CN108441483A - 一种促进牛鼻气管炎病毒复制的方法 - Google Patents
一种促进牛鼻气管炎病毒复制的方法 Download PDFInfo
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Abstract
本发明公开了一种促进牛鼻气管炎病毒复制的方法，其包括用低温常压等离子体发生器照射牛肾细胞培养基，将经过照射处理的所述培养基加入牛肾细胞中，并加入牛鼻气管炎病毒孵育。本发明通过等离子体间接处理细胞，处理均匀且强度可控。本发明处理时间只需2min，操作简单、快速，处理后的DMEM培养基与牛鼻气管炎病毒和牛肾细胞一同孵育1h就能显著提高牛鼻气管炎病毒在牛肾细胞中的复制，得到的高浓度牛鼻气管炎病毒经灭活处理可用于疫苗生产，显著提高疫苗生产效率。
Description
技术领域
本发明属于疫苗生产技术领域，具体涉及一种促进牛鼻气管炎病毒复制的方法。
背景技术
牛鼻气管炎病毒(Infectious bovine rhinotracheitis virus，IBRV)是双链DNA病毒，疱疹病毒科成员，是牛的重要传染病病原；牛传染性鼻气管炎(Infectious bovinerhinotracheitis，IBR)已成为全球性疫病，给养牛业造成重大经济损失。目前IBR在我国具有较高流行率，由于临床上治疗效果并不理想，因此常用疫苗接种来预防该病。
目前商业化的常规疫苗主要有两种：灭活苗和减毒苗，如何提高疫苗产量、降低疫苗生产成本成为疫苗生产的重点难题，因此，如何促进牛鼻气管炎病毒复制，提高疫苗的生产效率，是现有技术有待解决的技术问题。
发明内容
本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊，而这种简化或省略不能用于限制本发明的范围。
因此，本发明克服现有技术中存在的不足，提供一种促进牛鼻气管炎病毒复制的方法。
为解决上述技术问题，本发明提供了如下技术方案：一种促进牛鼻气管炎病毒复制的方法，其包括，用低温常压等离子体发生器照射牛肾细胞培养基，将经过照射处理的所述培养基加入牛肾细胞中，并加入牛鼻气管炎病毒孵育。
作为本发明所述促进牛鼻气管炎病毒复制的方法的一种优选方案：所述培养基包括DMEM培养基。
作为本发明所述促进牛鼻气管炎病毒复制的方法的一种优选方案：所述用低温常压等离子体发生器照射DMEM培养基，控制氦气流量为1slm/min，通过调节变压器控制高频电源的输出电压为0.96～1.24kV。
作为本发明所述促进牛鼻气管炎病毒复制的方法的一种优选方案：所述用低温常压等离子体发生器照射DMEM培养基，包括将所述DMEM培养基放在低温常压等离子体发生器正下方，等离子体束流尾端与DMEM培养基的距离控制为1cm。
作为本发明所述促进牛鼻气管炎病毒复制的方法的一种优选方案：所述照射，时间为2min。
作为本发明所述促进牛鼻气管炎病毒复制的方法的一种优选方案：还包括，在用低温常压等离子体发生器照射牛肾细胞培养基的前一天将牛肾细胞铺于六孔板中，每孔细胞数量50万。
作为本发明所述促进牛鼻气管炎病毒复制的方法的一种优选方案：所述将经过照射处理的培养基加入牛肾细胞中，其中，每孔加入1mL经过照射处理的所述培养基。
作为本发明所述促进牛鼻气管炎病毒复制的方法的一种优选方案：所述加入牛鼻气管炎病毒孵育，包括，加入牛鼻气管炎病毒后放入37℃培养箱中孵育，时间为1h。
作为本发明所述促进牛鼻气管炎病毒复制的方法的一种优选方案：还包括，加入牛鼻气管炎病毒孵育后，用含2％胎牛血清的1640培养所述牛肾细胞，继续培养至细胞全部脱落，收集所述牛肾细胞。
本发明的有益效果：本发明通过等离子体间接处理细胞，处理均匀且强度可控。本发明处理时间只需2min，操作简单、快速，处理后的DMEM培养基与牛鼻气管炎病毒和牛肾细胞一同孵育1h就能显著提高牛鼻气管炎病毒在牛肾细胞中的复制，得到的高浓度牛鼻气管炎病毒经灭活处理可用于疫苗生产，显著提高疫苗生产效率。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。其中：
图1为本发明低温常压等离子体处理培养基，孵育细胞的流程图。
图2为牛鼻气管炎病毒荧光定量PCR检测结果图，0s为对照组，120s表示低温常压等离子体处理时间为2min。
图3为牛鼻气管炎病毒TCID50法测定结果图。0s为对照组，120s表示低温常压等离子体处理时间为2min。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用其他不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受下面公开的具体实施例的限制。
其次，此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
实施例1：
细胞铺板：提前一天将长满的牛肾细胞用0.25％的胰酶消化离心重悬后计数，铺于六孔板中，每孔50万细胞。
向干净的12孔板中加入空白的待处理的DMEM培养基，每孔1mL，放在低温常压等离子体发生器正下方，等离子体束流尾端与培养基的距离(D)控制为1cm，喷射口垂直对准12孔板小孔的中央，控制氦气流量(F)为1slm/min，通过调节变压器控制高频电源的输出电压(U)为0.96～1.24KV，等离子体照射DMEM培养基2min。将单独照射后的培养基转入离心管中，按照每毫升培养基加入2uL的量加入牛鼻气管炎病毒。混匀。将6孔板中培养基轻轻吸出，加入上述含病毒培养基，每孔1mL；对照组加入1mL含病毒的未处理的DMEM培养基。将上述6孔板放入37℃培养箱中孵育1h后，将6孔板拿出换成含2％胎牛血清的1640维持液(图1)，再继续放入培养箱中培养至细胞全部脱落，收集对照组和实验组的培养液。一部分用荧光定量PCR(qPCR)检测病毒含量，一部分用TCID50法测定病毒滴度。
荧光定量PCR(qPCR)检测：采用天根病毒基因组DNA/RNA提取试剂盒(DP315)提取培养液病毒基因组DNA，提取步骤参照试剂盒说明书。将上述基因组DNA作为荧光定量PCR检测的模板，进行实时定量PCR检测。荧光定量PCR检测体系为10uL：5uLSYBR Green PCRMaster Mix，上、下游引物各0.4uL，煮沸后的上清模板2uL，ddH2O2uL。反应条件为：95℃预变性30s，95℃10s，60℃30s，72℃20s，40个循环，溶解曲线分析条件为：95℃15s，60℃30s，95℃15s。结果显示，低温常压等离子体照射后的培养基与牛鼻气管炎病毒和牛肾细胞一同孵育1h后，qPCR法检测牛鼻气管炎病毒产量较对照组提高2.6倍(图2，0s为对照组，120s表示低温常压等离子体处理时间为2min)。
TCID50法测定病毒滴度：提前一天将牛肾细胞消化铺板至96孔板中，每孔1万细胞。37℃培养待用。参考测定方法，将上述收集的对照组与实验组的病毒液体用1640培养基10倍梯度稀释6个梯度。吸去96孔板中的培养液，将上述两组不同稀释度的病毒加入孔中，每个稀释度8个重复，一同孵育1h后换成维持液。每天观察记录细胞病变(CPE)产生情况。观察CPE，计算病毒滴度：
按照Reed-Muench两氏法以每列所有平行中出现CPE样品达到50％作为分界线，取刚好病变率超过50％和低于50％的列做计算：距离比例＝(高于50％病变率的百分数-50％)/(高于50％病变率的百分数-低于50％病变率的百分数)lgTCID50＝距离比例×稀释度对数之间的差+高于50％病变率的稀释度的对数，结果显示，通过TCID50法测定病毒滴度与qPCR法结果基本一致，低温常压等离子处理后与对照相比病毒滴度提高2.19倍(图3，0s为对照组，120s表示低温常压等离子体处理时间为2min)。
本发明通过使用低温等离子体发生装置电离氦气来产生等离子体射流处理培养基。在这个处理过程中，等离子体射流暴露在空气中并与培养基接触，一些活性物质通过等离子体射流进入到培养基中，在培养基中产生大量的活性氧和活性氮。向处理后的培养加入病毒，再将混合液加入细胞一同孵育，发现这种经过处理的培养基对能够促进牛鼻气管炎病毒在牛肾细胞(MDBK细胞)的复制。相对于直接用低温常压等离子体处理细胞存在的安全隐患，本发明间接的处理方法更安全可靠，操作方便。
本发明通过等离子体间接处理细胞，处理均匀且强度可控。本发明处理时间只需2min，操作简单、快速，处理后的DMEM培养基与牛鼻气管炎病毒和牛肾细胞一同孵育1h就能显著提高牛鼻气管炎病毒在牛肾细胞中的复制，得到的高浓度牛鼻气管炎病毒经灭活处理可用于疫苗生产，显著提高疫苗生产效率。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
Claims (9)
1.一种促进牛鼻气管炎病毒复制的方法，其特征在于：包括，
用低温常压等离子体发生器照射牛肾细胞培养基，将经过照射处理的所述培养基加入牛肾细胞中，并加入牛鼻气管炎病毒孵育。
2.如权利要求1所述的方法，其特征在于：所述培养基包括DMEM培养基。
3.如权利要求2所述的方法，其特征在于：所述用低温常压等离子体发生器照射DMEM培养基，控制氦气流量为1slm/min，通过调节变压器控制高频电源的输出电压为0.96～1.24kV。
4.如权利要求2或3所述的方法，其特征在于：所述用低温常压等离子体发生器照射DMEM培养基，包括将所述DMEM培养基放在低温常压等离子体发生器正下方，等离子体束流尾端与DMEM培养基的距离控制为1cm。
5.如权利要求2或3所述的方法，其特征在于：所述照射，时间为2min。
6.如权利要求2或3所述的方法，其特征在于：还包括，在用低温常压等离子体发生器照射牛肾细胞培养基的前一天将牛肾细胞铺于六孔板中，每孔细胞数量50万。
7.如权利要求6所述的方法，其特征在于：所述将经过照射处理的培养基加入牛肾细胞中，其中，每孔加入1mL经过照射处理的所述培养基。
8.如权利要求7所述的方法，其特征在于：所述加入牛鼻气管炎病毒孵育，包括，加入牛鼻气管炎病毒后放入37℃培养箱中孵育，时间为1h。
9.如权利要求1～3、7～8任一所述的方法，其特征在于：还包括，加入牛鼻气管炎病毒孵育后，用含2％胎牛血清的1640培养所述牛肾细胞，继续培养至细胞全部脱落，收集所述牛肾细胞。
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