CN107137704B - 一种鸭2型腺病毒灭活疫苗 - Google Patents
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Abstract
本发明提供一种鸭2型腺病毒灭活疫苗，其中抗原为灭活后的GD株病毒；GD株病毒于2016年6月5日保藏在武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC No：V201633。本发明制备的鸭2型腺病毒灭活疫苗安全性良好，未出现由疫苗引起的任何局部和全身不良反应。保存期试验中经过性状、安全性试验、效力试验数据的分析，各项指标均稳定有效；利用血清学方法和免疫攻毒法评估疫苗的免疫效果，结果显示，本发明中制备的灭活疫苗对鸭群能够提供有效的免疫保护，具有很好的商品化开发前景。
Description
技术领域
本发明属于兽用生物制品技术领域，具体涉及一种鸭2型腺病毒灭活疫苗。
技术背景
腺病毒是家禽常见的传染性病原体。禽腺病毒分为三个群：Ⅰ群是从鸡、火鸡、鹅和鸭的呼吸道感染分离出来的禽腺病毒，有共同的群特异性抗原，可分为A、B、C、D、E5个种和12个血清型；Ⅱ群包括火鸡出血性肠炎病毒、雉大理石皮病病毒和鸡大脾病病毒；Ⅲ群是从鸡产蛋综合征和鸭分离到的腺病毒。鸭2型腺病毒属于Ⅰ群禽腺病毒，是1977年法国匈牙利人从患病番鸭上分离出来的，感染鸭主要表现精神沉郁，下痢，消瘦等症状。病毒一旦带入鸭群很难清除，主要有传播方式决定的，即水平传播和垂直传播，给饲养业造成严重的经济损失。
通过对Ⅰ群鸭2型腺病毒的流行病学调查，发现该病在我国鸭群中发病率日益上升趋势。基于目前，国内外没有预防该病的商品化灭活疫苗，该病还在不断蔓延。研制安全有效的灭活疫苗迫在眉睫，必须通过创新技术克服这一问题。
发明内容
本发明的目的是提供一种鸭2型腺病毒灭活疫苗，用于预防Ⅰ群鸭2型腺病毒疾病，从而弥补现有技术的不足。
本发明提供一种鸭2型腺病毒灭活疫苗，其中抗原为灭活后的GD株病毒；
所使用的鸭2型腺病毒GD株，于2016年6月5日保藏在武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC No：V201633。
所述的疫苗为灭活疫苗。
上述的灭活疫苗，其中病毒的灭活采用甲醛灭活；
本发明的灭活疫苗的制备方法是将GD株病毒液中加入终浓度0.2％的甲醛，37℃搅拌灭活16h，灭活的病毒液与油佐剂1:2混合乳化，得到鸭2型腺病毒灭活疫苗。
本发明制备的鸭2型腺病毒灭活疫苗安全性良好，未出现由疫苗引起的任何局部和全身不良反应。保存期试验中经过性状、安全性试验、效力试验数据的分析，各项指标均稳定有效；利用血清学方法和免疫攻毒法评估疫苗的免疫效果，结果显示，本发明中制备的灭活疫苗对鸭群能够提供有效的免疫保护，具有很好的商品化开发前景。
附图说明
图1：筛选病毒的接种图。
具体实施方式
下面结合实施例对本发明进行详细的描述。
实施例1
GD株的分离与鉴定
1、流行病学调查 自2014年以来，广东、浙江等地区的部分番鸭出现了一种以死亡率高的Ⅰ群鸭2型腺病毒疾病。对病死的番鸭剖检后，主要的病理变化如下：肝脏肿大、肝坏死，脾脏肿大出血点，胰腺坏死等多器官发生病变为特点的疾病，经临床调查和实验室检测，初步诊断为Ⅰ群鸭2型腺病毒。2015年发明人成功从广东某养鸭场的鸭群中分离出一株病毒。
2、病毒分离 取病鸭的肝脏、脾脏、心包液、胰腺、法氏囊的组织50～100g，用研钵研磨组织，其间不断加入液氮，直至研磨成粉末状(无明显的可见颗粒)，按1:5(w/v)比例与无菌生理盐水匀浆，反复冻融3次后6000r/min离心10分钟，取上清液加入青霉素和链霉素各10000IU/ml，4℃过夜，经0.22μm微孔滤器过滤，无菌检验合格后保存备用。将含1％病毒过滤液接种鸭胚成纤维细胞，无血清的M199培养液进行培养，37℃静置培养60～78h，可出现明显的细胞病变，如图1所示，收毒。
3、病毒的鉴定
3.1 血凝特性鉴定 无菌采集SPF鸡和鸭血液，制备成0.8％、1％和2％浓度血红细胞，4℃保存备用。按常规方法检测分离毒株是否具有凝集这些红细胞的特性。同时设Ⅲ群鸭1型腺病毒(EDSV-76)为凝集反应阳性对照。结果：分离毒不能凝集SPF鸡和鸭红细胞，即使改变红细胞的浓度，也不能使之凝集。Ⅲ群鸭1型腺病毒(EDSV-76)能够凝集鸡、鸭的红细胞。
3.2 理化特性检验 病毒液分别经盐酸(pH3)、氢氧化钠(pH10)、温度(60℃、1h)、氯仿、乙醚处理后，接种鸭胚成纤维细胞(0.2ml/T25方瓶)，另设生理盐水处理组作为对照。接种72h后观察细胞病变。结果：分离毒经乙醚、氯仿和盐酸(pH3)处理的毒株，不影响病毒在细胞中的增殖，出现明显的细胞病变，PCR检测结果阳性。表明病毒没有囊膜，对乙醚和氯仿有抵抗力，耐酸。而经氢氧化钠(pH10)和温度(50℃、1h)处理后，接种细胞，无细胞病变，PCR检测阴性。表明分离毒株的核酸类型为DNA，病毒不耐碱，对热敏感，60℃，1h可被灭活。
3.4 PCR检测 取病鸭的肝脏、脾脏、心包液、胰腺、法氏囊的组织无菌研磨，反复冻融3次，利用组织细胞基因组DNA快速提取试剂盒提取病毒DNA，进行PCR检测。1％琼脂糖凝胶电泳观察结果。
PCR反应体系：总体积25ul
在0.5m1PCR管中依次加入
10×Taq聚合酶缓冲液------------5u1
2.5mmo1 4dNTP-------------------4u1
上游引物(20pmol)---------------1u1
下游引物(20pmol)----------------1u1
Taq DNA聚合酶(1-2U)----------0.3ul
DNA--------------------------------4ul
用灭菌去离子水9.7ul补足至总体积25u1，PCR反应在PTC-100基因扩增仪内进行。PCR反应参数：预变性条件为95℃，5min，变性条件为94℃，30Sec，退火温度和时间为53℃，30Sec，延伸温度为72℃延伸35Sec，35个循环，最后72℃延伸10min。
经扩增，分离毒株扩增出了相应的目的片段，与预期的目的片段大小相符。对扩增的片段进行测序、拼接后，通过NCBI BLAST比对分析，分离毒与Ⅰ群禽腺病毒属于同一分支，是鸭2型腺病毒序列。与NCBI公布的序列同源性最接近，但也存在序列上的差异。
实施例2
GD株毒种的制备
挑选已长满单层的鸭胚成纤维细胞，弃掉原培养液，加入终浓度1％毒种(GD株原始毒种第3代)无血清的M199维持液，37℃静置培养60～78h，可出现明显的细胞病变，当细胞病变达80％以上时收获。按此方法连传10代，分别标记为C3～C10代。测定每代次病毒含量。将检验无菌且病毒含量≥106.0TCID50病毒液混合，定量分装，冻干保存。
实施例3
GD株抗原的制备
1 制苗材料选择 鸭胚成纤维细胞、GD株病毒液的制备。
2 细胞制备 取11～13日龄的SPF鸭胚，用0.25％胰酶进行消化，加入适量含10％血清的M199培养液进行培养。
3 接毒 挑选已长满单层的鸭胚成纤维细胞，弃掉原培养液，加入终浓度为1％毒种的维持液，置37℃培养箱继续培养。
4 收获 接毒后，每日观察2次，记录细胞病变情况。当细胞病变达80％以上时收获，冻融2次，收获的细胞毒灭活前在-20℃保存。
实施例4
GD株病毒液的灭活及半成品检验与疫苗的制备
1 灭活 将GD细胞病毒液导入灭活罐内，计量加入10％甲醛溶液，开启搅拌机搅拌，使其充分混合，甲醛的最终浓度为0.2％。加甲醛溶液后导入另一灭活罐中，以避免罐口附近粘附的病毒未能接触灭活剂。37℃灭活16小时(以罐内温度达到37℃开始计时，开启搅拌机连续搅拌)后取出，放2～8℃保存，应不超过1个月。
2 半成品检验
2.1 无菌检验 取灭活的病毒液，按现行《中国兽药典》附录进行检验，应无菌生长。
2.2 灭活检验 将灭活后的病毒液作10倍稀释，接种已长满单层的鸭胚成纤维细胞(24孔细胞板)4孔，每孔0.2ml，补充维持液至2.0ml；同时设未接种的鸭胚成纤维细胞作空白对照，37℃、5％CO2培养箱培养，观察168小时。将培养物收获反复冻融后盲传一代，继续培养120小时。观察是否出现CPE。
2.3 病毒含量测定 将毒种用细胞维持液进行10倍系列稀释，取10－6、10－7、10－8 3个稀释度，分别接种于已长满单层的鸭胚成纤维细胞(96孔细胞板)，每个稀释度接种6孔，每孔0.1ml。同时设病毒阳性对照孔和细胞阴性对照孔，37℃培、5％CO2培养箱培养和观察168小时，出现CPE者判为感染，计算TCID50。
3 灭活疫苗的制备
3.1 油相制备：
取矿物油94份，硬脂酸铝2份(g)，边加边搅拌，直到完全透明为止，再加入司班-80(Span-80)6份(mL)，充分混匀后，121℃高压蒸气灭菌备用。
3.2 水相制备：
在96份(mL)灭活的抗原液中加入4份(mL)灭菌吐温-80(Tween-80)，充分振摇，直到Tween-80彻底溶解。
3.3 乳化
取油相2份导入乳化罐中，开动电机低速搅拌，同时徐徐加入水相1份后，1000r/min，乳化30分钟，即成油乳剂灭活疫苗。
4 分装 定量分装，加盖密封，并粘贴标签，置2～8℃保存。
实施例5
疫苗成品检验
1 性状
外观 乳白色乳剂。
剂型 油包水型。取一清洁吸管，吸取少量疫苗滴入冷水中，除第1滴外，均应不扩散。
稳定性 吸取疫苗10ml加入离心管中，以3000r/min离心15分钟，管底析出的水相应不多于0.5ml。
黏度 按现行《中国兽药典》附录进行，应符合规定。
2 装量检查 按现行《中国兽药典》附录进行检查，应符合规定。
3 无菌检验 按现行《中国兽药典》附录进行检验，应无菌生长。
4 安全检验 皮下免疫21日龄SPF鸭10只，每只颈部皮下分点注射疫苗1.0ml，观察21d，生长发育均正常，精神状态良好，剖检注射部位疫苗吸收良好，无红肿、组织坏死等炎症反应。
实施例6
疫苗免疫效果检测
1 血清学方法 用21日龄SPF鸭20只，10只各颈部皮下或肌肉注射疫苗0.2ml，另10只不免疫作对照。免疫后7日～6个月，每只鸭分别采血，分离血清，用琼扩试验检测免疫鸭和对照鸭的鸭2型腺病毒抗体，记录抗体结果。
2 免疫攻毒法 用21日龄SPF鸭20只，10只各颈部皮下或肌肉注射疫苗0.2ml，另10只不免疫作对照。免疫后21～28日，所有免疫鸭和对照鸭，肌肉注射病毒液(约含106.0TCID50/0.1ml)，每只0.2ml。观察并详细记录免疫鸭和对照鸭的发病情况。
结果显示：5批疫苗免疫组21日及以后琼扩抗体均≥8/10阳性，对照组鸭全部为阴性；攻毒保护结果：5批疫苗免疫鸭攻毒后观察14日，保护数均不少于9只；对照鸭攻毒7日内全部发病，死亡9只，死亡鸭(死亡后剖检)及发病鸭(攻毒后7日剖检)剖检病理变化：均出现典型的肝脏肿大和坏死，脾脏、肾脏等多个器官坏死。详见下表1、2。
而且，对比实验表明，用目前市场上出售的Ⅰ群鸭2型腺病毒疫苗进行免疫，再用本发明筛选的GD株进行攻毒实验；结果表明目前市场上出售的疫苗的免疫效果远低于本发明的GD株制备的灭活疫苗的效果；推测是由于GD株基因发生变异导致目前疫苗的免疫效果不好。
表1：疫苗免疫后抗体效价检测结果
表2：5批疫苗效力检验结果
综上，本发明筛选的GD株疫苗一种免疫效果比较理想的灭活苗，能够有效的预防Ⅰ群鸭2型腺病毒的发生。
Claims (2)
1.一种鸭2型腺病毒灭活疫苗，其特征在于，所述的疫苗，其中抗原为灭活后的GD株病毒；所述的GD株病毒的保藏编号为CCTCC No：V201633。
2.如权利要求1所述的灭活疫苗，其特征在于，所述的GD株病毒采用甲醛进行灭活。
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