非洲猪瘟病毒D250R蛋白生物信息学分析及多克隆抗体制备

【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蛋白的理化性质及蛋白结构，在大肠杆菌表达系统中实现了ASFV D250R蛋白的高效表达，纯化的D250R蛋白免疫小鼠制备的多克隆抗体具有较高的特异性，为深入探讨ASFV D250R蛋白的生物学功能及ASFV相关诊断试剂的研发奠定了基础。

Bioinformatics Analysis of African Swine Fever Virus D250R Protein and Preparation of Polyclonal Antibodies

【Objective】 The aim of this study was to analyze the potential biological function of Africa swine fever virus (ASFV) D250R protein and prepare polyclonal antibodies against it, and provide materials for the development of ASFV D250R 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 D250R protein.The recombinant D250R 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 D250R 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 D250R 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 D250R 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 D250R protein contained a Nudix sequence with a dehydrolytic enzyme activity. The structure prediction results showed that the secondary structure of D250R 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-32a(＋) to obtain the pET-32a-D250R recombinant plasmid, which was transformed into E.coli BL21(DE3) receptor cells, and D250R protein was expressed in two forms of supernatant soluble and inclusion body under the induction conditions of 16 ℃ and 1 mmol/L IPTG.The protein supernatant was purified by nickel affinity chromatography column and molecular sieve chromatography to obtain a high purity protein.Western blotting results showed that the recombinant protein had good reactivity.The results of indirect ELISA showed that the antibody titer of D250R protein reached 1:256 000, and the polyclonal antibody was successfully prepared.The IFA test showed that the polyclonal antibody had good specificity.【Conclusion】 In this study, the physical and chemical properties and protein structure of D250R protein were analyzed at the molecular level, and the high-efficiency expression of ASFV D250R protein was realized in the E.coli expression system.The polyclonal antibody prepared by immunizing mice with the purified D250R protein had high specificity, which laid a foundation for further exploring the biological function of ASFV D250R protein and the development of ASFV related diagnostic reagents.