛋白含有Nudix序列,具有解水解酶活性。蛋白结构预测结果显示,D250R蛋白二级结构中α-螺旋、β-转角、延伸链、无规则卷曲占比分别为49.20%、7.60%、15.20%和28.00%,三维空间结构存在较多α-螺旋,与二级结构预测结果基本一致。将ASFV D250R基因克隆至pET-32a(+)获得pET-32a-D250R重组质粒,转化大肠杆菌BL21(DE3)感受态细胞,在16℃、1 mmol/L IPTG诱导下,D250R蛋白以可溶性和包涵体2种形式表达,蛋白大小约44 ku,蛋白上清经镍亲和层析柱和分子筛层析纯化得到纯度较高的蛋白;Western blotting检测结果显示,重组蛋白具有良好的反应原性;间接ELISA检测结果显示,D250R蛋白抗体效价达到1∶256 000,成功制备多克隆抗体;IFA检测结果表明该多克隆抗体具有良好的特异性。【结论】本研究在分子层面分析了D250R蛋白的理化性质及
【Objective】 The aim of this study was to analyze the potential biological function of Africa swine fever virus(ASFV) D250 R protein and prepare polyclonal antibodies against it, and provide materials for the development of ASFV D250 R protein function and related diagnostic reagents.【Method】 Bioinformatics softwares were applied to analyze the physicochemical properties, signal peptide, transmembrane structure, phosphorylation site, biological function and protein structure of D250 R protein.The recombinant D250 R protein was expressed by E.coli expression system, purified using nickel affinity chromatography column and molecular sieve, and the reactogenicity of the purified protein was detected by Western blotting.BALB/c mice were immunized with purified D250 R protein to prepare polyclonal antibody.The titer of the polyclonal antibody was determined by indirect ELISA,and the specificity of the polyclonal antibody was detected by indirect immunofluorescence assay(IFA).【Result】 The bioinformatics analysis showed that D250 R protein consisted of 250 amino acids with a theoretical molecular mass of 29 822.54 u, the theoretical isoelectric point was 8.96,it was a hydrophilic protein with no signal peptide region and no transmembrane region.The modification site prediction results showed that D250 R protein might had 15 phosphorylation modification sites, including 6 serine(Ser),6 tyrosine(Tyr) and 3 threonine(Thr),and had 2 N-glycosylation modification sites at positions 61 and 197,respectively.Biological function prediction showed that D250 R protein contained a Nudix sequence with a dehydrolytic enzyme activity. The structure prediction results showed that the secondary structure of D250 R contained 49.20% alpha helices, 7.60% beta turns, 15.20% extended strands and, 28.00% random coils, and more alpha helices existed in the three-dimensional spatial structure, which was basically consistent with the secondary structure prediction results. ASFV D250R gene was cloned into pET-32 a(+) to obtain the pET-3
作者
许春梅
邵明珠
王心悦
林佳迪
郭建雄
张祥胤
童德文
梁瑞英
赵晓民
XU Chunmei;SHAO Mingzhu;WANG Xinyue;LIN Jiadi;GUO Jianxiong;ZHANG Xiangyin;TONG Dewen;LIANG Ruiying;ZHAO Xiaomin(College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China;Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China;Hebei Normal University of Science&Technology,Qinhuangdao 066604,China)
出处
《中国畜牧兽医》
CAS
北大核心
2022年第12期1645-1655,共11页
China Animal Husbandry & Veterinary Medicine
基金
陕西省重点研发计划项目(2018ZDXM-NY-064)
国家自然科学基金(31972645)。
