CN105861444B - 一株抗b亚群禽白血病病毒的细胞系及其应用 - Google Patents
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Abstract
本发明提供一株抗B亚群禽白血病病毒(ALV‑B)的细胞系及其应用。将ALV‑B env基因插入表达载体,将构建的转移载体在鸡胚成纤维细胞系DF‑1中表达,通过Zeocin药物筛选获得有Zeocin抗性、稳定表达ALV‑B env基因的鸡胚成纤维细胞系DF‑1/B,该细胞系保藏编号为CCTCC No.C201609。对DF‑1/B细胞系进行检测和抗病毒感染分析,发现DF‑1细胞系中表达的ALV‑B env蛋白对ALV‑B病毒具有超感染抗性,能抵抗滴度为104TCID50的病毒感染复制量,本发明的DF‑1/B细胞系可用于不同亚群禽白血病病毒感染的鉴别诊断,临床诊断价值显著,应用前景广阔。
Description
技术领域
本发明涉及免疫学领域,具体地,涉及一株抗B亚群禽白血病病毒的细胞系及其应用。
背景技术
禽白血病病毒(ALV)属于反转录病毒科禽α型反转录病毒群。禽白血病病毒与肉瘤病毒紧密相关,常统称为禽白血病/肉瘤病毒。根据细胞感染范围、干扰谱和囊膜抗原特征,可将禽白血病/肉瘤病毒群进一步区分为A到J等10个亚群,其中A、B、C、D、E、J亚群见于鸡等。A、B、J亚群是常见的外源性病毒,C、D亚群是极少报道的外源性病毒,其中E亚群为内源性病毒。禽白血病病毒囊膜蛋白(env基因编码的病毒糖基化蛋白)是该类病毒的主要表面蛋白,它决定病毒感染的宿主范围,能诱导产生中和抗体。该蛋白与靶细胞表面特异性受体识别、结合,是病毒进入细胞,复制所必需的。感染特定亚群ALV后,细胞表面病毒受体能被该病毒产生的env基因编码的病毒糖基化蛋白封闭,表现出强大的超感染抗性,能限制相应病毒的再次感染。
目前禽白血病对我国养禽业危害严重,并出现了髓细胞瘤型和血管瘤型等多种病理表现型,在临床表现上还容易与网状内皮组织增生症、马立克氏病等肿瘤性疾病相混淆。该病主要为垂直传播,控制本病有效的措施是降低种鸡群的感染率和建立无外源性禽白血病病毒感染的种鸡群,其主要手段是通过对鸡群定期进行外源性ALV检测,及时淘汰带毒阳性鸡,以净化和消除鸡群中的ALV。在外源性ALV中,以J亚群、A亚群和B亚群禽白血病病毒对鸡有明显的致病性,对养禽生产的危害也大。
国内外对于禽白血病病毒的检测和鉴定方法主要有细胞培养、ELISA、间接免疫荧光试验、PCR等,其中ELISA在临床上应用最广,其优点是该法操作相对简便,有商品化的检测试剂盒可以利用,其不足之处是不能区分内源性和外源性ALV;间接免疫荧光试验比较直观,但是往往需要用到特异性抗体(单抗)。能够区分不同ALV亚群的PCR引物及其工作体系已经有报道。
在外源性禽白血病病毒的分离上,目前常用的细胞有鸡胚成纤维细胞(CEF)和源于0系鸡的成纤维细胞系DF-1。美国ADOL实验室基于DF1细胞系,建立了抗J亚群禽白血病病毒的DF-1/J细胞系。尽管美国ADOL实验室有一些特定的鸡品系,其产生的原代细胞可以分别限制特定外源性禽白血病病毒生长,但是这些品系鸡的稀有性限制了其效能的发挥。目前还没有可特异性地限制B亚群禽白血病病毒的细胞系的相关报道。
发明内容
本发明的目的在于提供一株特异性的抗B亚群禽白血病病毒的细胞系。
本发明的另一目的在于提供上述抗B亚群禽白血病病毒的细胞系的应用。
本发明的抗B亚群禽白血病病毒细胞系的构建方法,包括如下步骤:
(1)利用带KpnI和NotI酶切位点的引物从ALV-B(CD08毒株)基因组PCR扩增env片段,用限制性内切酶KpnI和NotI分别对扩增的env基因及真核表达质粒pcDNA3.1/Zeo(+)进行双酶切,分别纯化后将B亚群禽白血病病毒env基因片段连接到pcDNA3.1/Zeo(+)真核表达质粒,构建成重组质粒pcDNA-env-B;
(2)转移载体pcDNA-env-B在DF-1鸡胚成纤维细胞系表达的基础上,通过Zeocin药物多次筛选,得到具有Zeocin抗性、可稳定表达ALV-B env蛋白的鸡胚成纤维细胞系DF-1/B,即抗B亚群禽白血病病毒的细胞系。
本发明筛选得到的抗B亚群禽白血病病毒鸡胚成纤维细胞命名为DF-1/B,于2016年1月21日在中国典型培养物保藏中心(地址:中国武汉武汉大学,中国典型培养物保藏中心,简称CCTCC,邮编430072)保藏,保藏编号CCTCC No.C201609。
本发明提供了保藏编号为CCTCC No.C201609的抗B亚群禽白血病病毒的细胞系在制备B亚群禽白血病病毒诊断试剂中的应用。
本发明提供了保藏编号为CCTCC No.C201609的抗B亚群禽白血病病毒的细胞系在制备B亚群禽白血病病毒抗体检测试剂中的应用。
本发明保藏编号为CCTCC No.C201609的细胞系可用于制备ALV-B单克隆抗体。
进一步,本发明提供一种ALV-B单克隆抗体,其由保藏编号为CCTCC No.C201609的细胞系制备得到。本发明提供了保藏编号为CCTCC No.C201609的抗B亚群禽白血病病毒的细胞系在制备B亚群禽白血病病毒生物制品中的应用。
所述生物制品为疫苗。
本发明提供了保藏编号为CCTCC No.C201609的抗B亚群禽白血病病毒的细胞系在制备抗B亚群禽白血病转基因鸡中的应用。
本发明还提供了一种用于检测B亚群禽白血病病毒的试剂,含有保藏编号为CCTCCNo.C201609的抗B亚群禽白血病病毒的细胞系。
本发明的抗B亚群禽白血病病毒的细胞系可应用于B亚群禽白血病病毒的诊断。
本发明提供了一种用于检测抗B亚群禽白血病病毒的细胞系的特异性引物对,其上下游引物序列为:
上游引物:CGGGGTACCGCCACCATGGAAGCCGTCATAAAGATGAGGCGAGCCCTCTTTTTGC;
下游引物:AAGGAAAAAAGCGGCCGCCTATACTGCTCTTTCGGGCTGCTTA。
目标片段大小约1845bp。
含有上述特异性引物对的试剂盒属于本发明的保护范围。
本发明从env蛋白超感染抗性出发,将B亚群禽白血病病毒env基因插入pcDNA3.1/Zeo(+)真核表达载体构建成转移载体pcDNA-env-B,然后将载体pcDNA-env-B转染鸡胚成纤维细胞系DF-1,通过药物Zeocin筛选,获得抗Zeocin、稳定表达ALV-B env蛋白的抗B亚群禽白血病病毒的细胞系。所得到的抗B亚群禽白血病病毒的细胞系可以稳定传代,长期保存;能抵抗滴度为10 4TCID50的病毒感染复制量,特异性强,本发明的DF-1/B细胞系可用于B亚群禽白血病病毒的诊断。
附图说明
图1为抗B亚群禽白血病病毒的细胞系构建的技术路线
图2为正常DF-1细胞状态:纤维状,梭形,贴壁生长。
图3为PCR检测DF-1/B中env基因结果,用本发明设计的引物在DF-1/B中成功检测到1845bp的env基因,而DF-1中没有。
图4为间接免疫荧光(IFA)检测env基因表达情况结果,其中图4A为DF-1细胞(阴性对照)无绿色荧光,图4B为DF-1/B细胞有绿色荧光,说明外源env基因片段在DF-1/B中成功表达。
图5为Western-blot检测env基因的表达结果,DF-1/B中有env蛋白表达,而DF-1中没有,说明外源env片段在DF-1/B中成功表达。Actin为内参蛋白,env蛋白为目的蛋白。
图6为抗B亚群禽白血病病毒(ALV-B)实验结果,CD08能在DF-1上正常繁殖,但在DF-1/B上,CD08不能正常繁殖,只有在病毒滴度为105TCID50时出现复制,但病毒复制明显受到抑制,显著低于在DF-1上的复制。结果说明,DF-1/B具有很强的抗ALV-B侵染细胞的能力。
图7为抗J亚群禽白血病病毒(ALV-J CHN06)实验结果,ALV-J在DF-1/B和DF-1上都能正常繁殖,繁殖情况没有显著差别,说明DF-1/B对ALV-J没有抗性。
图8为临床病毒分群诊断实例结果,DF-1/B的S/P值显著低于DF-1,说明DF-1/B对该ALV有抗性。
具体实施方式
以下实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
本发明的DF-1细胞系为0系鸡胚成纤维细胞,CD08为B亚群禽白血病病毒,均来自依托华南农业大学的农业部兽用疫苗创制重点实验室。本发明所用的限制性内切酶Kpn I和Not I购自NEB公司。pMD18-T载体购自TaKaRa公司、真核表达质粒pcDNA3.1/Zeo(+)购自Invitrogen公司。ALV-A/B特异性单克隆抗体,美国ADOL实验室馈赠。FITC标记的山羊抗鼠IgG抗体及HRP标记的山羊抗鼠IgG抗体均购自Simga公司。SQ Tissue组织DNA提取试剂盒,胶回收试剂盒DNA Gel Extraction Kit,去内毒素质粒抽提试剂盒Plasmid Miniprep Kit为OMEGA公司产品。ALV抗原检测试剂盒(Avian Leukosis Virus Antigen Test Kit)为美国IDEXX公司产品。高糖DMEM培养基粉、0.25%胰酶(含EDTA)、胎牛血清为美国GIBCO公司产品;二甲基亚砜(DMSO)为Sigma公司产品。POLOdeliverer3000转染试剂盒购自上海锐赛生物技术有限公司。
实施例1抗B群禽白血病病毒(ALV-B)细胞系DF-1/B的建立
本发明扩增ALV-B env基因的引物由发明人自行设计:
上游引物:CGGGGTACCGCCACCATGGAAGCCGTCATAAAGATGAGGCGAGCCCTCTTTTTGC
下游引物:AAGGAAAAAAGCGGCCGCCTATACTGCTCTTTCGGGCTGCTTA
反应体系(50μL):LA Taq 25μL、上下游引物各1μL、ddH2O21μL、模板2μL。
反应程序:94℃预变性3min;94℃变性30s,55℃退火1min,72℃延伸2min,30个循环;72℃后延伸8min。
用自行设计的上述引物从ALV-B(CD08毒株)基因组中扩增出env片段,按照胶回收试剂盒DNA Gel Extraction Kit说明书进行回收;按照NEB公司限制性内切酶KpnI、NotI说明书分别对真核表达质粒pcDNA3.1/Zeo(+)及pMD18-T-env片段进行双酶切;分别回收酶切产物,并按照NEB公司T4连接酶说明书对回收产物进行连接,构建重组质粒pcDNA-env-B;转化感受态细胞DH5α,按照OMEGA公司去内毒素质粒抽提试剂盒说明书,进行重组质粒pcDNA-env-B抽提。质粒送广州英潍捷基(Invitrogen)公司测序。
测序正确的质粒按照上海锐赛生物技术有限公司POLOdeliverer3000转染试剂盒说明书进行转染。
消化前期准备的DF-1细胞,铺六孔细胞培养板,10%FBS,5%CO2、37℃培养。24h内进行转染,Corning公司六孔细胞培养板每孔转染体系为:2μg pcDNA-env-B质粒和10μL脂质体分别用稀释并孵育5min,然后再混合,共同孵育15min,将200μL质粒-脂质体复合物加入到准备好的六孔细胞培养皿中。转染后5h换细胞培养液(DMEM+10%FBS),24h后将六孔板的一个孔的细胞消化下来,铺24孔板,每孔500μL细胞培养液(DMEM+15%FBS+200μg/mL zeocin),每隔3-4天换一次同样体系的细胞培养液,10-15d形成单细胞集落,三周左右,单细胞集落长大,消化下来置于6孔板内培养,贴壁长满之后转入25cm2细胞瓶中培养,此时细胞培养物为(DMEM+10%FBS+200μg/mL zeocin)。用药物zeocin对细胞连续筛选30代,从核酸及蛋白水平对细胞内的env进行检测,并进行抗病毒实验以及病毒类型的应用。
细胞状态:纤维状,梭形,贴壁生长。(如图2所示)
PCR检测DF-1/B中env基因:抽提DF-1/B的DNA,按照前述扩增env基因的引物、体系及反应程序扩增env基因,结果如图3所示,env基因扩增片段大小为1845bp。
间接免疫荧光(IFA)检测env基因表达情况:铺满单层DF-1/B细胞的24孔板内,培养5d后,底层细胞用PBS洗3次,用4%多聚甲醛固定20min。再用PBS洗涤3次,加入1:100稀释的ALV-B单抗,4℃孵育过夜。PBS洗涤3次,加上1:200稀释的羊抗鼠IgG-FITC荧光抗体,37℃作用1h,PBS洗涤3次后,加1滴体积分数50%甘油覆盖细胞,在荧光显微镜下观察实验结果。设立一个DMEM阴性对照。结果如图4所示,在荧光显微镜下可观察到DF-1/B细胞的胞浆和表面有特异荧光,DF-1细胞没有绿色荧光。说明外源env片段在DF-1/B中成功表达。
Western-blot检测env基因的表达:DF-1/B和对照DF-1培养5d后提细胞总蛋白,用Bio-Rad垂直电泳仪进行电泳,浓缩胶电压为80V,分离胶电泳电压为100V,电泳缓冲液为1×Tris-甘氨酸电泳缓冲液,待溴酚蓝指示剂达到分离胶底部后停止电泳。切胶转膜后,将NC膜用1×TBS洗涤3次,每次5min。将NC膜置于含5%的脱脂奶粉的TBS中,室温封闭2h,1×TBST洗涤3次,每次5min。加蛋白和Actin单抗,4℃孵育12h。抗体稀释浓度为env(1:500),Actin(1:1000)。一抗孵育之后,将膜用TBST洗涤3次,每次5min,将NC膜置于1:10000HRP标记的山羊抗鼠IgG二抗中,37℃孵育1h,1×TBST洗涤3次,每次5min。在暗室中,向NC膜上加入ECL免疫化学发光液孵育5min,用保鲜膜包好NC膜置于感光胶片上,显影,定影,晾干之后图片扫描,保存。结果如图5所示,DF-1/B中有env蛋白表达,而DF-1中没有。说明外源env片段在DF-1/B中成功表达。
本发明筛选得到的抗B亚群禽白血病病毒鸡胚成纤维细胞命名为DF-1/B,于2016年1月21日在中国典型培养物保藏中心(地址:中国武汉武汉大学,中国典型培养物保藏中心,简称CCTCC,邮编430072)保藏,保藏编号CCTCC No.C201609。
实施例2抗B群禽白血病病毒(ALV-B)细胞系DF-1/B的生物学活性实验
抗病毒实验:分别用DMEM将ALV-B毒株CD08从100到105进行倍比稀释。分别消化DF-1/B细胞和DF-1细胞,各接种于一块24孔细胞培养板中,每孔接种1mL细胞悬液,细胞浓度为1.7×105cells/mL。
将CD08各病毒稀释梯度的毒液分别接种于两板细胞悬液中,每孔接种100μL毒液,每个病毒稀释梯度做三个重复,并做一个已知阳性毒株的对照和DMEM阴性对照,5%CO2、10%FBS、37℃培养箱中培养。待接种毒液后第二天,细胞长满单层后,换为1%FBS的细胞维持液进行培养。连续培养6d,期间持续监测细胞的生长情况,并在第6天收集细胞培养物,冻融三次后离心收集细胞上清。收集的细胞上清液用同一个批次的ELISA试剂盒进行p27抗原ELISA检测。结果如图6所示,CD08能在DF-1上正常繁殖,但在DF-1/B上,CD08不能正常繁殖,只有在病毒滴度为105TCID50时出现复制,但病毒复制明显受到抑制,显著低于在DF-1上的复制。结果说明,DF-1/B具有很强的抗ALV-B侵染细胞的能力。
用同样的方法将ALV-J毒株CHN06接种DF-1/B细胞和DF-1细胞。结果如图7显示,ALV-J在DF-1/B和DF-1上都能正常繁殖,繁殖情况没有显著差别,说明DF-1/B对ALV-J没有抗性。
临床病毒分群鉴别诊断实例:如图8所示,将临床分离的ALV同时接种DF-1和DF-1/B,培养7天后,冻融三次后离心收集细胞上清,ELISA检测,DF-1/B的S/P值明显低于DF-1,说明DF-1/B对该ALV有抗性,初步判断该ALV为ALV-B。抽提接种该ALV的DF-1细胞DNA,扩增gp85,回收后送测序,证明该毒株确为ALV-B。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (1)
1.一株抗B亚群禽白血病病毒的细胞系,其为鸡胚成纤维细胞,名为DF-1/B,其保藏编号为CCTCC No.C201609。
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