CN117965585B - 猪圆环病毒2d型佐剂嵌合病毒样颗粒疫苗及其制备方法 - Google Patents
猪圆环病毒2d型佐剂嵌合病毒样颗粒疫苗及其制备方法 Download PDFInfo
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Abstract
猪圆环病毒2d型佐剂嵌合病毒样颗粒疫苗及其制备方法，属于生物制药技术领域。解决了PCV2d型病毒样颗粒在大肠杆菌中难以高效和可溶性表达，抗体产生时间较长等问题。本发明通过在PCV2d的ORF2基因优化序列的N端添加C3d特异性表位基因序列来提高猪圆环病毒2d型佐剂嵌合病毒样颗粒疫苗的免疫效果，基于该方法获得了核苷酸序列SEQ2。进一步，通过将PCV2d的ORF2基因序列优化并在N端添加C3d特异性表位基因序列，插入pET28a载体，再转化到大肠杆菌BL21(DE3)中获得表达蛋白rC3d‑Cap。最后，疫苗的制备方法是将所述蛋白rC3d‑Cap通过硫酸铵沉淀技术、AKTA蛋白纯化系统和分子筛进行纯化，在与水包油佐剂进行乳化，获得的一种猪圆环病毒PCV2d病毒样颗粒疫苗。本发明适用于疫苗生产加工技术领域。
Description
技术领域
本发明属于生物制药技术领域，具体涉及猪圆环病毒疫苗及其制备技术。
背景技术
猪圆环病毒（Porcine circovirus，PCV）是目前发现的最小哺乳动物病毒，直径17-20nm，病毒粒子呈二十面体对称。猪圆环病毒主要有4个基因型：PCV1~PCV4，基因组编码2个主要的开放阅读框（Open reading frame，ORF）：ORF1和ORF2，ORF1编码复制酶相关蛋白（Rep），ORF2编码唯一的衣壳蛋白Cap，Cap蛋白是PCV2d主要的保护性抗原，能够诱导产生中和性抗体，是制备亚单位疫苗和病毒样颗粒（Virus like particle，VLP）的重要蛋白。
PCV2是引起断奶仔猪多系统衰竭综合征(Postweaning multisystemic wastingsyndrome，PMWS)的主要病原，该病呈世界流行性。其临床症状包括仔猪渐进性消瘦、贫血、腹泻、黄疸、淋巴结肿大等。此外，PCV2易发生基因突变，破坏感染猪只的免疫系统，并引起严重的免疫抑制。PCV2对养猪业的影响不可忽视，近几年的研究表明，我国PCV2d亚型阳性率可达80%，说明国内PCV2优势流行毒株已开始从PCV2b亚型转变为PCV2d亚型。因此，制备安全有效的针对PCV2d的疫苗对于PCV2的防控有重要的意义。VLP疫苗因为有与病毒相似的形态结构且不具备复制能力而受到青睐。目前针对PCV2d的VLP疫苗的研究已经取得了一定的进展，但是现有圆环病毒疫苗抗体产生时间较长，并且存在PCV2d型VLP在大肠杆菌中难以高效和可溶性表达等难点问题。
发明内容
本发明解决了PCV2d型病毒样颗粒在大肠杆菌中难以高效和可溶性表达，抗体产生时间较长等问题。
本发明提供如下技术方案：
本发明第一个方案，一种猪圆环病毒2d型佐剂嵌合病毒样颗粒疫苗的制备方法，所述制备方法是在制备所述病毒样颗粒疫苗中将PCV2d的ORF2基因序列优化并在N端添加分子佐剂C3d特异性表位基因。
优选地，所述将PCV2d的ORF2基因序列优化并在N端添加分子佐剂C3d特异性表位基因获得的基因的核苷酸序列如SEQ2所示。
优选地，所述疫苗制备方法步骤如下：
步骤一：将核苷酸序列SEQ2插入pET28a载体，再转化到大肠杆菌BL21 (DE3)中，表达rC3d-Cap蛋白。
步骤二：用硫酸铵沉淀技术对rC3d-Cap蛋白进行初步纯化，收集上清液，用0.22 μm过滤器过滤收集的上清液，然后利用AKTA蛋白纯化系统进行纯化，收集蛋白样品，再利用分子筛进行纯化，获得纯化后的rC3d-Cap蛋白，纯化后的rC3d-Cap蛋白自组装成病毒样颗粒，命名为PCV2d-C3d-VLP；
步骤三：将PCV2d-C3d-VLP用0.22 μm过滤器过滤后，与水包油佐剂进行乳化，获得PCV2d-C3d-VLP疫苗。
本发明的第三个方案，一种猪圆环病毒2d型佐剂嵌合病毒样颗粒疫苗，其是采用上述所述方法制备获得。
本发明的有益效果：
与现有技术相比，本发明所述的一种猪圆环病毒2d型佐剂嵌合病毒样颗粒疫苗的制备方法的有益效果是：
1、本发明通过对PCV2d的ORF2基因进行密码子优化和对Cap蛋白编码基因进行改造，大大提高了PCV2d主要保护性抗原Cap蛋白在大肠杆菌中的表达水平和组装成病毒样颗粒的产量。
本发明在PCV2d的ORF2基因优化序列的N端添加C3d特异性表位基因序列，C3d是一种源自补体C3的B细胞表位，可以形成C3d标记的病原体或抗原，其又能够结合B细胞和滤泡树突状细胞(DC)上的CR2 (CD21)，从而发挥调理素作用。C3d标记的病原体能同时与CD21和BCR相互作用，从而降低B细胞活化所需的抗原阈值，因此本发明所述的疫苗具有较好的免疫应答能力，提高了疫苗免疫效果。
2、本发明制备的佐剂嵌合病毒样颗粒疫苗能诱导机体产生更高水平的抗体，具有良好的免疫原性。
3、本发明制备的佐剂嵌合病毒样颗粒疫苗能诱导机体产生抗体的时间与同类商品苗相比更早。本发明制备的PCV2d-C3d-VLP疫苗在21天内诱导产生高水平抗体，同类商品化疫苗对照产生的高水平抗体在二次免疫后一周左右。且本案的PCV2d-C3d-VLP疫苗诱导的抗体持续在较高水平，直至试验结束。
4、本发明针对新型亚单位疫苗的生产加工技术提出了一种全新的设计思路。
附图说明
图1为重组蛋白的可溶性分析情况示意图：M泳道是蛋白质分子质量标准；1：IPTG诱导后的重组大肠杆菌总细胞裂解；2：离心后的细胞裂解液上清；3：离心后的细胞裂解液沉淀；箭头指示位置表示rC3d-Cap蛋白。
图2为重组蛋白的纯化情况示意图：箭头指示位置表示rC3d-Cap蛋白。
图3为重组蛋白的Western blotting鉴定情况示意图：箭头指示位置表示rC3d-Cap蛋白。
图4为重组蛋白自组装形成病毒样颗粒的电镜观察图。
图5为攻毒前仔猪PCV2d特异性抗体ELISA检测结果，S/P情况示意图，其中包括不免疫不攻毒空白的对照组；PCV2d-C3d-VLP疫苗组；同类商品化疫苗对照组。
图6为免疫后到剖杀前仔猪PCV2d特异性抗体ELISA检测结果，S/P情况示意图，其中包括不免疫不攻毒的空白对照组；攻毒组；PCV2d-C3d-VLP疫苗组；同类商品化疫苗对照组。
具体实施方式
实施例1，一种猪圆环病毒2d型佐剂嵌合病毒样颗粒疫苗的制备方法，具体方法包括：
一、重组蛋白的获取，
1、质粒构建：
通过分离毒株PCV2d，得到ORF2序列并测序，命名为SEQ1，由金斯瑞生物技术有限公司对SEQ1基因进行密码子优化并添加C3d特异性表位基因序列，获得序列SEQ2，并由该公司完成基因SEQ2与pET28a的连接工作，重组质粒命名为pET28a-rC3d-Cap。
2、重组蛋白表达：
将重组质粒pET28a-rC3d-Cap转化到大肠杆菌中进行表达，表达的蛋白命名为rC3d-Cap。将重组质粒pET28a-rC3d-Cap转化到大肠杆菌BL21 (DE3)后，在含有50 μg/mL卡那霉素的平板上挑取单克隆菌落，将其在含有50 μg/mL卡那霉素的LB培养基中培养12h，取4mL菌液放到200mL含有50 μg/mL卡那霉素的LB培养基中培养2.5小时。用0.1 mM IPTG在25℃下诱导融合蛋白表达20小时。5000 g离心10分钟，将菌体沉淀用含有0.02 M-phosphatebuffer，0.2 mM-EDTA，1%-Tween80，pH 7.5破碎缓冲液Buffer ET重悬，比例为1g菌： 10 mL破碎缓冲液。用超声细胞破碎仪破碎细菌，破碎后的菌液12000g离心10 min，收集总细胞裂解液，离心，离心后的细胞裂解液上清和沉淀分别采用SDS-PAGE评价上清液中可溶性rC3d-Cap蛋白的表达量，并进行考马斯亮蓝染色。检测结果见图1。
3、重组蛋白纯化：
用硫酸铵沉淀技术对rC3d-Cap蛋白进行初步纯化。将饱和硫酸铵溶液逐滴加入步骤2中的细胞裂解的上清液中，用磁力搅拌器充分混匀，使混合液中最终的硫酸铵百分饱和度达到50%。12000 g离心30 min，收集蛋白沉淀。将蛋白沉淀重悬于含有0.02 M-phosphatebuffer，pH 7.5重悬缓冲液中，12000 g离心30 min，收集上清液。用0.22μm过滤器过滤收集的上清液，利用AKTA蛋白纯化系统进行纯化。具体操作步骤如下：用含有0.02 M-phosphatebuffer，pH 7.5重悬缓冲液平衡层析柱，将上清液上样到DEAE弱阴离子交换层析柱上，先用含有0.4 M NaCl，0.02 M-phosphate buffer，pH 7.5缓冲液I洗涤层析柱，然后用含有1 MNaCl，0.02 M-phosphate buffer，pH 7.5缓冲液II洗脱rC3d-Cap蛋白，收集蛋白样品。将蛋白样品进行分子筛纯化，用平衡液预平衡分子筛层析柱，直至电导趋于稳定。将蛋白样品上样分子筛层析柱，上样量最大不超过柱床体积的5%，流速为0.5-1ml/min，紫外检测到起峰后分管接样，获得纯化后的rC3d-Cap蛋白。用鲎试剂检测内毒素水平，利用SDS-PAGE分析各管蛋白情况，检测结果见图2，结果为无可见杂质，证明获得的纯化后的rC3d-Cap蛋白纯度较高。
4、对上述获得的纯化后的rC3d-Cap蛋白的Western Blot分析 ：
纯化后的rC3d-Cap蛋白经12% SDS-PAGE分离，转移到PVDF膜上。将膜用5%脱脂奶粉封闭，将以兔抗PCV2d血清1：500为一抗，以荧光标记的山羊抗兔抗体1：10000为二抗，在近红外荧光扫描成像系统上扫描PVDF膜进行Western Blot鉴定。检测结果见图3，在目的条带30kDa位置出现一条单一的印迹，这说明纯化后的rC3d-Cap蛋白流穿液可被抗PCV2d阳性血清特异性识别，具有较好的反应原性。
5、对上述获得的纯化后的rC3d-Cap蛋白自组装成病毒样颗粒PCV2d-C3d-VLP的电镜观察：
取纯化后的rC3d-Cap蛋白悬液10微升滴到铜网上，待液体被充分吸收后，用10微升磷酸钨负染色液染色，待完全干燥后置于透射电镜下观察纯化后的rC3d-Cap蛋白自组装成PCV2d-C3d-VLP的情况。观察结果见图4，在TEM下可以清晰地观察到纯化后的rC3d-Cap蛋白成功自组装成直径大约为20 nm、均匀完整的高纯度的病毒样颗粒PCV2d-C3d-VLP。
二、PCV2d-C3d-VLP疫苗的制备：
将PCV2d-C3d-VLP用0.22 μm过滤器过滤后，与水包油佐剂乳化制备成PCV2d-C3d-VLP疫苗，使其蛋白浓度为50μg/mL，放置4℃存储。
实施例2，动物免疫、攻毒实验及试验猪PCV2特异性抗体检测
动物免疫实验：
选取12头4周龄的PCV2抗原和抗体检测均阴性的仔猪，随机分为4组，采用颈部肌肉注射进行免疫试验，分组和剂量见下表。
试验仔猪免疫和攻毒情况
第一次免疫后3周，按相同途径和相同剂量第二次免疫仔猪，于免疫后0、7、14、21、28、35天采集血液，分离血清，ELISA检测特异性抗体，检测结果见图5。
PCV2d-C3d-VLP疫苗免疫后，检测抗体的产生情况。根据抗体检测结果可以看出，该疫苗免疫猪后抗体产生快，在初次免疫后21日内，抗体效价均能达到较高水平，具有较好的免疫效果。而同类商品化疫苗在二次免疫后一周左右时间产生较高水平抗体。
攻毒实验：
第一次免疫后5周，1~3组经滴鼻和肌肉注射4×105TCID50的PCV2d，4组作为空白对照。所有仔猪在攻毒后4天和7 天在腋下和臀部2个部位注射4 mL经弗氏不完全佐剂乳化的钥匙孔血蓝蛋白乳剂，在攻毒后4天、7天、11天和19天腹腔注射10 mL巯基乙酸培养基。分别在攻毒后7、14、21、28天时采血，分离血清，用ELISA检测特异性抗体。检测结果见图6。根据图5和6的实验结果，本案制备的PCV2d-C3d-VLP疫苗在21天内天诱导产生高水平抗体，同类商品疫苗对照产生的高水平抗体在二次免疫后一周左右。且本案的PCV2d-C3d-VLP疫苗诱导的抗体持续在较高水平，直至试验结束。
试验猪PCV2d特异性抗体检测：
用猪圆环病毒2型ELISA抗体检测试剂盒检测猪血清中的抗体。将血清样品按1：100进行稀释，以血清稀释液、阴性对照血清和阳性对照血清为一抗，以PCV2d HPR标记抗体为二抗，通过TMB底物显色并使用450nm波长测量吸光度OD值进行血清抗体检测，并计算S/P值。
结果显示：PCV2d-C3d-VLP疫苗和商品化VLP疫苗均能不同程度地刺激仔猪产生针对PCV2d的特异性抗体，而且PCV2d-C3d-VLP疫苗组的血清抗体水平在攻毒前和攻毒后均显著高于同类商品化VLP疫苗组并且PCV2d-C3d-VLP疫苗能诱导机体产生抗体的时间与同类商品苗相比更早。结果证明，大肠杆菌原核表达系统表达的PCV2d病毒样颗粒具有良好的免疫原性，能诱导仔猪产生体液免疫应答，且效果优于同类商品疫苗。
Claims (2)
1.一种猪圆环病毒2d型(PCV2d)佐剂嵌合病毒样颗粒疫苗的制备方法，其特征在于，所述制备方法是在大肠杆菌中制备所述病毒样颗粒疫苗时将PCV2d的ORF2基因序列优化并在N端添加分子佐剂C3d特异性表位基因；
将PCV2d的ORF2基因序列优化并在N端添加分子佐剂C3d特异性表位基因获得的基因的核苷酸序列如SEQ2所示；
疫苗制备方法步骤如下：
步骤一：将核苷酸序列SEQ2插入pET28a载体，再转化到大肠杆菌BL21(DE3)中，表达rC3d-Cap蛋白；
步骤二：用硫酸铵沉淀技术对rC3d-Cap蛋白进行初步纯化，收集上清液，用0.22μm过滤器过滤收集的上清液，然后利用AKTA蛋白纯化系统进行纯化，收集蛋白样品，再利用分子筛进行纯化，获得纯化后的rC3d-Cap蛋白，纯化后的rC3d-Cap蛋白自组装成病毒样颗粒，命名为PCV2d-C3d-VLP；
步骤三：将PCV2d-C3d-VLP用0.22μm过滤器过滤后，与水包油佐剂进行乳化，获得PCV2d-C3d-VLP疫苗。
2.一种猪圆环病毒2d型佐剂嵌合病毒样颗粒疫苗，其特征在于，其是采用权利要求1所述方法制备获得。
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| A candidate DNA vaccine encoding a fusion protein of porcine complement C3d-P28 and ORF2 of porcine circovirus type 2 induces cross-protective immunity against PCV2b and PCV2d in pigs;Hou, ZM等;《Virology Journal》;20190502;第16卷;第1-8页 * |
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