CN113913461A - 牛病毒性腹泻e0-e2基因重组腺病毒疫苗构建方法 - Google Patents
牛病毒性腹泻e0-e2基因重组腺病毒疫苗构建方法 Download PDFInfo
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Abstract
本发明公开了一种牛病毒性腹泻E0‑E2基因重组腺病毒疫苗构建方法。本发明国内首次选用腺病毒载体来构建牛病毒性腹泻疫苗,运用重叠延伸PCR进行BVDV E0、E2双基因进行融合重组表达,构建重组腺病毒疫苗以预防牛病毒性腹泻疾病。
Description
技术领域
本发明涉及生物技术领域,尤其是基于预防牛病毒性腹泻疾病所构建牛病毒性腹泻E0-E2重组基因腺病毒载体疫苗的方法。
背景技术
自1946年P.Olafson在纽约首次发现牛病毒性腹泻,现该病逐渐分布流行全世界,随着养牛业的集约化、规模化养殖和新品种的交换引进使该病的发病率也随之增加,该病在全世界均有流行,对于该病的防控措施中目前最有效的仍然是疫苗免疫,但是该病毒的高变异性和传统疫苗的使用局限性疫苗的免疫失败促使该病愈发严重。故此需研制新型疫苗预防牛病毒性腹泻。
发明内容
本发明所要解决的技术问题是以预防牛病性腹泻疾病提供一种牛病毒性腹泻E0-E2基因重组腺病毒载体疫苗构建方法。
本发明是这样实现的:牛病毒性腹泻E0-E2基因重组腺病毒疫苗构建方法,运用重叠延伸PCR扩增双基因融合表达,新型重组腺病毒穿梭载体构建,重组腺病毒共转染包装,包含如下步骤进行:
(1)以克隆、鉴定成功的BVDV E0和BVDV E2基因组为模板应用引物BVDV E0-F/R和BVDV E2-F/R按照反应体系分别加入到离心管中,吹打混匀后瞬时离心以便去除气泡;将混匀后的EP管依次放入PCR仪中按照反应条件进行扩增BVDV E0、BVDV E2基因;
(2)重组穿梭载体构建与验证:以回收的E0、E2基因片段为模板,用E0-F,E2-R合成引物进行重叠延伸PCR扩增,回收E0-E2融合基因;通过对E0-E2融合基因与pDC316-mCMV-EGFP用Hind III酶、Sal I酶进行双酶切后回收产物,用T4连接酶将回收的E0-E2融合基因片段和pDC316-mCMV-EGFP载体线性化片段连接;并转入到感受态细胞Top 10培养后提取质粒,经双酶切和PCR进行验证;
(3)重组腺病毒包装:将验证好的穿梭质粒pDC316-E0-E2-EGFP与AdMax腺病毒系统的骨架质粒pBHGlox(delta)E1、3re混匀以后共转染至HEK293T细胞中于37℃、5%、CO2细胞培养箱中过夜培养。观察细胞生长形态直至细胞出现CPE,待一半细胞悬浮其余均有荧光时转移至2mL的冻存管中。-80℃/37℃水浴反复冻融3次,每次10min、涡旋1min,释放出腺病毒颗粒。4℃、12000g离心5min,0.22μm过滤收获上清液。验证完成腺病毒包装,将其命名为Ad5-E0-E2;
(4)重组腺病毒Ad5-E0-E2表达鉴定:采用SDS-PAGE和Western-blot方法进行鉴定;用重组腺病毒感染HEK293T细胞,待细胞产生大量病变且尚未脱落时收集细胞使其沉淀,使用裂解液使其裂解释放腺病毒;取上清液进行12%分离胶SDS-PAGE电泳以分离蛋白。经转膜后用TBST配置好的5%脱脂奶粉37℃封闭2h,取膜用TBST洗涤10min共3次;1:1000的牛BVDV阳性血清作为一抗,4℃孵育过夜;取膜用TBST配置好的HRP标记的山羊抗鼠IgG按1:1000倍数稀释后作二抗37℃孵育2h。用ECL发光试剂盒进行显色曝光并拍照;
(5)腺病毒滴度的测定:将100μL,1×105密度HEK293T细胞传于96孔细胞培养板中。用完全培养基对扩增的腺病毒做连续8个梯度的10倍稀释;弃去对应培养孔中旧培养基加入90μL梯度稀释的病毒溶液。感染12h后加入完全培养基100μL于37℃、5%、CO2细胞培养箱中培养,观察细胞生长形态直至出现细胞病变情况;计数倒数第二孔中细胞荧光数,最后一孔中细胞荧光数。 计算病毒滴度。
重叠延伸PCR扩增BVDV E0、E2双基因融合;根据合成的BV DV E2、E0基因片段设计特异性PCR引物扩增目的基因,BVDV E0-F:粗体划线为Sal I酶切位点;BVDV E0-R:5’-TCAGGTTTGCAATGCAAGTCTGCATAGGCTCCAAACCA-3'设计用于扩增BVDV E0基因;BVDV E2-F:5’-CATGGTTTGGAGCCTATGCAGACTTGCATTGCAAACCT-3'BVDV E2-R; 粗体划线为HindIII酶切位点,回收的E0、E2基因片段为模板,用E0-F,E2-R合成引物按反应体系进行重叠延伸PCR扩增,回收E0-E2融合基因。
重组穿梭载体构建;通过对E0-E2融合基因与pDC316-mCMV-EGFP用Hind III酶、Sal I酶进行双酶切以后回收产物,用T4连接酶将回收的E0-E2融合基因片段和pDC316-mCMV-EGFP载体线性化片段连接构建穿梭质粒pDC316-E0-E2-EGFP。
重组腺病毒包装;重组腺病毒包装将验证好的穿梭质粒pDC316-E0-E2-EGFP与AdMax腺病毒系统的骨架质粒pBHGlox(delta)E1、3re混匀以后共转染至HEK293T细胞中于37℃、5%、CO2细胞培养箱中过夜培养,观察细胞生长形态直至细胞出现CPE,待一半细胞悬浮其余均有荧光时转移到2mL的冻存管中,-80℃/37℃水浴反复冻融3次,每次10min、涡旋1min,释放出腺病毒颗粒,4℃、12000g离心5min,0.22μm过滤收获上清液,验证完成腺病毒包装。
结果
1.目的基因的扩增结果
将保存C24标准毒株进行解冻增殖后提取总RNA以cDNA为模板设计引物扩增BVDVE0、E2基因,获得一条大小为681bp、一条大小为1122bp的目的条带,两条特异性条带大小与预期结果一致。
2.重组穿梭载体构建与验证结果
通过用Hind III+Sal I酶对E0-E2融合基因与pDC316-mCMV-EGFP双酶切酶切后凝胶电泳并回收产物,用T4连接酶将回收的E0-E2融合基因片段和pDC316-mCMV-EGFP载体线性化片段连接。转入到感受态细胞Top 10培养后提取质粒,经PCR和双酶切进行验证,1%琼脂糖凝胶电泳中检测到一条1989bp和穿梭质粒5879bp的目的条带。目的条带和穿梭质粒条带出现的位置与预期相符。将目的条带胶回收后进行基因测序鉴定,测序结果与E0-E2融合基因序列完全重合,证明共表达E0-E2融合基因及pDC316-mCMV-EGFP,穿梭质粒载体pDC316-E0-E2-EGFP构建成功。
3.穿梭载体与腺病毒共转染包装检测结果
将构建好的穿梭质粒载体pDC316-E0-E2-EGFP与腺病毒系统骨架质粒混匀后转染HEK293细胞,转染17-20天后观察到细胞后产生串联、空泡、圆缩并脱离培养皿底壁等CPE现象,镜检细胞呈现绿色荧光与预期结果相符,证明重组腺病毒包装成功将其命名为Ad5-E0-E2。
4.重组腺病毒表达蛋白Western-blot检测结果
用重组腺病毒感染长成单层的293细胞,60小时后收获细胞,提取细胞总蛋白进行SDS-PAG电泳,将SDS-PAG电泳条带转移至NC膜上,分别以牛BVDV阳性血清作为一抗为一抗,山羊抗鼠IgG-HRP为二抗进行westen boltting检测。被Ad5-E0-E2感染的HEK293T细胞在约65ku处出现特异性条带。
5.腺病毒滴度的测定结果
Ad5-E0-E2滴度=1.1×1010pfu/mL
6.ELISA检测结果
稀释倍数 | P/N(肌注) | P/N(皮下) | 阳性对照 | 阴性 |
10<sup>1</sup> | 6.504 | 4.376 | 4.054 | 1.337 |
10<sup>2</sup> | 5.568 | 4.130 | 3.774 | 0.871 |
10<sup>3</sup> | 4.487 | 3.033 | 2.775 | 0.365 |
10<sup>4</sup> | 3.086 | 2.295 | 2.185 | 0.172 |
10<sup>5</sup> | 1.943 | 1.285 | 2.085 | 0.117 |
根据ELISA检测结果表明:将血清稀释到10-1至10-4后经过肌肉注射,皮下注射均能在小鼠体内检测到较好的抗体水平。而在10-5及以后抗体水平较差,在同等浓度梯度下肌肉注射的免疫效果较皮下注射组抗体水平较高。表明本实验构建的Ad5-E0-E2免疫小鼠产生抗体最低浓度为10-4抗体水平平均值为1:10000。对照组抗体检测均为阴性。
由于采用了上述技术方案,与现有技术相比,本发明国内首次选用腺病毒载体来构建牛病毒性腹泻疫苗,运用重叠PCR进行BVDV E0、E2双基因进行融合重组表达,构建重组腺病毒疫苗以预防牛病毒性腹泻疾病。
附图说明
图1为E0、E2扩增结果;
图1中,M.2000bp DNALaddder 1.E0 PCR产物2.E2 PCR产物
图2为E0-E2融合基因扩增结果。
图2中,M.2000bp DNA Laddder 1.E0-E2 PCR产物。
图3为pDC316-E0-E2-EGFP酶切鉴定结果;
图3中,M.5000bp DNA Laddder 1.pDC316-E0-E2-EGFP酶切产物。
2.pDC316-mCMV-EGFP载体3.阴性对照
图4为重组穿梭质粒pDC316-E0-E2-EGFP在293T细胞中转染情况;
图4中,A.重组穿梭质粒转染细胞荧光图;B.重组穿梭质粒转染细胞未激发荧光图。
图5为重组腺病毒的Western-blot验证。
图5中,M.蛋自质分子质量标准1.2.3.Ad5-E0-E2蛋白。
具体实施方式
本发明的实施例:牛病毒性腹泻E0-E2基因重组腺病毒疫苗构建方法,运用重叠延伸PCR扩增双基因融合表达,新型重组腺病毒穿梭载体构建,重组腺病毒共转染包装,包含如下步骤进行:
(1)以克隆、鉴定成功的BVDV E0和BVDV E2基因组为模板应用引物BVDV E0-F/R和BVDV E2-F/R按照反应体系分别加入到离心管中,吹打混匀后瞬时离心以便去除气泡;将混匀后的EP管依次放入PCR仪中按照反应条件进行扩增BVDV E0、BVDV E2基因;
(2)重组穿梭载体构建与验证:以回收的E0、E2基因片段为模板,用E0-F,E2-R合成引物进行重叠延伸PCR扩增,回收E0-E2融合基因;通过对E0-E2融合基因与pDC316-mCMV-EGFP用Hind III酶、Sal I酶进行双酶切后回收产物,用T4连接酶将回收的E0-E2融合基因片段和pDC316-mCMV-EGFP载体线性化片段连接;并转入到感受态细胞Top 10培养后提取质粒,经双酶切和PCR进行验证;
(3)重组腺病毒包装:将验证好的穿梭质粒pDC316-E0-E2-EGFP与AdMax腺病毒系统的骨架质粒pBHGlox(delta)E1、3re混匀以后共转染至HEK293T细胞中于37℃、5%、CO2细胞培养箱中过夜培养。观察细胞生长形态直至细胞出现CPE,待一半细胞悬浮其余均有荧光时转移至2mL的冻存管中。-80℃/37℃水浴反复冻融3次,每次10min、涡旋1min,释放出腺病毒颗粒。4℃、12000g离心5min,0.22μm过滤收获上清液。验证完成腺病毒包装,将其命名为Ad5-E0-E2;
(4)重组腺病毒Ad5-E0-E2表达鉴定:采用SDS-PAGE和Western-blot方法进行鉴定;用重组腺病毒感染HEK293T细胞,待细胞产生大量病变且尚未脱落时收集细胞使其沉淀,使用裂解液使其裂解释放腺病毒;取上清液进行12%分离胶SDS-PAGE电泳以分离蛋白。经转膜后用TBST配置好的5%脱脂奶粉37℃封闭2h,取膜用TBST洗涤10min共3次;1:1000的牛BVDV阳性血清作为一抗,4℃孵育过夜;取膜用TBST配置好的HRP标记的山羊抗鼠IgG按1:1000倍数稀释后作二抗37℃孵育2h。用ECL发光试剂盒进行显色曝光并拍照;
(5)腺病毒滴度的测定:将100μL,1×105密度HEK293T细胞传于96孔细胞培养板中。用完全培养基对扩增的腺病毒做连续8个梯度的10倍稀释;弃去对应培养孔中旧培养基加入90μL梯度稀释的病毒溶液。感染12h后加入完全培养基100μL于37℃、5%、CO2细胞培养箱中培养,观察细胞生长形态直至出现细胞病变情况;计数倒数第二孔中细胞荧光数,最后一孔中细胞荧光数。 计算病毒滴度。
重叠延伸PCR扩增BVDV E0、E2双基因融合;根据合成的BV DV E2、E0基因片段设计特异性PCR引物扩增目的基因,BVDV E0-F:粗体划线为Sal I酶切位点;BVDV E0-R:5’-TCAGGTTTGCAATGC AAGTCTGCATAGGCTCCAAACCA-3'设计用于扩增BVDV E0基因;BVDV E2-F:5’-CATGGTTTGGAGCCTATGCAGACTTGCATTGCAAACCT-3'BVDV E2-R; 粗体划线为HindIII酶切位点,回收的E0、E2基因片段为模板,用E0-F,E2-R合成引物按反应体系进行重叠延伸PCR扩增,回收E0-E2融合基因。
重组穿梭载体构建;通过对E0-E2融合基因与pDC316-mCMV-EGFP用Hind III酶、Sal I酶进行双酶切以后回收产物,用T4连接酶将回收的E0-E2融合基因片段和pDC316-mCMV-EGFP载体线性化片段连接构建穿梭质粒pDC316-E0-E2-EGFP。
重组腺病毒包装;重组腺病毒包装将验证好的穿梭质粒pDC316-E0-E2-EGFP与AdMax腺病毒系统的骨架质粒pBHGlox(delta)E1、3re混匀以后共转染至HEK293T细胞中于37℃、5%、CO2细胞培养箱中过夜培养,观察细胞生长形态直至细胞出现CPE,待一半细胞悬浮其余均有荧光时转移到2mL的冻存管中,-80℃/37℃水浴反复冻融3次,每次10min、涡旋1min,释放出腺病毒颗粒,4℃、12000g离心5min,0.22μm过滤收获上清液,验证完成腺病毒包装。
1.将构建的Ad5-E0-E2以大腿内侧肌肉注射接种100μL(1.1×1010pfu/mL)于体重18-20g雌性10只小鼠经首免和一次加强免疫后,采集免疫小鼠血液于使用ELISA试剂盒检测小鼠体内的抗体水平,结果免疫小鼠均能产生抗体,产生抗体最低浓度为10-4。
2.将构建的Ad5-E0-E2以背部皮下多点注射接种100μL(1.1×1010pfu/mL)于体重18-20g雌性10只小鼠经首免和一次加强免疫后,采集免疫小鼠血液于使用ELISA试剂盒检测小鼠体内的抗体水平,结果免疫小鼠均能产生抗体,产生抗体最低浓度为10-4。
3.将构建的Ad5-E0-E2以鼻部粘膜滴鼻接种30μL(1.1×1010pfu/mL)于体重18-20g雌性10只小鼠经首免和一次加强免疫后,采集免疫小鼠血液于使用ELISA试剂盒检测小鼠体内的抗体水平,结果免疫均能产生抗体,产生抗体最低浓度为10-2。
牛病毒性腹泻病(Bovine viraldiarrhea/mucosal disease,BVD)是由牛病毒性腹泻病毒(Bovine viral diarrheavirus,BVDV)所引起的牛重症性传染性疾病,于1946年P.Olafson在纽约首次发现,现该病逐渐分布流行全世界,随着养牛业的集约化、规模化养殖和新品种的引进使该病的发病率也随之增加,病死率高达35%,对全世界的养殖经济造成损失。对于该病的防控措施中目前最有效的仍然是疫苗免疫,在售的商品疫苗有减毒疫苗和灭活疫苗但是由于该病毒的高变异性和传统疫苗的局限性疫苗的免疫失败从而促使该病愈发严重。腺病毒载体疫苗是携带具有保护性抗原基因从而使免疫宿主产生免疫反应,腺病毒载体疫苗不整合到宿主基因、无需佐剂但是刺激机体产生强烈的体液和细胞免疫反应等优势。BVD腺病毒载体疫苗仅1999年国外Elahi等利用BVDV C蛋白基因构建重组腺病毒,将其免疫小鼠后,能够产生特异性免疫反应,但未做进一步研究。国内对于BVD腺病毒载体疫苗未曾报道研究,BVD的E0、E2基因蛋白是囊膜糖蛋白和主要保护性抗原,E0基因具有高度保守性和在病毒正常生理活性方面发挥着重要作用。E2基因具有保守的糖基化位点是BVDV的主要保护性抗原蛋白且是主要抗原区域与宿主细胞识别、吸附的重要部位。因此,本文首次使用以人腺病毒5型为载体将牛病毒性腹泻E0-E2基因进行融合重组表达,构建重组腺病毒疫苗。为构建牛病毒性腹泻病毒(BVDV)E0-E2基因重组腺病毒以及评价其免疫效果。通过特异性PCR方法扩增出BVDV的E0、E2基因,然后进行重叠延伸PCR扩增E0-E2融合基因。经酶切后构建pDC316-E0-E2-EGFP,通过与腺病毒骨架质粒pBHGlox(delta)E、3re共转染HEK293T细胞进行包装重组腺病毒Ad5-E0-E2。用SDS-PAGE检测重组Ad5-E0-E2在HEK293T细胞中的表达情况,并使用Western-blot验证其反应原性。另将100μL 1.1×1010pfu/mL剂量的Ad5-E0-E2分别以肌肉注射和皮下注射的方式接种SPF级小鼠,经2次免疫后,采取免疫小鼠血液于使用ELISA试剂盒检测小鼠体内的抗体水平,验证重组的Ad5-E0-E2重组腺病毒疫苗免疫效果。结果显示,PCR扩增出了681bp的E0蛋白基因片段和1122bp的E2蛋白基因片段;对pDC316-E0-E2-EGFP穿梭质粒进行双酶切后采用PCR和凝胶电泳方法验证,本试验构建出了pDC316-E0-E2-EGFP穿梭质粒,SDS-PAGE验证构建的重组腺病毒能够表达65ku的重组蛋白,Western-blot验证重组腺病毒具有较好的反应原性。通过肌肉注射及皮下注射两种免疫方式均能产生针对BVDV的特异性抗体。结果表明,试验成功构建Ad5-E0-E2重组腺病毒,该重组腺病毒能够诱导小鼠产生针对BVDV的特异性抗体。
序列表
<110> 贵州大学
<120> 牛病毒性腹泻E0-E2基因重组腺病毒疫苗构建方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> DNA
<213> 牛病毒性腹泻病毒(Bovine viraldiarrhea/mucosal disease)
<400> 1
gagagctccg cgaaaacata acacagtgga a 31
<210> 2
<211> 38
<212> DNA
<213> 牛病毒性腹泻病毒(Bovine viraldiarrhea/mucosal disease)
<400> 2
tcaggtttgc aatgcaagtc tgcataggct ccaaacca 38
<210> 3
<211> 38
<212> DNA
<213> 牛病毒性腹泻病毒(Bovine viraldiarrhea/mucosal disease)
<400> 3
catggtttgg agcctatgca gacttgcatt gcaaacct 38
<210> 4
<211> 26
<212> DNA
<213> 牛病毒性腹泻病毒(Bovine viraldiarrhea/mucosal disease)
<400> 4
acaagcttct gaggccttct gttctg 26
Claims (4)
1.一种牛病毒性腹泻E0-E2基因重组腺病毒疫苗构建方法,其特征在于运用重叠延伸PCR扩增双基因融合表达,新型重组腺病毒穿梭载体构建,重组腺病毒共转染包装,包含如下步骤进行:
(1)以克隆、鉴定成功的BVDV E0和BVDV E2基因组为模板应用引物BVDV E0-F/R和BVDVE2-F/R按照反应体系分别加入到离心管中,吹打混匀后瞬时离心以便去除气泡;将混匀后的EP管依次放入PCR仪中按照反应条件进行扩增BVDV E0、BVDV E2基因;
(2)重组穿梭载体构建与验证:以回收的E0、E2基因片段为模板,用E0-F,E2-R合成引物进行重叠延伸PCR扩增,回收E0-E2融合基因;通过对E0-E2融合基因与pDC316-mCMV-EGFP用Hind III酶、Sal I酶进行双酶切后回收产物,用T4连接酶将回收的E0-E2融合基因片段和pDC316-mCMV-EGFP载体线性化片段连接;并转入到感受态细胞Top 10培养后提取质粒,经双酶切和PCR进行验证;
(3)重组腺病毒包装:将验证好的穿梭质粒pDC316-E0-E2-EGFP与AdMax腺病毒系统的骨架质粒pBHGlox(delta)E1、3re混匀以后共转染至HEK293T细胞中于37℃、5%、CO2细胞培养箱中过夜培养;观察细胞生长形态直至细胞出现CPE,待一半细胞悬浮其余均有荧光时转移至2mL的冻存管中;-80℃/37℃水浴反复冻融3次,每次10min、涡旋1min,释放出腺病毒颗粒;4℃、12000g离心5min,0.22μm过滤收获上清液;验证完成腺病毒包装,将其命名为Ad5-E0-E2;
(4)重组腺病毒Ad5-E0-E2表达鉴定:采用SDS-PAGE和Western-blot方法进行鉴定;用重组腺病毒感染HEK293T细胞,待细胞产生大量病变且尚未脱落时收集细胞使其沉淀,使用裂解液使其裂解释放腺病毒;取上清液进行12%分离胶SDS-PAGE电泳以分离蛋白。经转膜后用TBST配置好的5%脱脂奶粉37℃封闭2h,取膜用TBST洗涤10min共3次;1:1000的牛BVDV阳性血清作为一抗,4℃孵育过夜;取膜用TBST配置好的HRP标记的山羊抗鼠IgG按1:1000倍数稀释后作二抗37℃孵育2h。用ECL发光试剂盒进行显色曝光并拍照;
2.根据权利要求1所述的牛病毒性腹泻E0-E2基因重组疫苗构建方法,其特征在于:重叠延伸PCR扩增BVDV E0、E2双基因融合;根据合成的BVDV E2、E0基因片段设计特异性PCR引物扩增目的基因,BVDV E0-F:5’-GAGAGCTCCGCGAAAACATAACACAGTGGAA-3',粗体划线为SalI酶切位点;BVDV E0-R:5’-TCAGGTTTGCAATGCAAGTCTGCATAGGCTCCAAACCA-3'设计用于扩增BVDV E0基因;BVDV E2-F:5’-CATGGTTTGGAGCCTATGCAGACTTGCATTGCAAACCT-3'BVDV E2-R;5’-ACAAGCTTCTGAGGCCTTCTGTTCTG-3',粗体划线为HindIII酶切位点,回收的E0、E2基因片段为模板,用E0-F,E2-R合成引物按反应体系进行重叠延伸PCR扩增,回收E0-E2融合基因。
3.根据权利要求1所述的牛病毒性腹泻E0-E2基因重组疫苗构建方法,其特征在于:重组穿梭载体构建;通过对E0-E2融合基因与pDC316-mCMV-EGFP用Hind III酶、Sal I酶进行双酶切以后回收产物,用T4连接酶将回收的E0-E2融合基因片段和pDC316-mCMV-EGFP载体线性化片段连接构建穿梭质粒pDC316-E0-E2-EGFP。
4.根据权利要求1所述的牛病毒性腹泻E0-E2基因重组疫苗构建方法,其特征在于:重组腺病毒包装;重组腺病毒包装将验证好的穿梭质粒pDC316-E0-E2-EGFP与AdMax腺病毒系统的骨架质粒pBHGlox(delta)E1、3re混匀以后共转染至HEK293T细胞中于37℃、5%、CO2细胞培养箱中过夜培养,观察细胞生长形态直至细胞出现CPE,待一半细胞悬浮其余均有荧光时转移到2mL的冻存管中,-80℃/37℃水浴反复冻融3次,每次10min、涡旋1min,释放出腺病毒颗粒,4℃、12000g离心5min,0.22μm过滤收获上清液,验证完成腺病毒包装。
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