CN112010949B - 一种重组pcv2病毒样颗粒蜂胶疫苗及其制备方法 - Google Patents
一种重组pcv2病毒样颗粒蜂胶疫苗及其制备方法 Download PDFInfo
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Abstract
本发明提供一种重组PCV2病毒样颗粒疫苗制备方法，所述的制备方法是通过对猪圆环病毒2型Cap蛋白进行优化，并利用重组无内毒素大肠杆菌高密度发酵，获得高表达量的猪圆环病毒2型病毒样颗粒。本发明的猪圆环病毒2型Cap蛋白，其相比原始序列增加了一对二硫键，并对相应的序列片段进行优化，试验结果表明，通过所述序列的优化增加了病毒样颗粒疫苗的免疫原性，试验结果表明，通过高密度发酵与高效表达实现了Cap蛋白的高效表达，10倍浓缩表达菌可获得1:64‑1:128的琼扩效价；相比未优化的序列和其他佐剂的疫苗。
Description
技术领域：
本发明属于分子生物学领域，涉及一种重组PCV2病毒样颗粒蜂胶疫苗及其制备方法，尤其涉及，一种利用优化的PCV2 Cap蛋白基因和无致病性内毒素的大肠杆菌高密度发酵与高效表达技术实现高产量、低成本制备PCV2病毒样颗粒蜂胶疫苗的方法、以及一种安全性更高、免疫效果更好的PCV2病毒样颗粒蜂胶疫苗，既可以用于猪圆环病毒2型病免疫预防，又可以用于猪圆环病毒2型病紧急防治。
背景技术：
猪圆环病毒病（PCVD）是猪圆环病毒2型（PCV2）引起的一种重要传染病，该病毒是一种小的致病性的单链DNA病毒，与猪皮炎与肾病综合征、先天性震颤、后期流产和渗出性皮炎等疾病密切相关，给全球养猪业造成了严重的经济损失。2009-2012年间，我国由PCV2感染所致的经济损失高达150亿元。猪圆环病毒疫苗是控制该病流行和减少养猪业损失的重要手段，2006年，在北美获批第一种猪圆环病毒疫苗，我国自2009年开始陆续有猪圆环病毒2型灭活疫苗疫苗上市销售，该灭活疫苗有全病毒和基因工程疫苗灭活疫苗两种，但猪2型圆环病毒在体外培养的增值力不强，增殖滴度较低，其制备费用远远高于其他疫病的灭活疫苗，且生产中批间质量较难统一，生产工艺需要进一步改善。
Cap蛋白是PCV2病毒主要的衣壳蛋白，含有很多保护性抗原决定簇，是基因工程疫苗设计的靶标蛋白。目前市场上销售的PCV基因工程疫苗有三类: 一类由杆状病毒/昆虫细胞表达系统表达制备PCV2类病毒颗粒疫苗，该类疫苗有利于保证疫苗的稳定性和一致性，同时容易将免疫动物与感染动物区分开，便于疫苗效果的评价，但工艺要求较高，其价格昂贵，没有市场竞争力，产品中可能会混有杆状病毒颗粒，产生副反应，存在一定的生物安全风险。第二类是采用真核酵母菌进行表达，例如复旦大学2016年申请的发明专利CN201610806538.0公开了一种猪圆环病毒II型衣壳蛋白重组表达的马克斯克鲁维酵母工程菌，将猪圆环病毒II型衣壳蛋白基因克隆到表达载体中，并在马克斯克鲁维酵母中重组表达获得猪圆环病毒II型病毒样颗粒。发明专利CN201510091995.1也公开了采用毕赤酵母表达重组猪圆环病毒病毒样颗粒(VLP)。第三类是将PCV2的ORF2基因(Cap蛋白)插入大肠杆菌原核载体中，利用大肠杆菌表达PCV 2 Cap蛋白，制备亚单位疫苗，大肠杆菌表达系统具有生长速度快、易培养、表达蛋白产量高、成本低廉等特点，但使用的佐剂为化学物质，不能激发良好的细胞免疫；不能用于紧急防治。
发明内容：
针对目前猪圆环病毒2型病毒样颗粒疫苗及其制备方法存在的问题，本发明提供一种重组PCV2病毒样颗粒疫苗制备方法，所述的制备方法是通过对猪圆环病毒2型Cap蛋白进行优化，并利用重组无致病性内毒素的大肠杆菌高密度发酵与高效表达技术，获得高表达量的猪圆环病毒2型病毒样颗粒，为控制PCV2提供更加有效的抗原。
一种猪圆环病毒2型Cap蛋白，其特征在于，其氨基酸序列如SEQ ID No：1所示。
一种编码所述猪圆环病毒2型Cap蛋白的核酸分子，其特征在于，其碱基序列如SEQID NO.2所示。
含有所述的核酸分子的载体，所述载体为重组表达载体或重组克隆载体，所述载体为由所述核酸分子插入pET28a载体得到。
含有所述重组表达载体的重组菌。
所述重组菌由所述的重组表达载体转化大肠杆菌得到。优选的，所述大肠杆菌为大肠杆菌ClearColiTMBL21（DE3）。
一种制备PCV2病毒样颗粒疫苗的方法，其特征在于，包括：培养所述的重组菌，从培养产物中分离纯化得到所述Cap蛋白。
一种制备PCV2病毒样颗粒疫苗的方法，其特征在于，包括：
（1）将SEQ ID No：2所述的编码所述猪圆环病毒2型Cap蛋白的核酸分子插入到pET28a表达载体，得到重组载体；
（2）将重组载体转化大肠杆菌ClearColiTMBL21（DE3），构建重组表达菌；
（3）利用高密度发酵方法，对PCV2 Cap蛋白进行高效可溶表达；
（4）收集培养发酵的菌，分离纯化获得PCV2 Cap病毒样颗粒；
（5）以所述PCV2病毒样颗粒为活性成分，制备PCV2 Cap病毒样颗粒疫苗。
所述分离纯化包括：所收集的培养发酵的菌，高压均质破碎后，利用亲和层析柱纯化PCV2 Cap病毒样颗粒。
所述的重组表达菌培养所用培养基为市售的培养基。
所述的PCV2病毒样颗粒疫苗中PCV2 Cap病毒样颗粒为活性成分或者活性成分之一。
所述的PCV2病毒样颗粒在制备预防和/或治疗PCV2引发的疾病的药物和或疫苗中的应用。
一种重组PCV2病毒样颗粒疫苗制备使用的佐剂是经纯化的天然佐剂蜂胶。其使用量为5-10mg/ml干物质。
基于以上技术方案，本发明具有如下优点和有益效果：
针对目前猪圆环病毒2型病毒样颗粒疫苗及其制备方法存在的问题，本发明提供一种重组PCV2病毒样颗粒疫苗制备方法，所述的制备方法是通过对猪圆环病毒2型Cap蛋白进行优化，并利用重组无内毒素大肠杆菌高密度发酵，获得高表达量的猪圆环病毒2型病毒样颗粒。本发明的猪圆环病毒2型Cap蛋白，其相比原始序列增加了一对二硫键，并对相应的序列片段进行优化，试验结果表明，通过所述序列的优化增加了病毒样颗粒疫苗的免疫原性，试验结果表明，通过高密度发酵与高效表达实现了Cap蛋白的高效表达，10倍浓缩表达菌可获得1:64-1:128的琼扩效价；相比未优化的序列和其他佐剂的疫苗，本发明具有的优点如下：
1.本发明使用天然佐剂蜂胶代替传统的化学物质作为疫苗佐剂，不影响食品安全，注射局部无不良反应，能全面启动机体的免疫中枢系统，引起良好的细胞免疫和体液免疫应答。同时，蜂胶具有促生长作用，可以有效抵消因猪圆环病毒感染引起的生长缓慢损失。
2.本发明的Cap蛋白病毒样颗粒蜂胶疫苗可于7天更快的刺激机体产生特异性高效价保护抗体，且在免疫后期也能达到商品化疫苗相当的保护水平；在首免后14天即可产生1:20的中和滴度和1:800以上的ELISA抗体效价，高于商品化的疫苗和未优化的蛋白。同时，产生较好的细胞免疫，符合猪圆环病毒2型的免疫学特点。是一种既可以用于免疫预防，又可以用于紧急防治的新型疫苗。
附图说明：
图1：pET28a重组质粒双酶切鉴定图：M，DL2000 marker；1，重组载体BamH I和Hind III 双酶切。
图2：pET28a重组质粒菌液PCR鉴定：M，DL2000 marker；1-3，菌液PCR扩增结果。
图3：SDS-PAGE检测重组蛋白的表达：M，蛋白Marker；1，阴性对照；2，诱导后阳性菌全菌体；3，诱导超声后的沉淀；4，诱导超声后的上清。
图4：重组蛋白的Western blot鉴定：M，蛋白Marker；1，阴性对照；2，诱导后阳性菌全菌体。
图5：表达产物超声后的琼扩效价结果。
图6：PCV2 Cap蛋白病毒样颗粒电镜结果。
图7：不同表达菌株所制备颗粒疫苗的体温试验结果。
图8：血清抗体水平ELISA检测结果。
图9：中和抗体测定结果。
具体实施方式：
下面结合实例及附图对本发明作进一步详细的描述，以证明本发明的优势，但本发明的实施方式不限于此。
实施例1：重组大肠杆菌的构建
1.1 序列优化与重组载体的构建
根据测定的山东省滨州畜牧兽医研究院分离的PCV2 毒株的基因序列（Genbank登录号：JF272498）中ORF2的编码序列，对编码Cap蛋白的ORF2的进行优化，得到SEQ ID No：1所示的蛋白序列，在此基础上，依据密码子偏好设计ORF2的编码序列SEQ ID No：2，由上海生物工程有限公司合成并构建基于pET-28a(+)的PCV-2 Cap蛋白的原核表达质粒pET-BZCap，具体将ORF2的编码序列SEQ ID No：2插入pET-28a(+)的BamHⅠ和Hind III位点之间，经双酶切验证，结果见图1。
重组质粒pETBZCap的转化
将重组质粒pET-BZCap转化至大肠杆菌ClearColiTMBL21（DE3）中，涂布含有卡那霉素的平板，37℃倒置培养过夜。
重组菌的诱导表达
参照载体说明书Novagen公司的pET System Manual • 11th Edition，挑取阳性单个菌落，接种至适量的含50 μg/ml卡那霉素的LB培养基中，于37℃振荡培养至OD600达到0.5左右，加IPTG至终浓度为1mmol/L，继续培养5小时。
检验：
离心收集200ml菌体，PBS(10 mmol/L , pH7.2)洗涤，加入PBS缓冲液按1/ 10 体积比浓缩。超声波破碎，12000rpm高速离心后，收集上清。
取100 μL上清加入等体积的2×凝胶上样缓冲液（100 mmol/ L Tris-HCl pH6.8，4 % SDS ，0.1 %溴酚蓝，30 %甘油），金属浴100℃加热10分钟。SDS-PAGE电泳检测，分离胶的丙烯酰胺浓度为12%。考马斯亮蓝染色，脱色液脱色至条带清晰为止，同时设立不加IPTG诱导的培养物作为对照。结果见图3。
实施例2：重组菌的诱导表达及重组蛋白纯化
2.1 无致病性内毒素的大肠杆菌高密度发酵与高效表达Cap蛋白
将PCV2 Cap蛋白无致病性内毒素的大肠杆表达菌ClearColiTMBL21（DE3）-pET-BZCap按照2%的接种量接种到200L高效表达培养基中，于37℃培养至OD600达到10左右，加IPTG至终浓度为1mmol/L，继续诱导培养5小时，溶氧控制在30%-50%，pH控制在7.2-7.8。
重组蛋白病毒样颗粒的制备
将诱导表达后的菌液经管式离心机10000r/min离心30分钟，收集菌体，PBS(10mmol/L , pH7.2)洗涤，加入PBS缓冲液按1/ 10 体积比浓缩，高压均质机破碎，12000rpm高速离心后，收集上清。
2.2.1中空纤维过滤：将0.65μm中空纤维柱安装到中空纤维柱控制设备中，用无菌注射用水循环浸润中空纤维柱10 min。破碎液经中空纤维柱过滤澄清，获得上样液。
2.2.2 SP Bestarose FF阳离子交换层析
2.2.2.1系统预处理
2.2.2.1.1缓冲液：0.1mol/L NaOH；平衡缓冲液（Buffer A）：0.2M PB Buffer，PH8.0；洗脱缓冲液（Buffer B）：0.2M PB Buffer + 1M NaCl，PH8.0。
2.2.2.1.2将阳离子交换填料SP Bestarose FF捣匀装入EC0200-GI0500H柱中，离子交换柱组装完毕后测定柱效。
2.2.2.1.3用无菌0.1mol/L NaOH处理层析柱2个柱体积(CV)，再用注射用水清洗至pH7.5，然后用Buffer A平衡离子交换柱至电导率柱前后一致(柱前和柱后电导率在6.5-6.6ms/cm)，pH稳定在8.0，紫外UV280基线平稳。
2.2.3纯化过程
将上样液上样，设定上样线性流速为0.2L/min，压力控制小于3.00bar，上样量一个柱体积(6.28L或14.28L )，经离子交换柱上样完成后，用Buffer A洗涤未结合的杂蛋白。待UV280值下降至低于 0.100AU，用高盐溶液Buffer B洗脱猪圆环病毒Cap蛋白。待紫外UV280值增加时开始收集洗脱后样品，待UV280值下降至低于0.100AU时停止收集蛋白，继续洗脱至抗原液全部流出柱体。收集的洗脱样品即为猪圆环病毒Cap蛋白纯化洗脱液。保存生产记录报告。
2.2.4 琼扩效价及内毒素测定
测定Cap蛋白纯化液内毒素含量，应≤1.0万EU/ml。通过琼扩试验测定效价，应≧1:32，若效价<1:32，继续依照2.4步骤进行操作。
2.2.5超滤浓缩
依据琼扩效价将上清液用100KD超滤浓缩2-10倍，从浓缩液出口取浓缩液测定内毒素含量，应≤10000EU/mL；测定蛋白浓度≥2.0 mg/ml;琼脂扩散试验测定效价≧1:32。
2.3 重组蛋白的western-blot分析
重组蛋白作SDS-PAGE电泳结束后，电转移至硝酸纤维素膜。用半干式转印仪转印，具体转印显色等操作均按硝酸纤维膜产品说明书即Millipore公司的Immobilon-PTransfer MembraneUser Guide进行。然后用含3 %脱脂奶粉的PBST封闭过夜，以兔抗PCV-2阳性血清作为一抗室温孵育1小时，HRP标记的羊抗兔IgG为二抗室温孵育1小时，DAB显色试剂盒显色。结果见图4。
重组蛋白琼扩试验
称取1.0 g 琼脂糖、8.0g 氯化钠，加100 ml 蒸馏水或 Ph 7.0 0.0l mol/L 磷酸盐缓冲液，加热融化，分装于平皿，厚约3mm，按六角形打孔，中心一孔，周围六孔，将每一待测表达产物进行2倍倍比稀释，按顺时针方向依次加到周围六孔，中心孔加诊断血清，以测定表达产物的相对浓度。结果见图5，结果表明，通过高密度发酵与高效表达技术实现了Cap蛋白的高效表达，10倍浓缩表达菌可获得1:64的琼扩效价。
电镜观察
将2.1步骤中获得的超声后上清用于电镜病毒样颗粒分析。利用透射电镜观察，结果（如图6）显示，重组Cap蛋白能够自我组装成直径20~24nm大小的VLPs，形态结构类似于猪圆环病毒2型病毒粒子。
实施例3：重组PCV2病毒样颗粒疫苗的制备
3.1灭活
将蛋白液置于灭活容器内，计量加入甲醛溶液，随加随摇，使其充分混合，甲醛溶液的最终浓度为0.2%，37℃灭活16小时后取出，置2-8℃保存。
半成品检验
3.2.1 无菌检验按现行《中国兽药典》附录进行无菌检验应合格。
3.2.2 蛋白含量测定按Bradford法检测蛋白含量，测定蛋白浓度≥2.0 mg/ml。
3.2.3 用琼扩法检测病毒样颗粒抗原的效价在1:32以上。
3.2.4用鲎试剂法检测无致病性内毒素含量应≤10000EU/mL。
蜂胶佐剂的制备：取含量50%以上纯度的蜂胶，冷冻粉碎，溶解于75%乙醇中，反复搅拌48小时，用蜂胶提取器提取蜂胶，蒸馏回收乙醇。将纯化的蜂胶用甘油配制成蜂胶干物质含量为30mg/mL的佐剂溶液备用。
疫苗的制备
经过检验合格后的半成品PCV2-Cap重组蛋白抗原进行疫苗制备（以下配制中各液体成分按体积比计）。
重组蛋白的稀释：使用无菌生理盐水将半成品PCV2-Cap重组蛋白病毒样颗粒抗原稀释成150μg/ml（采用Bradford法检测蛋白含量），琼扩效价为1：32以上。
配苗：将PCV2-Cap重组蛋白的稀释液与蜂胶溶液按照适宜配比混合搅拌配成混悬液，无菌分装于疫苗瓶内，加塞封盖密封，于4℃保存备用。
疫苗的检验
3.4.1 无菌检验：按现行《中国兽药典》附录进行，符合规定。
安全检验：
用3-5日龄仔猪10头（对应母猪怀孕后期检测PCV2中和抗体效价≤1:4），每头耳后颈部肌肉注射疫苗1.0mL，同时设对照5只，在相同的条件下饲养，连续观察14日，记录试验猪采食、饮水及临床情况，具体结果参见表1。
表1 重组PCV2病毒样颗粒疫苗安全性检查结果
| 组别 | 仔猪数量 | 体温 | 食欲 | 精神 | 健康状况 | 死亡数量 |
| 试验组 | 10 | 正常 | 正常 | 正常 | 良好 | 0 |
| 对照组 | 5 | 正常 | 正常 | 正常 | 良好 | 0 |
实施例4：不同表达菌株所制备颗粒疫苗的体温试验
将原核表达质粒pET-BZCap转化到大肠杆菌BL21(DE3)后，用大肠杆菌BL21(DE3)和本发明的ClearColiTMBL21（DE3）相同条件下表达PCV2 Cap蛋白，分别选取相同菌数大肠杆菌离心，利用PBS 10倍浓缩重悬菌体，超声后离心取上清。选取15日龄仔猪9只，分3组，每组3头。实验组每头注射1ml 10倍稀释的上清，阴性对照组注射PBS 2ml，连续观察7天体温。具体结果参见图7。
基于图7的结果可知，采用BL21（DE3）作为表达用菌株所制备得到的疫苗上清中含有一定量的内毒素，因此，在注射7天内，仔猪都表现出较高的体温，而本申请选用ClearColiTMBL21（DE3）作为表达菌株，注射后仔猪的体温与对照组基本无差异，仅前两天表现出体温略有提高，以上结果表明，本申请采用ClearColiTMBL21（DE3）作为表达菌株解决了传统原核表达菌株含有内毒素导致免疫动物体温升高和食欲下降的问题。
实施例5：疫苗的免疫效力试验
5.1试验材料：
5.1.1 疫苗
试验苗：实施例3中制备得到的重组PCV2病毒样颗粒疫苗试制品；
对照苗1：商品化猪圆环病毒2型杆状病毒载体灭活疫苗；
对照苗2：根据测定的山东省滨州畜牧兽医研究院分离的PCV2 毒株的基因序列中ORF2的编码序列，氨基酸序列为SEQ ID No:3，基因序列为SEQ ID No:4，根据实施例1-3同样的方法制备得到基于PCV2-Cap蛋白的疫苗，其与试验苗的区别仅在于蛋白序列不同。
免疫效力试验方法
5.2.1 动物试验设计
用3-5日龄仔猪70头（对应母猪怀孕后期检测PCV2中和抗体效价≤1:4），随机分为4组，其中组1-3每组20头，分别试验苗组、对照苗1组、对照苗2组，组4为阴性对照组，共10头，其中试验苗和对照苗1、对照苗2每头耳后颈部肌肉注射相应疫苗1.0ml，阴性对照组耳后颈部肌肉注射生理盐水1.0ml。在相同的条件下饲养，连续观察14日，记录试验猪采食、饮水及临床情况，14d后分别进行第一次采血，并进行加强免疫，加强免疫的剂量和方法与首次免疫相同，在二免后第14天（即首免后28天）进行第二次采血，在首免后第42天进行第三次采血。
表2 动物试验分组情况
| 疫苗 | 免疫仔猪数量 | 接种情况 |
| 试验苗 | 20 | 实施例3中制备得到的重组PCV2病毒样颗粒疫苗试制品 |
| 对照苗1 | 20 | 猪圆环病毒2型杆状病毒载体灭活疫苗 |
| 对照苗2 | 20 | 氨基酸序列为SEQ ID No:3的疫苗试制品 |
| 阴性对照 | 10 | 生理盐水 |
5.2.2 效力试验
（1）血清抗体水平测定
采用Biochek 猪圆环病毒2型PCV2抗体ELISA检测试剂盒对血清进行抗体检测。
（2）中和抗体检测
待检血清于56℃水浴30min灭活，22μm滤器过滤除菌，将处理好的血清用维持液进行倍比稀释，依次是1:4、1:8、1:16、1:32、1:64、1:128、1:256。将增殖的PCV2用维持液稀释到2000TCID50/mL，然后与不同稀释度的血清等体积混合，37℃水浴作用1h。弃掉长有PK15细胞的96孔板中的营养液，用纯DMEM洗涤细胞一遍，再将水浴作用后的血清病毒混合物加到孔中，每个血清稀释度设置3个重复，100μL每孔，37℃培养72h。同时设置阴性血清对照、阴性血清病毒对照和空白对照。72h后用间接免疫荧光法(IFA)测定血清中和抗体效价，以能够抑制50％PCV2感染的血清最大稀释度作为待检血清的中和抗体效价。
免疫效力试验结果
5.3.1血清抗体水平测定结果
血清抗体水平ELISA检测结果见图8。基于图8的结果可知，本发明的重组PCV2病毒样颗粒疫苗、商品化的对照苗1和对照苗2均能在首免后14天产生PCV2抗体，首免后28天和42天，均产生了较高水平的抗体，而阴性对照组没有产生相应的特异性抗体。基于本发明重组PCV2病毒样颗粒疫苗与对照苗1和对照苗2之间的比较可知，本发明的PCV2病毒样颗粒疫苗在首免后14天即可产生1:1000的滴度，高于商品化的对照苗1和对照苗2。在首免后28天和42天，其抗体滴度水平与商品化苗的对照苗1接近，但是高于对照苗2。由此可见，本发明通过对PCV2-Cap蛋白的优化，能够更快的刺激机体产生特异性抗体，在早期形成较高的抗体水平，且在免疫后期也能达到商品化疫苗基本相当的保护水平。
中和抗体测定结果
中和抗体测定结果如图9所示，首免后14天、28天和42天，疫苗接种组均产生了中和抗体，而阴性对照组没有产生中和抗体。基于本发明重组PCV2病毒样颗粒疫苗与对照苗1和对照苗2之间的比较可知，本发明的PCV2病毒样颗粒疫苗在首免后14天即可产生1:20的中和滴度，高于商品化的对照苗1和对照苗2。在首免后42天，其抗体滴度水平与商品化苗的对照苗1接近，但是高于对照苗2。
序列表
<110> 山东绿都生物科技有限公司
山东省滨州畜牧兽医研究院
<120> 一种重组PCV2病毒样颗粒蜂胶疫苗及其制备方法
<160> 4
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Claims (11)
1.一种猪圆环病毒2型Cap蛋白，其特征在于，其氨基酸序列如SEQ ID No：1所示。
2.一种编码权利要求1所述的猪圆环病毒2型Cap蛋白的核酸分子，其特征在于，其碱基序列如SEQ ID NO.2所示。
3.一种含有权利要求2所述的核酸分子的载体，所述载体为重组表达载体或重组克隆载体，所述载体为由所述核酸分子插入pET28a载体得到。
4.一种含有如权利要求3所述的重组表达载体的重组菌。
5.根据权利要求4所述的重组菌，其特征在于，由所述的重组表达载体转化大肠杆菌得到，所述大肠杆菌为无致病性内毒素的大肠杆菌ClearColiTMBL21（DE3）。
6.一种制备PCV2病毒样颗粒蜂胶疫苗的方法，其特征在于，包括：培养权利要求5所述的重组菌，从培养产物中分离纯化得到权利要求1所述的Cap蛋白。
7.根据权利要求6所述的制备PCV2病毒样颗粒蜂胶疫苗的方法，其特征在于，包括：
（1）将SEQ ID No：2所述的编码所述猪圆环病毒2型Cap蛋白的核酸分子插入到pET28a表达载体，得到重组载体；
（2）将重组载体转化无致病性内毒素的大肠杆菌ClearColiTMBL21（DE3），构建重组表达菌；
（3）应用高密度发酵与高效表达技术，使用市售培养基，对PCV2 Cap蛋白进行高效可溶表达；
（4）收集培养发酵的菌，分离纯化获得PCV2 Cap病毒样颗粒；
（5）以所述PCV2 Cap病毒样颗粒为活性成分，制备PCV2 Cap病毒样颗粒疫苗。
8.根据权利要求7所述的方法，所述分离纯化包括：所收集的培养发酵的菌，高压均质破碎后，利用亲和层析柱纯化PCV2 Cap病毒样颗粒。
9.根据权利要求7所述方法制备得到的PCV2 Cap病毒样颗粒疫苗，所述的PCV2 Cap病毒样颗粒疫苗中PCV2 Cap病毒样颗粒为活性成分或者活性成分之一。
10.根据权利要求7所述方法中制备得到的所述的PCV2 Cap病毒样颗粒在制备预防和/或治疗PCV2引发的疾病的药物中的应用。
11.根据权利要求7所述方法中制备得到的所述的PCV2 Cap病毒样颗粒在制备预防和/或治疗PCV2引发的疾病的疫苗中的应用。
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