CN116655747A - ASFV p49蛋白线性表位肽、其特异抗体、杂交瘤细胞及其应用 - Google Patents
ASFV p49蛋白线性表位肽、其特异抗体、杂交瘤细胞及其应用 Download PDFInfo
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Abstract
本申请公开了一种ASFV p49蛋白线性表位肽、其特异抗体、杂交瘤细胞及其应用,旨在解决ASFV血清型的检测诊断及ASFV预防和治疗试剂或药物不完善、不全面的技术问题。本发明鉴定得到一种ASFV p49线性B细胞表位:333YQTHYMENIVTLVPR347和/或383NNYIPKYTGGIGDSK397,并诱导制得能特异性识别该表位的单克隆抗体。该线性B细胞表位丰富了ASFV p49蛋白的免疫学功能,为后续研究p49蛋白的结构特性以及抗原特性提供参考,可应用于抗病毒药物及防疫疫苗等的研发。
Description
技术领域
本发明申请涉及分子免疫学领域,具体涉及ASFV p49蛋白线性表位肽、其特异抗体、杂交瘤细胞及其应用。
背景技术
非洲猪瘟(African swine fever, ASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)引起猪的一种出血性、致死性的高度接触性传染病。发病率和死亡率高达100%。世界动物卫生组织(OIE)将其列为法定报告动物疫病。由于目前没有可以有效治疗非洲猪瘟的相关药物,非洲猪瘟的传播与防治仍然是全球需要关注的事情。
ASFV为一种双链DNA病毒,它是非洲猪瘟病毒科(Asfarviridae)非洲猪瘟病毒属(Asfarvirus)的唯一成员。ASFV不同病毒株的基因组大小也有所差异,大小在170~190kbp之间,含150多个开放阅读框,编码多种蛋白。目前,全球已知的ASFV有24种基因型和8个血清群。非洲猪瘟病毒颗粒大约具有70种不同的多肽段,包含多种结构蛋白以及参与病毒转录的各种酶和因子。
p49蛋白是一种由B438L基因编码的结构蛋白,位于ASFV BA71V株的EcoRI B基因组片段内,大小约49.3 kDa,在感染后晚期表达,没有任何翻译后修饰的跨膜结构域。p49蛋白作为衣壳结构蛋白,参与病毒后期的组装过程,定位于病毒颗粒组装位点。在缺乏p49蛋白时,病毒颗粒会形成管状结构而失去自身的二十面体对称结构,说明p49蛋白在衣壳组装过程中发挥着重要作用,有望作为疫苗开发的靶蛋白,因此针对ASFV p49蛋白单克隆抗体的制备及其B细胞表位的研究能够为ASF相关诊断试剂、预防和治疗药物的研究及应用提供重要技术支持,并为蛋白结构解析及表位疫苗制备奠定基础。
公开于该背景技术部分的信息仅用于加深对本公开的背景技术的理解,而不应当被视为承认或以任何形式暗示该信息构成本领域技术人员所公知的现有技术。
发明内容
基于现有研究的不足和现实需要,本申请旨在提供一种ASFV p49蛋白线性B细胞表位,并诱导产生特异性中和抗体,以建立、完善ASFV 血清型的鉴定方法,并为ASF相关诊断试剂、预防和治疗药物的制备研究提供技术支撑。
为解决上述技术问题,本申请采用如下技术方案:
基于长期、大量的试验研究,鉴定得到一种ASFV p49线性B细胞表位:333YQTHYMENIVTLVPR347和/或383NNYIPKYTGGIGDSK397。
本申请以原核系统表达的ASFV p49蛋白作为免疫原免疫小鼠,利用细胞融合技术,以ASFV p49蛋白作为检测原,通过间接ELISA筛选出抗ASFV p49蛋白的单克隆细胞株,分别命名为3B12、6F1和7C5。
筛选的单克隆细胞株所产生的单克隆抗体分别能够特异性的识别并结合ASFVp49蛋白上的333YQTHYMENIVTLVPR347和383NNYIPKYTGGIGDSK397序列区域。
本申请将表达的ASFV p49蛋白作为免疫原免疫小鼠,经间接ELISA测定免疫后的血清效价能够达到1:102400。制备的单克隆抗体3B12、6F1和7C5的效价可高达1:128000、1:128000和1:1024000,并具有良好的特异性。
本申请实施例中提供的一个或多个技术方案,至少具有如下技术效果或优点:
1. 本申请综合利用分子生物学、病毒学和免疫学等相关技术,筛选、鉴定得到一种ASFV p49蛋白的线性B细胞表位,为ASFVp49蛋白在疫苗研发上的应用提供技术支持;并进一步利用诱导制备的三株单克隆抗体对含有ASFV p49蛋白线性B细胞表位的多肽、p49蛋白、ASF灭活病毒进行识别,表明该ASFV p49线性B细胞表位能被特异性识别。
2. 本申请鉴定得到的ASFV p49蛋白线性B细胞表位丰富了ASFV p49蛋白的免疫学功能,为后续研究p49蛋白的结构特性以及抗原特性提供参考,可应用于抗病毒药物研发。
3. 筛选得到的单克隆细胞株所产生的单克隆抗体分别能够特异性的识别并结合ASFV p49蛋白上的333YQTHYMENIVTLVPR347和383NNYIPKYTGGIGDSK397序列区域。
4. 所诱导制备的单克隆抗体可特异识别ASF灭活病毒,可为ASFV的鉴别诊断提供新的技术手段。
附图说明
图1为本申请一实施例中 SDS-PAGE鉴定;其中,M为Marker;1-3依次为诱前全菌、诱后超声上清、诱后超声沉淀。
图2为本申请一实施例中Western Blot鉴定;其中,M为Marker;1为空载蛋白;2为诱后全菌。
图3为本申请一实施例中SDS-PAGE鉴定;其中,M为Marker;1-2依次为目的蛋白纯化前、纯化后复性。
图4为本申请一实施例中Western Blot鉴定;其中,M为Marker;1为目的蛋白与阳性猪血清的反应。
图5为本申请一实施例中p49重组蛋白免疫小鼠融合前血清效价。
图6为本申请一实施例中间接ELISA筛选融合后阳性杂交瘤细胞株。
图7为本申请一实施例中IFA实验验证ASFV p49蛋白与单克隆抗体3B12、6F1以及7C5的特异性结合。
图8为本申请一实施例中制备的3B12、6F1和7C5单克隆抗体的腹水效价。
图9为本申请一实施例中ASFV p49全长蛋白截短示意图。
图10为本申请一实施例中单克隆抗体3B12、6F1和7C5与设计合成的多肽片段(P1-P10)的Peptide-ELISA和Dot-ELISA实验结果。
图11为本申请一实施例中筛选的表位肽P1和P6与阳性猪血清的Peptide-ELISA和Dot-ELISA实验结果。
具体实施方式
下面结合附图和实施例来说明本发明的具体实施方式,但以下实施例只是用来详细说明本发明,并不以任何方式限制本发明的范围。
在以下实施例中所涉及的仪器设备如无特别说明,均为常规仪器设备;所涉及的试剂如无特别说明,均为市售常规试剂;所涉及的试验方法,如无特别说明,均为常规方法。
实施例1:免疫原制备
ASFV p49蛋白定位于“病毒工厂”,能够在动物体内产生免疫应答反应,因此利用原核表达系统对ASFV p49蛋白的全长序列进行表达。
1.1引物设计
根据NCBI(http://www.ncbi.nlm.nih.gov)上的ASFV p49蛋白的基因序列信息(GenBank accession No. QIE07029.1),利用SnapGene软件设计引物,选取BsaI和XhoI为酶切位点(划线部分),并添加保护碱基(斜体部分),扩增ASFV p49基因,引物序列见表1。
表1 引物序列表
。
1.2 p49基因的PCR扩增
以合成的ASFV p49基因序列为模板进行PCR扩增。PCR扩增体系如表2所示。
表2 PCR扩增体系
。
1.3 纯化回收PCR产物
目的基因扩增结束后,通过1%琼脂糖凝胶电泳验证PCR扩增产物。使用DNA纯化回收试剂盒对目的条带位置的扩增产物进行切胶回收,使用Nano Drop测定回收的DNA溶液浓度,做好标记,-20℃保存备用。
1.4 目的基因和载体的双酶切
(1)将p49基因/ pE-SUMO载体进行双酶切,酶切反应体系见表3。
表3 双酶切反应体系
。
(2)各组分混合均匀后放置于37℃金属浴上双酶切3 h。
1.5 酶切产物的回收
使用DNA纯化回收试剂盒回收双酶切产物,吸取1 µL的回收产物测定回收的DNA浓度,做好标记,-20℃保存备用。
1.6 重组质粒的构建
(1)p49基因与pE-SUMO载体连接
在200 µL EP管中加入下列组分:
表4 连接体系
。
将离心管中的上述溶液混匀后,置于16℃连接仪中进行过夜连接。
(2)转化:在超净工作台里,将10 μL的连接产物加入100 μL DH5α感受态细胞中,温和混匀,冰上静置40 min。静置后,将离心管在42℃金属浴中热击90 s,然后在冰水混合物中静置4 min,向离心管加入900µL LB液体培养基,37℃,220 r/min摇床培养40 min,8000rpm离心2 min。离心后,弃上清,用100 µL LB液体培养基重悬,并吸取50 µL涂布到LB固体培养基(氨苄抗性)上,倒置于37 ℃恒温培养箱中过夜培养。
(3)菌液PCR筛选阳性克隆:挑取圆滑的单个菌落接种到LB液体培养基(氨苄抗性)中,在37℃,220 r/min摇床上培养至浑浊,吸取浑浊的菌液作为菌液PCR的模板进行验证。
在200 µL EP管中加入表5中所列组分。
表5 菌液PCR反应体系
。
将各个组分混匀后,置于PCR仪中进行PCR扩增。菌液PCR结束后将产物用1%琼脂糖凝胶进行鉴定,选择条带位置与目的基因位置相符的单克隆菌株送至上海生工生物股份有限公司测序。
(4)重组质粒的提取:选则测序成功的阳性菌株,接种至20 mL LB液体培养基(氨苄抗性)中,37℃,220 rpm摇床过夜培养。用天根质粒小提试剂盒,按照试剂盒说明书操作步骤提取pE-SUMO-p49重组质粒。
1.7 目的蛋白的表达、纯化及鉴定
(1)目的蛋白的表达
a. 将pE-SUMO-p49重组质粒转化大肠杆菌Rosetta感受态细胞中,构建pE-SUMO-p49-Rosetta表达菌。
b. 将构建的pE-SUMO-p49-Rosetta表达菌1:100接种于80 mL LB液体培养基(氨苄抗性)进行二活。
c. 接种后,置于37℃摇床里220 rpm培养2 h左右,当OD600值在0.6~0.8时,加入诱导剂IPTG(0.2 mmol/L),在16℃摇床里220 rpm继续培养至次日。
d. 次日,收菌,用菌体破碎缓冲液(20 mmol/L Tris,100 mmol/L NaCl,pH 8.0)重悬进行超声破碎。
e. 超声破碎后,4℃、12000 rpm离心10 min,分别取40 μL超声上清和沉淀加入10μL 5×SDS上样缓冲液煮沸10 min进行SDS-PAGE及Western Blot鉴定,如图1、图2所示,p49重组蛋白以包涵体的形式表达。
(2)目的蛋白的纯化及鉴定
超声破碎后收集超声沉淀,用洗涤缓冲液洗(20 mmol/L Tris,100 mmol/L NaCl,2 mol/L Urea,pH 8.0)三次,每次常温搅拌30 min,在4℃条件下12000 rpm离心10 min。最后将溶解缓冲液(20 mmol/L Tris,100 mmol/L NaCl,8 mol/L Urea,pH 8.0)加入沉淀,室温下搅拌溶解30 min后,放于4℃冰箱中。次日,取出溶解液,4℃、12000 rpm离心10 min,分离上清,利用镍亲和层析柱进行纯化,详细纯化步骤如下:
a. 平衡Ni填料:用包涵体变性液冲洗亲和层析柱,冲洗的流穿缓冲液可循环使用。
b. p49重组蛋白与Ni料结合:平衡柱子后,将变性后离心上清用0.45µm滤头过滤,以1 mL/min的速度过柱,重复过柱2次,并用包涵体变性缓冲液冲洗层析柱。
c. 蛋白洗脱:分别用含有20、50、100、200、500 mmol/L浓度咪唑的包涵体变性缓冲液逐步冲洗层析柱,每个梯度的洗脱液用蛋白质指示剂指示,当蛋白质指示剂颜色不发生变化时,替换下一梯度。
d. 将纯化后收集的样品,通过SDS-PAGE进行分析。
e. 将纯化的目的蛋白在含有不同浓度尿素(6、3、1.5、0 mol/L)的包涵体复性液(20 mmol/L Tris,100 mmol/L NaCl,pH 8.0)中低温梯度透析,最后在含有20 mmol/LTris和150 mmol/L NaCl 的缓冲液中低温透析,从而使目的蛋白能够充分复性,复性后通过SDS-PAGE及Western Blot鉴定,如图3、图4所示,纯化后p49重组蛋白浓度达到90%以上且与阳性猪血清反应。
实施例2:p49重组蛋白小鼠免疫及免疫效果分析
2.1小鼠免疫及免疫效果初步评价
取4只6-8周龄的Balb/c小鼠,以p49重组蛋白作为免疫原免疫小鼠,每只免疫50 μg,共免疫三次,每隔两周免疫一次,首免用弗氏完全佐剂和p49重组蛋白混合乳化,二免和三免均用弗氏不完全佐剂和p49重组蛋白混合乳化。在首次免疫后14 d、28 d、42 d采集小鼠尾部静脉,通过间接ELISA测定小鼠免疫后血清效价。
2.2 间接ELISA测定p49重组蛋白免疫小鼠血清效价
将p49重组蛋白用CBS稀释到2 μg/mL后包被96孔反应板,每孔100 μL,37℃包被2h;弃去包被液,PBST洗涤三次,用5%脱脂奶在37℃封闭2 h;弃去5%脱脂奶,PBST洗涤三次;首孔加入1:100稀释的免疫后小鼠血清,同时以未免疫小鼠血清作为阴性对照,从左到右梯度倍比稀释,37℃孵育1 h;PBST洗涤三次,加入1:5000稀释的HRP标记的羊抗鼠,37℃孵育1h;PBST洗涤三次,每孔加入100 μL TMB显色液,避光显色5 min后,每孔加入100 μL 2 mol/L H2SO4终止显色;测定OD450nm值,分析免疫效果。如图5所示,针对p49重组蛋白产生较好的免疫效果。
实施例3:p49重组蛋白单克隆抗体的制备与鉴定
3.1细胞融合
细胞融合前3 d,选择效价最好的小鼠吸取100 μg p49重组蛋白采用腹腔注射对小鼠进行超免。超免3 d后进行细胞融合。首先选取一只健康的昆明鼠,引颈处死后浸泡在75%的酒精中,将昆明鼠固定在解剖板上,在超净台里用无菌的剪刀镊子剪开昆明鼠的表皮,用无菌注射器吸取20 mL含HAT和10%胎牛血清的RPMI-1640培养基,刺穿腹膜注射进昆明鼠的腹腔,然后吸出与80 mL培养基混匀铺在96孔细胞培养板中,每孔100 μL。次日,采取超免小鼠的眼球血,将血液在37℃恒温箱中放置2 h后,4000 rpm离心10 min,吸取上清作为阳性对照,分装后保存于-20℃冰箱备用。接着将超免小鼠引颈处死,浸泡在75%的酒精中,5 min后固定在解剖板上,在超净台里用无菌的剪刀镊子剪开皮毛,更换另一套无菌的剪刀镊子剪开小鼠的腹膜,然后取出小鼠的脾脏放置在200目无菌的尼龙网上,用剪刀研磨,期间不断滴加GNK冲洗,使脾细胞单个滤入下方的无菌烧杯中。将脾细胞转入50 mL无菌离心管中,与培养的SP2/0细胞一起离心,1000 rpm离心10 min。离心后,弃上清,温和弹散细胞,各加20 mLGNK重悬后计数。将脾细胞和SP2/0细胞以8:1的个数混合在一起,然后1000rpm离心10 min。离心后弃上清,温和弹散底部细胞,将离心管置于37℃水浴中,向离心管底部滴加1 mL 50%PEG1500,90 s内加完,静止90 s。然后缓慢加入15 mL GNK终止,继续静置5min,接着补加25 mL GNK,1000 rpm离心10 min。离心后弃上清,轻轻弹散底部细胞,加100mL培养基轻轻混匀,每孔100 μL铺在含有饲养层细胞的96孔细胞培养板里,每孔终体积200μL,在细胞培养箱里培养7d。
3.2 杂交瘤细胞阳性孔的筛选
细胞融合7d后,观察细胞团大小,然后用间接ELISA检测细胞上清。以融合小鼠的眼球血血清作为阳性对照。用p49重组蛋白和空载菌表达蛋白作为检测原,用CBS稀释成2 μg/mL每孔包被50 μL,37℃包被2 h。弃掉包被液,PBST洗板3次,每孔加入200 μL 5%脱脂奶37℃封闭2 h。吸取细胞上清,每孔50 μL,37℃孵育1 h。弃掉细胞上清,PBST洗板3次,每孔加入50 μL 1:5000稀释的HRP标记羊抗鼠作为二抗,37℃孵育1 h。弃掉二抗,PBST洗板3次,每孔加入50 μL TMB显色液,避光显色5 min后,每孔加入50 μL 2 mol/L H2SO4终止反应,选择OD450nm读值高的细胞孔扩大培养,然后通过稀释法对阳性杂交瘤细胞孔进行亚克隆,通过多次亚克隆直至获得稳定分泌单克隆抗体的杂交瘤细胞株,结果如图6所示,筛选出3株阳性杂交瘤细胞株分别命名为3B12、6F1和7C5。
3.3 间接免疫荧光(IFA)实验
每孔2×104个293T细胞铺于96孔细胞培养板上,每孔100 μL,当细胞密度达到80%时,将pcDNA3.1-p49以及pcDNA3.1-GFP质粒转染293T细胞,置于细胞培养箱中培养48 h。48h后,弃去细胞上清,使用4%组织细胞固定液固定细胞,每孔100 μL固定20 min。吸出固定液,PBS洗板3次,用5%脱脂奶封闭2 h。吸出5%脱脂奶,PBS洗板3次,每孔加入100 μL含有0.3% TritonX-100的细胞上清,37℃孵育1 h,以融合小鼠的眼球血血清(1:100)和未免疫小鼠血清(1:100)分别作为阳性和阴性对照。吸出上清,PBS洗板3次,每孔加入100 μL GoatAnti-Mouse IgG/FITC(1:200)作为二抗,37℃避光孵育1 h。吸出二抗,PBS洗板3次,每孔加入50 μL DAPI溶液,用倒置显微镜观察荧光,并拍照保存。结果如图7所示,结果显示单克隆抗体3B12、6F1和7C5与真核表达的ASFV p49蛋白同样可以发生反应。
3.4 单克隆抗体的大量制备及腹水的纯化
亚克隆后同样采用间接ELISA进行筛选,将筛选的阳性杂交瘤细胞株3B12、6F1和7C5扩大培养。在经产Balb/c小鼠腹腔中注射500 μL不完全弗式佐剂,注射一周后,将扩大培养的阳性杂交瘤细胞用500 μL RPMI-1640基础培养基稀释后计数,每只小鼠腹腔注射1.0×106个杂交瘤细胞。注射后每天留意小鼠腹部变化,当小鼠腹部不断变大且行动困难时,将小鼠引颈处死,剪开腹腔收集腹水。
3.5 单克隆抗体效价测定
按照Protein A琼脂糖纯化树脂说明书纯化腹水,通过间接ELISA测定单克隆抗体的效价。用CBS将p49重组蛋白稀释至2 μg/mL,每孔100 μL,37℃包被2 h。包被后,每孔200μL 5%脱脂奶37℃封闭2 h。PBST洗板3次,以纯化后的单克隆抗体1:500稀释后作为首孔,然后从左到右依次梯度倍比稀释,同时以未免小鼠血清作为阴性对照,每孔100 μL,37℃孵育1 h。PBST洗板3次,每孔加入100 μL 1:5000稀释的HRP标记羊抗鼠,37℃孵育1 h。PBST洗板3次,每孔加入100 μL TMB显色液,避光显色5 min,每孔加入100 μL 2 mol/L H2SO4终止反应,并读取OD450nm,结果如图8所示。
实施例4:ASFV p49蛋白线性B细胞表位的定位
4.1 ASFV p49蛋白氨基酸序列的截短表达纯化及鉴定
采用重叠多肽法对ASFV p49蛋白进行截短,截短情况见图9所示,共截短8段,采用上述同样的方法将其构建到pET-32a载体的BamHI和XhoI酶切位点之间,转化大肠杆菌Rosetta感受态细胞。采用上述同样的方法进行诱导表达,最后按照镍亲和层析柱说明书对表达的蛋白进行纯化。用CBS分别将纯化的截短蛋白和空载菌表达蛋白稀释至2 μg/mL,每孔50 μL 37℃包被2 h。PBST洗板3次,每孔200 μL 5%脱脂奶37℃封闭2 h。PBST洗板3次,每孔加入50 μL 1:1000稀释的单克隆抗体作为一抗,37℃孵育1 h。PBST洗板3次,每孔加入50μL 1:5000稀释的HRP标记羊抗鼠作为二抗,37℃孵育1 h。PBST洗板3次,每孔加入50 μLTMB显色液,避光显色5 min,每孔加入50 μL 2 mol/L H2SO4终止反应,并读取OD450nm,结果见表6所示,最终确定3B12、6F1单克隆抗体识别333~390位氨基酸区域,7C5单克隆抗体识别381~438位氨基酸位点。
4.2 ASFV p49蛋白多肽的合成及鉴定
基于上述实验结果,对截短片段B7(aa 333-390)和B8(aa381-438)进一步截短合成多肽P1-P10(吉尔生化),见表7所示。将合成的10段多肽用PBS稀释成4 mg/mL,每条多肽按照每孔4 μg包被,包被p49重组蛋白作为阳性对照原,PBS作为阴性对照原,以1:1000稀释的单克隆抗体作为一抗,1:5000稀释的HRP标记羊抗鼠作为二抗。结果如图10所示,Peptide-ELISA和Dot-ELISA显示3B12和6F1单克隆抗体与P1发生反应,7C5单克隆抗体与P6发生反应。
表6 截短蛋白的鉴定
。
表7 截短多肽的氨基酸序列
。
4.3 ASFV p49蛋白表位肽的鉴定
将筛选的表位肽采用上述方法进行包被,其中以1:1000稀释的阳性猪血清作为一抗,1:5000稀释的HRP标记羊抗猪作为二抗,结果如图11所示,Peptide-ELISA和Dot-ELISA显示P1和P6均与阳性猪血清发生反应,这是首次报道的B细胞表位。
上面结合附图和实施例对本申请作了详细的说明,但是,所属技术领域的技术人员能够理解,本申请的实施方式并不受上述实施例的限制,其它的任何未背离本申请发明构思的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本申请的保护范围之内。
Claims (8)
1.一种ASFV p49蛋白线性B细胞表位肽,其氨基酸序列为YQTHYMENIVTLVPR和/或NNYIPKYTGGIGDSK。
2.一种ASFV p49蛋白线性多肽,用以激活B细胞产生特异性细胞免疫,含有权利要求1所述表位肽,其氨基酸序列为333YQTHYMENIVTLVPR347和/或383NNYIPKYTGGIGDSK397。
3.权利要求1所述ASFV p49蛋白线性B细胞表位肽或权利要求2所述ASFV p49蛋白线性多肽在制备ASF诊断试剂、预防疫苗或治疗药物中的应用。
4.一种抗体或其抗原结合片段,其特异性结合ASFV p49蛋白线性B细胞表位肽,且所述表位肽氨基酸序列为YQTHYMENIVTLVPR和/或NNYIPKYTGGIGDSK。
5.根据权利要求4所述抗体或其抗原结合片段,其特征在于,其为单克隆抗体3B12、6F1和7C5中的至少一种。
6.权利要求4所述抗体或其抗原结合片段在制备ASF诊断试剂或治疗药物中的应用。
7.一种杂交瘤细胞,其分泌的单克隆抗体能特异性结合权利要求1所述ASFV p49蛋白线性B细胞表位肽。
8.权利要求7所述杂交瘤细胞在制备ASF诊断试剂或治疗药物中的应用。
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