CN116042536B - 一种牛疱疹病毒4型毒株及其培养方法和应用 - Google Patents
一种牛疱疹病毒4型毒株及其培养方法和应用 Download PDFInfo
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Abstract
一种牛疱疹病毒4型毒株及其培养方法和应用，命名为DN01，该病毒已于2021年12月28日在中国典型培养物保藏中心进行的保藏，保藏编号为CCTCC NO:V202193。该病毒对犊牛具有较强致病力，能引起肾脏、肝脏、肺脏、脾脏、阴道和子宫等器官的病变；同时具备良好的免疫原性，可诱发母牛产生抗体；根据本发明中的病毒制备的高滴度的血清抗体，具有较好的血清效价。
Description
技术领域
本发明涉及分子生物技术领域，特别是一种牛疱疹病毒4型毒株及其培养方法和应用。
背景技术
牛疱疹病毒(Bovine herpesvirus，BHV-4)属于疱疹病毒科，丙型疱疹病毒亚科，猴病毒属，在国内和国外牛场中均有蔓延。这种病毒在患有结膜炎、肺炎和上呼吸道炎症、肠炎、皮肤病损、乳房皮炎、产后子宫炎、慢性子宫炎的牛身上皆有分离到。另外，BHV-4还与流产相关，在流产胎儿中有同时发现牛病毒性腹泻病毒(BVDV)和BHV-4。该病多为牛的隐性感染，结合其他病原一起发病，可能使临床症状加剧，且表现多样化，给诊断和治疗带来较大困难，对养牛业构成严重威胁。
由于该病感染多为隐性感染，合并其他病原感染后的临床表现多样化，难以通过临床观察作出诊断，因此，需要依靠实验室检测进行确诊，如病毒的分离鉴定，血清学诊断，以及分子生物学检测。目前对于BHV-4的诊断研究的较少，因此寻找一个具有较好免疫原性的毒株对于BHV-4的预防措施及诊断方法的研制有重大的意义。
发明内容
解决的技术问题：为了克服现有技术的不足，本发明提供一种牛疱疹病毒4型毒株及其培养方法和应用。该病毒可在MAC-T细胞中增殖，通过该病毒制得的灭活和纯化病毒可诱发母牛产生抗BHV-4的抗体；且制备的血清抗体，具有较好的血清效价。
技术方案：一种牛疱疹病毒4型毒株，命名为DN01，该病毒已于2021年12月28日在中国典型培养物保藏中心进行的保藏，保藏编号为CCTCC NO:V202193。
编码所述病毒的DNA分子，核苷酸序列如SEQ ID NO.1所示。
上述牛疱疹病毒4型毒株的培养方法，所述病毒感染MAC-T细胞后，37℃，5％ CO2培养箱中培养，当细胞病变达80％以上时，收获病毒液。
一种牛疱疹病毒BHV-4疫苗，含有上述毒株。
一种牛疱疹病毒BHV-4疫苗，含有上述DNA分子。
如下(1)-(2)任一项在制备疫苗制剂中的用途：(1)所述的牛疱疹病毒4型毒株；(2)权利要求2所述的DNA分子。
有益效果：本发明中的病毒DN01可在MAC-T细胞中增殖，病毒浓度可达107TCID50/mL，无外源病毒污染；本发明中的病毒DN01对牛具有致病力，病毒感染了犊牛之后产生了肾脏、肝脏、肺脏、脾脏、阴道和子宫等器官的病变；根据本发明中的病毒DN01制备的灭活和纯化病毒可诱发母牛产生抗BHV-4的抗体；根据本发明中的病毒DN01制备的血清抗体，具有较好的血清效价。
附图说明
图1为本发明中BHV-4的电镜观察图。
图2为本发明中各器官组织病理学观察图。
具体实施方式
下面结合附图和具体实施方式对本发明作进一步详细说明。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
实施例1病毒的分离培养和鉴定
(1)病毒的分离
2020年底采集自河北某养殖场发病牛的粪便样本，低温运输至实验室。用MAC-T细胞(牛乳腺上皮细胞)培养法对样本进行病毒分离，培养三代后细胞出现细胞融合，进行病毒分离，获得病毒DN01,该病毒已于2021年12月28日在中国典型培养物保藏中心进行的保藏，保藏编号为CCTCC NO:V202193，保藏地址为中国湖北省武汉市武昌区八一路299号，武汉大学。
(2)病毒的鉴定
1.基因组序列分析
对该病毒的基因组序列进行分析，全基因序列为SEQ ID No.1所示的序列(见序列表)。
2.电镜检查
在电镜下观察牛疱疹病毒4型DN01，如图2所示，病毒颗粒直径范围在150nm左右，表面有囊膜。
3.无菌、支原体检测
按照《中国兽药典》方法对该病毒进行无菌、支原体检查，结果表明无支原体和其它微生物污染。
4.病毒滴度检测
采用微量滴定法测定该株病毒的病毒浓度，方法如下：用96孔细胞培养板培养MAC-T细胞，将病毒用细胞维持液从10-1倍比稀释至10-8，分别将每个稀释度的病毒液接种100μL/孔，每个稀释度接种8孔，将对数生长期的MAC-T细胞稀释至3×106个细胞/mL，每个细胞孔中接种100μL，同时设细胞对照孔。置37℃，5％CO2培养箱中培养，4-6天观察细胞感染病毒情况，计算病毒浓度，病毒浓度达107TCID50/mL。
实施例2DN01毒株的致病力
将4头试验犊牛随机分成2组，检测其牛疱疹病毒4型均为阴性。第1组接种107TCID50的病毒DN01，第2组接种4mL DMEM作为阴性对照组，接种方式为鼻腔接种。
接种后14天内，每天测量体温2次，记录临床症状，采集鼻拭子、眼拭子和肛门拭子进行病毒检测。在接种后第18天确定鼻腔停止排毒后，安乐死所有的实验牛，采集组织器官等样本，作组织病理学观察。结果显示对照组各器官正常，而实验组各器官病变程度如下(图2)：
肾脏：肾小球囊腔浆液和红细胞渗出，肾小管蛋白管型，间质结缔组织增生，肾小管上皮细胞变性坏死，淋巴细胞为主的炎性细胞浸润。
肝脏：肝细胞脂肪变性。
肺脏：肺脏支气管性肺炎，部分支气管黏膜坏死脱落，腔内大量细胞堆积。
脾脏：脾脏出血，并见有大量中性细胞浸润。
阴道：阴道黏膜坏死，血管充血，大量中性细胞、浆细胞、巨噬细胞、淋巴细胞等炎性细胞浸润。
子宫：子宫黏膜坏死，大量中性细胞、浆细胞、巨噬细胞、淋巴细胞等炎性细胞浸润，部分巨噬细胞空泡化，胞浆见有红染圆形蛋白物质。
实施例3母牛免疫原性试验
(1)病毒培养
复苏MAC-T细胞，经扩增，至细胞长至薄单层后，将病毒DN01接种至细胞瓶内，37℃，5％CO2培养箱中培养，每天观察细胞病变，当细胞病变达80％以上时，收获病毒液，-20℃保存备用。
(2)病毒灭活和纯化
病毒灭活前测定TCID50。将上述制备的病毒液用甲醛灭活24-48h。将病毒液按100mL加入42.5g硫酸铵的量加入硫酸铵，搅匀后至4℃冰箱搅拌沉淀过夜后，5000r/min离心40min，弃上清，沉淀用灭菌双蒸水悬浮，装于透析袋中，于PBS中搅拌透析过夜，即为纯化BHV-4病毒。病毒纯化后进行离心：10000rpm下10min，取上清液并将其病毒浓度调整为107TCID50/mL，即可用于后续免疫原性试验。
(3)抗体中和实验
将上述制备的纯化后病毒免疫荷斯坦母牛，4mL/头/次，在整个试验过程中观察记录母牛的健康情况，第一次免疫后20天进行第二次免疫，并于一免前、二免前(一免后20天)、一免后40天、一免后60天采集母牛的血液，收集血清，进行抗体中和测定。
方法如下：在96孔细胞培养板中加入分离获得的母牛血清，用细胞生长液倍比稀释至一定浓度后(每个稀释孔4个重复)，加入事先稀释至200TCID50/mL的病毒液，混合均匀后放入37℃，5％CO2培养箱中中和1h-2h，然后加入细胞悬液，细胞浓度为2-3×105个/mL，置于37℃，5％CO2培养箱中进行培养。5-7天后观察细胞病变情况，其中以半数细胞出现病变的血清的最高稀释倍数为血清的中和效价，阳性判定指标为：中和效价大于1:8；阴性血清效价小于1:8。结果见下表1。
表1为母牛免疫试验中和抗体检查结果
| 采血时间 | 中和效价 |
| 免疫前(0d) | ＜8 |
| 免疫后(20d) | 8 |
| 免疫后(40d) | 32 |
| 免疫后(60d) | 16 |
从表1可以看出，使用本发明病毒DN01制备的灭活和纯化病毒可诱发母牛产生抗BHV-4的抗体，并随着免疫次数的增加而呈上升趋势。
实施例4以病毒DN01为免疫原制备多抗血清
病毒DN01经过感染细胞、培养、收获病毒液、测定TCID50、灭活和纯化后，与弗氏完全佐剂充分乳化，免疫3只新西兰大白兔。采取颈背部皮下多点注射，2mL/只。免疫两周后加强免疫，佐剂换成弗氏不完全佐剂，共加强免疫3次，最后一次免疫2周后采血，分离血清进行中和抗体效价测定。
中和抗体效价测定方法：在96孔细胞板内加入分离的兔血清，用细胞维持液进行倍比稀释，加入稀释好的100TCID50/mL的病毒DN01病毒液，中和2-3h后加入细胞悬液，细胞浓度为2-3×105个/mL，置37℃，5％CO2培养箱中进行培养。5-7天后观察细胞病变情况，其中以出现细胞病变的血清最高稀释度为血清中和效价，阳性判断标准为：中和效价大于1:8，阴性对照血清效价小于1:8。试验结果显示3只新西兰兔的抗DN01血清的效价均大于1:1024。该结果证实使用本发明的病毒DN01免疫新西兰兔可获得血清效价较好的兔抗DN01多抗血清。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (6)
1.一种牛疱疹病毒4型毒株，命名为DN01，该病毒已于2021年12月28日在中国典型培养物保藏中心进行的保藏，保藏编号为CCTCC NO:V202193。
2.编码权利要求1 所述病毒的DNA分子，其特征在于，核苷酸序列如SEQ ID NO.1 所示。
3.权利要求1所述牛疱疹病毒4型毒株的培养方法，其特征在于，所述病毒感染MAC-T细胞后，37℃，5% CO2培养箱中培养，当细胞病变达80%以上时，收获病毒液。
4.一种牛疱疹病毒BHV-4疫苗，其特征在于，含有权利要求1所述毒株。
5.一种牛疱疹病毒BHV-4疫苗，其特征在于，含有权利要求2所述DNA分子。
6.如下（1）-（2）任一项在制备疫苗制剂中的用途：（1）权利要求1所述的牛疱疹病毒4型毒株；（2）权利要求2所述的DNA分子。
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