CN113604439A - 一种抗猪萨佩罗病毒vp1蛋白杂交瘤细胞株、单克隆抗体及其应用 - Google Patents
一种抗猪萨佩罗病毒vp1蛋白杂交瘤细胞株、单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,公开了一种分泌猪萨佩罗病毒VP1蛋白单克隆抗体的杂交瘤细胞株,其中所述杂交瘤细胞株保藏编号CCTCC NO:C2021177,保藏于中国典型培养物保藏中心,保藏地址为中国武汉·武汉大学。本发明所涉及的杂交瘤细胞具有稳定的抗体分泌能力,所分泌的单克隆抗体与猪萨佩罗病毒VP1蛋白具有良好的反应特异性,其识别的抗原表位在猪萨佩罗病毒VP1蛋白第40~46位氨基酸,其多肽序列为40PALTAAE46,该表位尚未见报道。本发明为以后进行猪萨佩罗病毒病原学及其致病机理的研究奠定了良好的物质基础。
Description
技术领域
本发明属于生物技术领域,具体涉及表达猪萨佩罗病毒VP1蛋白单克隆抗体的杂交瘤细胞株。
背景技术
猪萨佩罗病毒(Porcine Sapelovirus,PSV)首次在英国发现,由于最初分离自腹泻猪的肠道中,故被命名为猪肠道病毒8型(porcine enterovirus 8,PEV8)。PSV主要经粪-口途径传播,可引起猪脑脊髓灰质炎、腹泻、肺炎、繁殖障碍、心肌炎等多系统综合征,同时也可表现为无明显特征的亚临床感染,临床上还会与猪瘟病毒、猪细小病毒、猪流行性腹泻病毒、猪繁殖与呼吸障碍综合征病毒、猪肠道病毒等发生混合感染,给养猪行业带来严重危害。
猪萨佩罗病毒是一种微球形,无囊膜包裹的单股正链RNA病毒,属于小RNA病毒科,萨佩罗病毒属。其基因组大小约7.5kb,仅编码一个开放阅读框,多聚蛋白结构与其他小RNA病毒相似,呈L-4-3-4分布。VP1蛋白是PSV病毒粒子最外部的表面蛋白,据小RNA病毒研究结果显示,VP1蛋白是肠道病毒诱导机体产生中和抗体及病毒维持感染力的主要蛋白,是口蹄疫病毒具有抗原性的关键成分,同时也是柯萨奇病毒诱导机体产生体液免疫应答的主要成分。此外,也有研究已经证明,VP1蛋白是PSV的主要免疫原性蛋白。
近年来,根据世界各国养猪业感染PSV情况的报道发现,疫情呈上升趋势。快速、准确的兽医临床诊断是降低PSV造成经济损失的关键。目前,PSV的检测仍然停留于病原分离鉴定及普通PCR检测上,敏感性、特异性均不十分理想,假阳性率高。因此,建立一种快速、特异的PSV检测方法对猪萨佩罗病毒病的防控具有重要意义。
单克隆抗体是由单一B细胞克隆产生的高度均一、仅针对某一特定抗原表位的抗体。通过单克隆抗体技术来获取杂交瘤细胞株,该技术已经应用到了兽医领域多个动物病毒的研究之中,如非洲猪瘟病毒、口蹄疫病毒、流感病毒等。目前仅有利用原核表达系统体外表达PSV重组VP1以及3C蛋白,尚无检测抗体的方法。
发明内容
本发明要解决的技术问题在于提供一种抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株、单克隆抗体及其应用,该方法所制备的杂交瘤细胞株可表达用于检测猪萨佩罗病毒病毒的单克隆抗体,其所分泌的单克隆抗体与猪萨佩罗病毒VP1蛋白具有良好的反应特异性。
本发明所解决的技术问题采用的技术方案是:
一种抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株,命名为杂交瘤细胞株PSV-VP1-33-2A,保藏于中国典型培养物保藏中心,保藏日期为2021年7月12日,保藏地址为中国武汉·武汉大学,保藏号为CCTCC NO:C2021177。
一种单克隆抗体,所述单克隆抗体由上述杂交瘤细胞株分泌产生。
进一步的,其重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1~3所示;轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.4~6所示。
进一步的,其重、轻链的氨基酸序列分别如SEQ ID NO:7和SEQ ID NO:8所示。
一种用于检测猪萨佩罗病毒的试剂、试纸条或试剂盒,其含有上述的单克隆抗体。
上述单克隆抗体或试剂、试纸条或试剂盒在非诊断用途的检测猪萨佩罗病毒方面的应用。
一种非诊断用途的检测猪萨佩罗病毒的方法,使用上述单克隆抗体或上述试剂、试纸条或试剂盒。
一种核酸分子,其特征在于,包含编码上述单克隆抗体的核苷酸。
进一步的,所述核酸分子编码抗体,的轻链可变区的核苷酸序列如SEQ ID NO:9所示,所述核酸分子编码抗体的重链可变区的核苷酸序列如SEQ ID NO:10所示。
一种抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株的制备方法,包括以下步骤:原核表达的重组猪萨佩罗病毒VP1蛋白与弗氏佐剂混合后注射BALB/C小鼠,并对IFA检测结果阳性的小鼠进行加强免疫;将加强免疫后的小鼠采集脾B淋巴细胞,并与杂交瘤细胞进行融合,挑选阳性克隆后进行亚克隆得到抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株。
一种抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株的制备方法,该方法由以下步骤组成:
(1)原核表达的重组猪萨佩罗病毒VP1蛋白与弗氏佐剂1:1混合后注射BALB/C小鼠,100ug/只。三次免疫后对IFA检测结果阳性的小鼠进行加强免疫,剂量50~100ug/只。
(2)IFA方法测定血清抗体稀释度在1:103的小鼠采集脾B淋巴细胞与杂交瘤细胞进行融合,挑选阳性克隆后进行亚克隆得到抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株。
本发明还提供一种表达猪萨佩罗病毒VP1蛋白的单克隆抗体,该单克隆抗体由上述方法得到的抗猪萨佩罗病毒病毒VP1蛋白杂交瘤细胞株表达。
本发明所取得的技术效果如下:
至今为止市面上没有PSV的血清学诊断产品,PSV的诊断局限于分子水平,这给PSV的后续研究带来极大不便。PSV的四种结构蛋白(VP4、VP2、VP3、VP1)构成了成熟病毒粒子的整个核衣壳,在病毒感染过程中承担着重要角色。在PSV的四种结构蛋白中,VP1蛋白位于PSV病毒粒子的最外部,小RNA病毒科中的病毒研究表明,如在柯萨奇病毒中,VP1蛋白被证明是能诱导抗体产生体液免疫的优势抗原;在猪肠道病毒中,VP1蛋白除了可以诱导产生中和抗体外,还含有细胞受体的结合位点,是病毒基因入侵细胞的关键蛋白。其次相较于真核表达系统而言,原核表达系统具有成本低廉、操作方法简单、表达水平高、生产周期短等优点,适合于目标蛋白的大规模生产。因此本发明公开了一种分泌PSV重组VP1蛋白单克隆抗体的杂交瘤细胞株,其所分泌的单克隆抗体与猪萨佩罗病毒VP1蛋白具有良好的反应特异性。并首次明确了PSV的一个线性B细胞表位,表明分泌单克隆抗体的杂交瘤细胞株可以识别大多数PSV毒株,为PSV新型诊断试剂的研究工作提供了有力的免疫学工具。本发明提供一种抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株的制备方法,该方法所制备的杂交瘤细胞株可表达用于检测猪萨佩罗病毒病毒的单克隆抗体。
附图说明
下面将结合附图及实施例对本发明作进一步说明,附图中:
图1是由杂交瘤细胞株分泌产生的单克隆抗体与PSV感染细胞的间接免疫荧光图,结果显示,单克隆抗体可特异性识别PSV,胞浆中出现绿色荧光,而未接毒细胞中未观察到荧光;
图2是由杂交瘤细胞株分泌产生的单克隆抗体的Western blot结果,结果显示,单克隆抗体可与PSV感染细胞样特异性结合,在32Kda处有一条清晰条带,而未接毒细胞样中未观察到条带;
图3是由杂交瘤细胞株分泌产生的单克隆抗体与PSV感染细胞的IP结果,结果表明单克隆抗体能在IP试验中特异性结合PSV感染细胞样品中的VP1蛋白;
图4是单克隆抗体抗原表位的初步鉴定Western blot结果;抗原表位的初步氨基酸范围为VP1蛋白第30~52位氨基酸,其多肽序列为30NGIMINQGDAPALTAAETGESDT52
图5是单克隆抗体抗原表位的精确鉴定Western blot结果;结果显示,单克隆抗体识别的线性表位为VP1蛋白第40~46位氨基酸,其多肽序列为40PALTAAE46,
图6是单克隆抗体识别的表位在PSV不同病毒株间的序列比对,表明单克隆抗体可以识别大多数PSV毒株;
图7是单克隆抗体抗原表位在蛋白三维结构模型上的定位,表明单克隆抗体所识别的抗原表位暴露于VP1蛋白的头部
图8是单克隆抗体的特异性鉴定,表明单克隆抗体只特异性识别PSV。
具体实施方式
下面,结合附图以及具体实施方式,对本发明做进一步描述,但应当理解本发明的保护范围并不受具体实施方式的限制。
实施例1单克隆抗体杂交瘤细胞株的建立
1材料和方法
1.1实验材料
1.1.1主要试剂
HAT、HT选择培养基购自北京博奥龙公司、HRP goat anti Mouse IgG购自Abcam生命科学有限公司、弗氏完全佐剂,弗氏不完全佐剂,PEG1450购自Sigma公司、DMEM培养基购自Gibco公司、胎牛血清购自杭州四季青公司,羊抗鼠IgG荧光抗体购自Abbkine,ProteinA/G免疫沉淀磁珠购自Bimake公司,荧光显微镜购自德国LeiCa公司。
1.1.2细胞、实验动物、毒株与血清
ST-R细胞(IFN-β受体敲除的猪睾丸细胞)、SP2/0骨髓瘤细胞为本发明人实验室冻存。雌性BALB/C小鼠购于扬州大学比较医学中心。HuN2株PSV。
1.重组猪萨佩罗病毒VP1抗原的制备
(1)重组质粒的构建:根据HuN2株VP1序列设计特异性引物,插入原核表达载体,经过酶切鉴定得到重组质粒。
(2)重组蛋白的表达与纯化:将阳性重组质粒转化BL21(DE3),进行蛋白的预表达。确认目的蛋白表达后,将菌种在2×YT液体培养基中大量培养、诱导表达,收集蛋白进行SDS-PAGE电泳,剥离分离胶并在预冷的18g/L KCl中染色10min,切下目的蛋白条带并切成小块。将胶块置入3500MW透析袋中,加入5mL PBS,120V电洗脱2h。弃去胶块,将透析袋置于含有200mL PBS的烧杯中,冰浴透析过夜。次日将透析袋包埋入PEG 8000粉末进行蛋白浓缩30min,收集透析袋内的蛋白溶液,分装后于-80℃保存备用。
2.获取杂交瘤细胞
(1)小鼠免疫:利用纯化的重组蛋白为免疫原,免疫6~8周龄雌性BALB/C小鼠。
第一次免疫:取等量的重组蛋白与弗氏完全佐剂1:1混合,以100ug/只的剂量皮下多点注射小鼠;
第二次免疫:间隔两周后,取等量的重组蛋白与弗氏不完全佐剂1:1混合,以100ug/只的剂量腹腔注射小鼠;
第三次免疫:再间隔两周后,取等量的重组蛋白与弗氏不完全佐剂1:1混合,以100ug/只的剂量腹腔注射小鼠;第三次免疫后7~10天,小鼠眼眶静脉丛采血用IFA方法测定血清抗体效价,选择效价高的小鼠在细胞融合前3天,腹腔注射50-100ug抗原加强免疫。
(2)免疫小鼠血清效价的测定:采用IFA方法检测免疫血清效价。将ST-R细胞铺于24孔板中,以MOI=0.01接种PSV病毒液,同时以未感染的ST-R细胞作为阴性对照。感染后约16-20h,弃去培养基,PBS洗3遍,加入4%多聚甲醛室温固定10min,弃固定液,PBS洗3遍,牛血清白蛋白(BSA)与Triton-X-1001:1混合封闭30min,PBS洗3遍,加入小鼠血清,37℃孵育1h,PBS洗3遍,再用Dylight488羊抗鼠二抗(1:2000)37℃避光孵育1h,PBS洗3次,倒置荧光显微镜观察结果。
(3)细胞融合:
小鼠脾细胞的准备
将冲击免疫的小鼠脱颈处死,浸泡于75%酒精5min,放置于平皿上,小心取出脾脏,转移到干净平皿中剔除多余组织,使用DMEM冲洗;
将脾转移到另一盛有DMEM的干净平皿中,用针头扎脾多次,然后将脾转移到50ml离心管上的细胞筛上,加入2mlDMEM润湿细胞筛,使用注射器推头部分研磨脾,使用DMEM冲洗细胞筛,最后将脾细胞悬于10mlDMEM中,取10ul悬液使用0.4%台盼蓝染色并计数。将离心管放置37℃培养箱备用。
小鼠饲养细胞的制备
将健康的小鼠脱颈处死,浸泡于75%酒精5min,放置于平皿上,小心剪开腹部被皮,暴露腹膜;
使用10ml注射器吸取5mlHAT培养基,一只手持镊子轻轻夹起腹膜,另一只手持注射器将HAT培养基推入腹腔,针头要避开器官尤其是肠;
轻轻晃动腹腔中的液体,使饲养细胞悬于HAT培养基,然后回抽注射器。将取出的饲养细胞悬液加入10ml离心管你,放置37℃培养箱备用。
SP2/0细胞准备
SP2/0细胞需处于对数期,细胞密度较高,细胞圆润、透亮(融合前一天传一代);
使用10mlDMEM将培养皿的SP2/0吹下,取10ul悬液用0.4%台盼蓝染色并计数;
按照脾细胞:SP2/0细胞数量比5:1~10:1的比例进行混合,制成总体积20ml的混合液。
细胞融合
将SP2/0-脾细胞混合液1000rpm离心5mim,倒去上清,轻轻拍打离心管底部,使沉淀的细胞均匀悬浮于残液,且没有细胞团块;
一边旋转离心管,一边沿壁滴加PEG1450,1min内加完,然后37℃水浴1min,最后在5min内补加DMEM至20ml,遵循先快后慢原则。全部完成后,放置37℃培养箱10min.
将融合细胞混合液1000rpm离心5min,倒去上清,轻轻拍打离心管,使沉淀的细胞均匀悬浮于残液,加入HAT培养基和饲养细胞悬液,使总体积为72ml,1ml/孔,铺于3个24孔板。
(4)杂交瘤细胞筛选与克隆:间接IFA法筛选细胞培养上清,选择效价高的阳性克隆杂交瘤细胞进行亚克隆,并用有限稀释法连续克隆化2~3次,直至100%阳性率,最后获得稳定分泌抗猪萨佩罗病毒VP1蛋白单克隆抗体细胞株,标记为PSV-33-2A。将克隆化后阳性率达100%的细胞扩增培养后液氮冻存。
3.杂交瘤细胞稳定性测定
在连续传代过程中冻存和复苏细胞,检测复苏后的细胞是否还分泌阳性抗体。
4.腹水的制备
选取8周龄的BALB/c小鼠4只,腹腔注射液体石蜡,500μL/只。7天后取106个阳性单克隆杂交瘤细胞悬于200μL DMEM,分别腹腔注射每只小鼠。观察小鼠生长状态,待腹部膨大时使用10mL注射器吸取腹水,5000rpm/mim离心10mim后将抗体于-80℃保存备用。
实施案例2单克隆抗体的特性鉴定
(1)单抗与PSV的间接免疫荧光试验
将ST-R细胞铺于24孔板中,以MOI=0.01接种PSV病毒液,同时以未感染的ST-R细胞作为阴性对照。感染后约16-20h,弃去培养基,PBS洗3遍,加入4%多聚甲醛室温固定10min,弃固定液,PBS洗3遍,牛血清白蛋白(BSA)与Triton-X-1001:1混合封闭30min,PBS洗3遍,加入VP1单抗,37℃孵育1h,PBS洗3遍,再用Dylight488羊抗鼠二抗(1:2000)37℃避光孵育1h,PBS洗3次,倒置荧光显微镜观察结果。
(2)Western blot鉴定单克隆抗体的反应性
在6孔板中接种ST-R细胞,待细胞生长状态良好并生长至80%满时,以PSV MOI=0.1感染细胞,当CPE达到50%时收样。进行SDS-PAGE,200mA恒流30min转印至PVDF膜。用含5%脱脂乳的TBST封闭2h,以1:1 000稀释的腹水作为一抗,4℃孵育过夜;TBST洗涤3次后,与HRP标记羊抗鼠IgG二抗(1:10 000)室温作用1h;TBST洗涤3次后,使用ECL发光试剂盒显色。同时设置未接毒的ST-R细胞样品作为阴性对照。
(3)Protein A/G IP试验鉴定单克隆抗体的反应性
在6孔板中接种ST-R细胞,待细胞生长状态良好并生长至80%满时,以PSV MOI=0.1感染细胞,37℃感染2h后弃去上清,换成维持培养基。当CPE达到50%时收样,收集上清放置冰上备用。取磁珠20μL,结合缓冲液洗涤2次,加入1:100稀释的VP1单抗腹水200μL,吸打混匀后室温翻转离心管15min,使抗体吸附到磁珠上。之后按说明书标准程序制备磁珠-抗体-抗原复合物,加入5×Loading Buffer煮沸,分离上清,进行WB检测。同时设置未接毒的ST-R细胞样品作为阴性对照,设置接种PSV的ST-R细胞总蛋白作为阳性对照。
实施案例3单克隆抗体抗原表位鉴定
(1)单克隆抗体抗原表位的初步鉴定
以PSV-HuN2株为模板扩增得到四段部分重叠的VP1蛋白基因片段A、B、C、D,各个片段通过酶切位点插入pEGFP-C3载体中,构建得到的阳性质粒送去南京擎科测序部测序。参照Entranster-H4000说明书,将各重组质粒转染至HEK293A细胞中,36h后收集细胞总蛋白,以单克隆抗体(1:1000)作为一抗,HRP Goat Anti-Mouse IgG作为二抗,通过Western Blot初步定位抗原表位的区域范围位于B片段上,氨基酸位置为30~75aa。同上所述,将B片段分为3段部分重叠的基因片段B1、B2、B3,设计相关引物并构建相关载体,如上所述进行第二轮Western Blot鉴定,抗原表位的区域范围位于B1片段上,氨基酸位置为30~52aa。
(2)单克隆抗体抗原表位的精确鉴定
为了精确的鉴定出单克隆抗体识别的区域,对鉴定出的片段进行N端和C端逐个氨基酸缺失。通过Western blot精确定位抗原表位的区域范围为40~46aa。
(3)单克隆抗体抗原表位的保守性分析
将鉴定出的表位序列与Genbank上已公布的国内外各地区的PSV毒株VP1氨基酸序列进行比对,结果显示表位40PALTAAE46在PSV毒株上较为保守,没有突变位点,能够识别大部分PSV毒株。
实施案例4单克隆抗体特异性鉴定
为了鉴定单克隆抗体的特异性,对猪萨佩罗病毒(PSV)、塞内卡病毒(SVV)、猪德尔塔冠状病毒(PDcov)、盖塔病毒(GETV)、猪副流感病毒5型(PIV5)进行WB分析。收集上述不同病毒株的细胞感染样品,用单克隆抗体PSV-VP1-33-2A作为一抗,HRP Goat Anti-MouseIgG作为二抗,进行WB鉴定。
实施案例5抗猪萨佩罗病毒VP1蛋白抗体的基因鉴定
通过RT-PCR法克隆Ig可变区基因。提取总RNA,合成单链cDNA,用Trizol法体取33-2A杂交瘤细胞株的总RNA,用反转录试剂盒将总RNA逆转为cDNA文库。
重链骨架区上游引物
VH-F1:SAGGTGMAGCTKCASSARTCWGG
VH-F2:ATGGRATGSAGCTGKGTMATSCTCT
VH-F3:ATGRACTTCGGGYTGAGCTKGGTTTT
VH-F4:ATGGCTGTCTTGGGGCTGCTCTTCT
重链可变区下游引物
VH-R:TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA
轻链前导肽上游引物
VL-F1:ATGGATTTTCAAGTGCAGATTTTCAG
VL-F2:ATGGAGACAGACACACTCCTGCTAT
VL-F3:ATGGAGWCACAKWCTCAGGTCTTTRTA
VL-F4:ATGKCCCCWRCTCAGYTYCTKGT
VL-F5:ATGAAGTTGCCTGTTAGGCTGTTG
轻链可变区下游引物
VL-R:GGATACAGTTGGTGCAGCATCAGCCCGTTT
配制PCR反应体系(50ul)如下:
反应条件:cDNA:2μl;上游引物(10μM):2μl;下游引物(10μM):2μl;PrimerSTARMax聚合酶:25μl;ddH 2O:补足至50μl。
98℃预变性3min;重复如下循环35次:98℃30s,57℃30s,72℃1min;最后,72℃延伸5min。
琼脂糖凝胶电泳分离并回收VL、VH片段。将回收后的VL、VH片段分别与pMD19-T载体(Takara公司)进行连接,连接体系如下:
VL PCR产物/VH PCR产物各7ul,
pMD19-T载体1μl,
10x T4 Ligase buffer 1μl
T4 Ligase 0.5μl
ddH2O补足至10μl,
4℃连接过夜。
连接产物转化入感受态细菌中,37℃过夜培养后,挑取单个菌落,37℃震摇2小时后进行菌液PCR鉴定,以对应抗体的cDNA为阳性对照。配制反应体系(20μl)如下:
菌液:1μl,
上游引物(10μM):1μl;
下游引物(10μM):1μl
2xHieff Robust PCR Master Mix:10μl
反应条件同前
选取菌PCR阳性的克隆扩大培养,用质粒提取试剂盒(Omega公司)提取阳性克隆质粒,送检测序。每个抗体的每条链至少送检5个克隆样品,至少三个样品测序结果相同为止。
抗体轻链可变区核苷酸序列:
ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAA(SEQ ID NO.9)
轻链可变区氨基酸序列:
METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK(SEQ ID NO.8)
轻链可变区CDR区(Ig blast/IMGT分析)
CDR1:RASKSVSTSGYSYMH(SEQ ID NO.4)
CDR2:YLVSNL(SEQ ID NO.5)
CDR3:QHIRELT(SEQ ID NO.6)
抗体重链可变区核苷酸序列:
CTGCTGACGGGGATCTCTAGAGATTCAGGTGCAGCTGCAGGAATCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATACCTGCGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAAAGTGATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCTCAAAGCATGCTCTATCTGCAAATGAACAACTTGAAAACGGAGGACACAGCCATGTATTACTGTGTGAGGTTAACTGGGGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACAGCCAATCGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCAT(SEQ ID NO.10)
重链可变区氨基酸序列:
VQLQESGGGLVQPKGSLKLSCAASGFTFNTCAMNWVRQAPGKGLEWVARIRSKSDNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRLTGDFWGQGTTLTVSSAKTTANRRPAGMQAWRNH(SEQ IDNO.7)
重链可变区CDR区(Ig blast/IMGT分析)
CDR1:AASGFTFNTCAMN(SEQ ID NO.1)
CDR2:VARIRSKSDNYATYYADSV(SEQ ID NO.2)
CDR3:VRLTGDF(SEQ ID NO.3);
猪萨佩罗病毒HuN2株VP1核苷酸序列:
GGAGATGTAAAGGATGAGGTTCAGGCCATTGTAAATAAAACACTGCAGAATGCACTCAACACAAAGCCTCAGAGGGAACAGTCCTCTAATGGTATTATGATCAACCAAGGAGATGCACCGGCACTGACAGCAGCTGAAACTGGAGAGTCTGATACCAATTCAGGGGGTTCTACCATGGAACTACAAGCTACAAATTGTACTTTTAGTTTAAAGGAGACAGATTTAGAATATTTAATGTCTAGGTATTCCCTTATGTATGAGGATAAATTAGATTATACTAATAATCAGGGTAACAGACATTTGAGATATAACTTGGATTTTAGGACTATAGGTAAATCAAGTAGTGATATTACTAAGTTTAAGGCTTTTACATATTGGAGATTTGATTTAGATGTAGTGGTAATGTTATTGGAGGACAAACCAGCTGCTGTTAAAAATTTAATGTTCCAGGTGCTGTACACACCTCACGGTGGTGTGGTGCCGGACAGACACAATTCACGTGTCTGGAATGCGCCCAATTCAACTAGTGTATATACAAGAGTAGGTGATTGTCCAGCCTCTTTTAGAATTCCATTTATGTCTGTTTGTAACTTTTATACATCCTTTTATGATGGTGATGGTAATTTTGACCGGAATGGTGCCTCTTATGGTATTAACCCCGGTAATTTTATAGGGACAATTGCTGTTAGGTTAGCTAATGATATTGTTACTGCAGAAGTTAGTGGTTCCTTTAGAGTTAAAATTTTTCTTAGGCCTGTAAACATAGAGGCTTATATGCCTAGACCCCTTATTGCTTATAAGGCTAATGGTGATGCTGTACAAGATAGTTCAACATACTACCCCGCAACCCAAATTGGGTTTTACTCAGCGGAGCAGTTG(SEQ ID NO.11);
猪萨佩罗病毒HuN2株VP1氨基酸序列:
GDVKDEVQAIVNKTLQNALNTKPQREQSSNGIMINQGDAPALTAAETGESDTNSGGSTMELQATNCTFSLKETDLEYLMSRYSLMYEDKLDYTNNQGNRHLRYNLDFRTIGKSSSDITKFKAFTYWRFDLDVVVMLLEDKPAAVKNLMFQVLYTPHGGVVPDRHNSRVWNAPNSTSVYTRVGDCPASFRIPFMSVCNFYTSFYDGDGNFDRNGASYGINPGNFIGTIAVRLANDIVTAEVSGSFRVKIFLRPVNIEAYMPRPLIAYKANGDAVQDSSTYYPATQIGFYSAEQL(SEQID NO.12);
本发明公开了一种分泌猪萨佩罗病毒VP1蛋白特异性单克隆抗体的杂交瘤细胞株,对本领域的技术人员来说,可以根据以上描述的技术方案以及构思,做出其它各种相应的改变以及形变,而所有这些改变以及形变都应该属于本发明权利要求的保护范围之内。
序列表
<110> 扬州大学
<120> 一种抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株、单克隆抗体及其应用
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Arg Ile Arg Ser Lys Ser Asp Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser
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Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met Leu
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Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr
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Cys Val Arg Leu Thr Gly Asp Phe Trp Gly Gln Gly Thr Thr Leu Thr
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Val Ser Ser Ala Lys Thr Thr Ala Asn Arg Arg Pro Ala Gly Met Gln
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Ala Trp Arg Asn His
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atggagacag acacactcct gttatgggta ctgctgctct gggttccagg ttccactggt 60
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atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 180
caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 240
ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 300
cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 360
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ctgctgacgg ggatctctag agattcaggt gcagctgcag gaatctggtg gaggattggt 60
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cgccatgaac tgggtccgcc aggctccagg aaagggtttg gaatgggttg ctcgcataag 180
aagtaaaagt gataattatg caacatatta tgccgattca gtgaaagaca ggttcaccat 240
ctccagagat gattctcaaa gcatgctcta tctgcaaatg aacaacttga aaacggagga 300
cacagccatg tattactgtg tgaggttaac tggggacttc tggggccaag gcaccactct 360
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taatcat 427
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ggagatgtaa aggatgaggt tcaggccatt gtaaataaaa cactgcagaa tgcactcaac 60
acaaagcctc agagggaaca gtcctctaat ggtattatga tcaaccaagg agatgcaccg 120
gcactgacag cagctgaaac tggagagtct gataccaatt cagggggttc taccatggaa 180
ctacaagcta caaattgtac ttttagttta aaggagacag atttagaata tttaatgtct 240
aggtattccc ttatgtatga ggataaatta gattatacta ataatcaggg taacagacat 300
ttgagatata acttggattt taggactata ggtaaatcaa gtagtgatat tactaagttt 360
aaggctttta catattggag atttgattta gatgtagtgg taatgttatt ggaggacaaa 420
ccagctgctg ttaaaaattt aatgttccag gtgctgtaca cacctcacgg tggtgtggtg 480
ccggacagac acaattcacg tgtctggaat gcgcccaatt caactagtgt atatacaaga 540
gtaggtgatt gtccagcctc ttttagaatt ccatttatgt ctgtttgtaa cttttataca 600
tccttttatg atggtgatgg taattttgac cggaatggtg cctcttatgg tattaacccc 660
ggtaatttta tagggacaat tgctgttagg ttagctaatg atattgttac tgcagaagtt 720
agtggttcct ttagagttaa aatttttctt aggcctgtaa acatagaggc ttatatgcct 780
agacccctta ttgcttataa ggctaatggt gatgctgtac aagatagttc aacatactac 840
cccgcaaccc aaattgggtt ttactcagcg gagcagttg 879
<210> 12
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Gly Asp Val Lys Asp Glu Val Gln Ala Ile Val Asn Lys Thr Leu Gln
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Asn Ala Leu Asn Thr Lys Pro Gln Arg Glu Gln Ser Ser Asn Gly Ile
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Glu Ser Asp Thr Asn Ser Gly Gly Ser Thr Met Glu Leu Gln Ala Thr
50 55 60
Asn Cys Thr Phe Ser Leu Lys Glu Thr Asp Leu Glu Tyr Leu Met Ser
65 70 75 80
Arg Tyr Ser Leu Met Tyr Glu Asp Lys Leu Asp Tyr Thr Asn Asn Gln
85 90 95
Gly Asn Arg His Leu Arg Tyr Asn Leu Asp Phe Arg Thr Ile Gly Lys
100 105 110
Ser Ser Ser Asp Ile Thr Lys Phe Lys Ala Phe Thr Tyr Trp Arg Phe
115 120 125
Asp Leu Asp Val Val Val Met Leu Leu Glu Asp Lys Pro Ala Ala Val
130 135 140
Lys Asn Leu Met Phe Gln Val Leu Tyr Thr Pro His Gly Gly Val Val
145 150 155 160
Pro Asp Arg His Asn Ser Arg Val Trp Asn Ala Pro Asn Ser Thr Ser
165 170 175
Val Tyr Thr Arg Val Gly Asp Cys Pro Ala Ser Phe Arg Ile Pro Phe
180 185 190
Met Ser Val Cys Asn Phe Tyr Thr Ser Phe Tyr Asp Gly Asp Gly Asn
195 200 205
Phe Asp Arg Asn Gly Ala Ser Tyr Gly Ile Asn Pro Gly Asn Phe Ile
210 215 220
Gly Thr Ile Ala Val Arg Leu Ala Asn Asp Ile Val Thr Ala Glu Val
225 230 235 240
Ser Gly Ser Phe Arg Val Lys Ile Phe Leu Arg Pro Val Asn Ile Glu
245 250 255
Ala Tyr Met Pro Arg Pro Leu Ile Ala Tyr Lys Ala Asn Gly Asp Ala
260 265 270
Val Gln Asp Ser Ser Thr Tyr Tyr Pro Ala Thr Gln Ile Gly Phe Tyr
275 280 285
Ser Ala Glu Gln Leu
290
Claims (10)
1.一种抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株,其特征在于,命名为杂交瘤细胞株PSV-VP1-33-2A,保藏号为CCTCC NO:C2021177。
2.一种单克隆抗体,其特征在于,所述单克隆抗体由权利要求1所述的杂交瘤细胞株分泌产生。
3.根据权利要求2所述的单克隆抗体,其特征在于,其重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1~3所示;轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.4~6所示。
4.根据权利要求3所述的单克隆抗体,其特征在于,其重、轻链的氨基酸序列分别如SEQID NO:7和SEQ ID NO:8所示。
5.一种用于检测猪萨佩罗病毒的试剂、试纸条或试剂盒,其含有权利要求2-4中任一项所述的单克隆抗体。
6.权利要求2~4中任一项所述的单克隆抗体或权利要求5所述的试剂、试纸条或试剂盒在非诊断用途的检测猪萨佩罗病毒方面的应用。
7.一种非诊断用途的检测猪萨佩罗病毒的方法,其特征在于,使用权利要求2~4中任一项所述的单克隆抗体或权利要求5所述的试剂、试纸条或试剂盒。
8.一种核酸分子,其特征在于,包含编码权利要求2~4中任一项所述的单克隆抗体的核苷酸。
9.根据权利要求8所述的核酸分子,其特征在于,所述核酸分子编码抗体,的轻链可变区的核苷酸序列如SEQ ID NO:9所示,所述核酸分子编码抗体的重链可变区的核苷酸序列如SEQ ID NO:10所示。
10.根据权利要求1所述的抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株的制备方法,其特征在于,包括以下步骤:原核表达的重组猪萨佩罗病毒VP1蛋白与弗氏佐剂混合后注射BALB/C小鼠,并对IFA检测结果阳性的小鼠进行加强免疫;将加强免疫后的小鼠采集脾B淋巴细胞,并与杂交瘤细胞进行融合,挑选阳性克隆后进行亚克隆得到抗猪萨佩罗病毒VP1蛋白杂交瘤细胞株。
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