MS2 Phage-mediated Preparation and Immunogenicity of Chimeric Nanoparticles with Linear Epitope of Reproductive and Respiratory Syndrome Virus
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摘要:
目的 以MS2噬菌体外壳蛋白为载体，构建展示猪繁殖与呼吸综合征病毒GP5蛋白上线性表位的嵌合纳米颗粒，并研究其免疫原性，为猪繁殖与呼吸综合征病毒或其他病毒表位展示提供新的方法和思路。 方法 利用重叠延伸PCR将GP5上优势线性表位基因序列插入到MS2噬菌体外壳蛋白基因上，构建重组载体，通过原核表达系统表达嵌合蛋白，目的蛋白经过硫酸铵沉淀和凝胶过滤层析纯化，用动态光散射和电镜对嵌合蛋白进行物理表征，通过蛋白印迹和动物免疫试验研究表位嵌合颗粒的免疫原性。 结果 成功将线性表位基因插入MS2噬菌体外壳蛋白基因，嵌合蛋白在原核表达系统中以水溶性表达，目的蛋白经过纯化，纯度达85%以上。嵌合蛋白在体外自组装形成了均一的、直径为25～31 nm的嵌合表位纳米颗粒，该嵌合颗粒免疫动物后，产生可以和灭活病毒反应的高水平抗体，具有良好的免疫原性。 结论 MS2噬菌体外壳蛋白可以耐受9个外源多肽（猪繁殖与呼吸综合征病毒GP5上线性表位）的插入，在体外自组装形成嵌合病毒样颗粒。该颗粒将外源多肽高密度展示在表面，免疫动物可产生针对该表位的抗体。该技术可为猪繁殖与呼吸综合征病毒其他表位或更长串联表位的展示奠定基础。 -
关键词:
- MS2噬菌体 /
- 线性表位 /
- 嵌合纳米颗粒 /
- 猪繁殖与呼吸综合征病毒
Abstract:Objective Using the protein on coat of MS2 bacteriophage as a carrier, chimeric nanoparticles with a linear epitope of porcine reproductive and respiratory syndrome virus, GP5, were constructed, and their immunogenicity determined. Method The dominant neutralizing epitope gene sequence on GP5 was inserted into the MS2 protein by overlapping extension PCR. The recombinant vector was to express the chimeric protein through a prokaryotic expression system. The target protein was purified by ammonium sulfate precipitation and gel filtration chromatography followed by characterization with dynamic light scattering and electron microscopy. The immunogenicity of the constructed chimeric nanoparticles was determined by Western blot and animal immunoassay. Result The linear epitope was successfully inserted into the target sequence, and the recombinant vector reached a purity greater than 85%. The purified target protein self-assembled in vitro to form uniform chimeric nanoparticles with a diameter of 25–31 nm. After immunizing animals, the chimeric particles produced a high-level of antibodies that reacted with inactivated viruses showing significant immunogenicity. Conclusion The MS2 phage coat protein allowed the insertion of 9 exogenous polypeptides (the linear epitope on GP5) and self-assembled in vitro to form the chimeric virus-like particles. Each particle carried the exogenous polypeptides on the surface generating high immunogenicity. The current technology provided a new venue for building other epitopes of porcine reproductive and respiratory syndrome virus or epitopes with longer tandem. -
图 1 上下游片段和全长片段CP-B1的扩增
注：M：DNA分子量标准；1：上游片段；2：下游片段；3：SOE-PCR扩增全长片段。
Figure 1. Gel electrophoresis on amplication products of PCR and SOE-PCR
Note: M: DNA Marker DL2000; Lane 1: upstream fragment; Lane 2: downstream fragment; Lane 3: SOE-PCR product of whole fragment.
图 2 重组质粒pET28a（+）/CP-B1双酶切鉴定
注：M：DNA分子量标准；1～2：两个质粒分别用Nhe Ⅰ 和 Bam HⅠ双酶切。
Figure 2. Identification of recombinant plasmids by restriction enzyme digestion
Note: M: DNA Marker DL2000; Lane 1–2: two recombinant plasmids pET28a（+）/CP-B1 was digested using restriction enzymes Nhe Ⅰ and Bam HⅠ, respectively.
图 3 嵌合重组蛋白表达、纯化SDS-PAGE图和分子筛检测
注：a：M：蛋白分子量标准；1：超声破碎后上清；2：硫酸铵沉淀后重悬上清；3：凝胶过滤层析第一个流出峰浓缩后；b：第一个峰为目的蛋白峰。
Figure 3. Chimeric recombinant protein expression, SDS-PAGE purification （a）, and gel filtration chromatography （b）
Note: a: M: Protein Marker; Lane 1: supernatant after sonication; Lane 2: resuspension after precipitation of ammonium sulfate; Lane 3:the first peak of gel filtration chromatography; b: the first peak is the target protein.
图 4 嵌合表位纳米颗粒的物理表征
注：a：电镜观察；b：DLS分析检测。
Figure 4. Physical characterization of chimeric epitope nanoparticles
Note: a: Electron microscopic observation. b: DLS detection.
图 5 嵌合表位纳米颗粒免疫原性检测结果
注：a：蛋白印迹；M：蛋白分子量标准；1：超声破碎后上清；2：硫酸铵沉淀后重悬上清；3：凝胶过滤层析第一个流出峰浓缩后；b：豚鼠抗血清抗体水平。
Figure 5. Immunogenicity of chimeric epitope nanoparticles on guinea pigs
Note: a: M: Protein Marker; Lane 1: supernatant after sonication; Lane 2: resuspension after precipitation of ammonium sulfate; Lane 3: the first peak of gel filtration chromatography; b: anti-serum antibody levels in guinea pigs.
表 1 PCR和OE-PCR中用到的引物序列
Table 1. Primer sequences used in PCR and OE-PCR
引物名称 Primer name 引物序列 Primer sequence（5′-3′） CP-F CTAGCTAGCCTTCTAACTTTACTC CP-R CGCGGATCCTGTTGTCTTCGACATGGGTA IN-F CGTCGACAATGGCGGATCCCATCTACAGCTGATTTACA IN-R ACAGTCACGTCGCCAGTCAAGTTGTAAATCAGCT 注：CP-F引物中序列斜体加粗部分为Nhe Ⅰ 酶切位点，CP-R引物中序列斜体加粗部分为Bam HⅠ 酶切位点。引物IN-F和IN-R中，加粗部分为MS2载体插入位点两侧序列，斜体部分为插入的线性表位B1（37～45）基因序列，斜体加下划线部分为12个碱基的互补片段。
Note: The bold part in italics of CP-F primers is the Nhe Ⅰ digestion site, The bold part in italics of CP-R primer is the Bam HⅠ digestion site. In the primers IN-F and IN-R, the bolded part is the sequence on both sides of the MS2 vector insertion site, and the italicized part is the inserted neutralizing epitope B1（37–45）gene sequence. The 12 italicized and underlined parts are complementary fragments.
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