CN111471104B - 一种prrsv广谱中和单克隆抗体及其应用 - Google Patents
一种prrsv广谱中和单克隆抗体及其应用 Download PDFInfo
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- CN111471104B CN111471104B CN202010355075.7A CN202010355075A CN111471104B CN 111471104 B CN111471104 B CN 111471104B CN 202010355075 A CN202010355075 A CN 202010355075A CN 111471104 B CN111471104 B CN 111471104B
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Abstract
本发明采用PRRSV病毒液‑SD16作为免疫原,免疫Balb/c小鼠。经细胞融合,病毒感染Marc145细胞筛选克隆,获得高效分泌单克隆抗体的阳性杂交瘤细胞系,获得鼠单克隆抗体MAB‑GP3。用ELISA技术测定该单克隆抗体MAB‑GP3的亚型为IgG2b型单克隆抗体。PRRSV‑I型和PRRSV‑II型病毒感染Marc145细胞,利用IFA技术用该单克隆抗体进行检测,证明单克隆抗体MAB‑GP3对PRRSV‑I和PRRSV‑II型病毒具有广谱反应性。随后用其进行病毒中和实验,利用Western blot技术和qPCR技术证明该单克隆抗体对PRRSV‑I和PRRSV‑II型病毒都具有中和活性,可阻止病毒入侵,保护机体免受感染。
Description
技术领域
本发明属于生物领域,具体涉及一种具有广谱中和活性的PRRSV单克隆抗体,能广谱中和I型和II型病毒。
背景技术
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndromevirus,PRRSV)引起的以母猪流产和仔猪呼吸障碍为主要特征的病毒性传染病,并能引起严重的免疫抑制。该病毒已在全球猪群中广泛传播,给世界养猪业造成了巨大的经济损失,已成为全球规模化猪场的主要疫病之一,也是全球猪病控制上的一大难题。PRRSV是有囊膜的单股正链RNA病毒,属于动脉炎病毒科,动脉炎病毒属。目前PRRSV有两种基因型:1型亦称欧洲型(Lelystad原型)和2型亦称北美型(VR2332原型),PRRSV-1和PRRSV-2株核苷酸序列约有60%相似性。其中PRRSV-II型病毒包括VR2385,VR2332,CH1a,SD16等, PRRSV-I型毒目前中国发现的有GZ11。
中和抗体是当病原微生物侵入机体时会产生相应的抗体。病原微生物入侵细胞时需要依赖病原体自身表达的特定分子与细胞上的受体结合,才能感染细胞,并进一步扩增。中和抗体是B淋巴细胞产生的某些抗体,能够与病原微生物表面的抗原结合,从而阻止该病原微生物黏附靶细胞受体,防止侵入细胞。中和抗体在抗猪繁殖与呼吸综合征病毒感染过程中起重要作用, 在自然感染PRRSV的过程中,中和抗体产生较晚,为病毒在宿主体内大量复制并感染其他易感动物提供了充裕的时间。然而被动输入PRRSV的中和抗体可预防PRRSV感染妊娠母猪,并且对母猪和仔猪都可起到消除性免疫作用。
然而由于不同PRRSV毒株的核酸序列差别较大存在抗原性存在差异,导致单一毒株致弱的PRRSV弱毒疫苗难以对核酸序列差别较大的野毒难以产生完整的保护。另一方面,临床上也有报道在猪群上也检测个别PRRSV感染猪个体的血清样本含有的多克隆血清可同时中和1型和2型PRRSV病毒的感染,提示不同来源的PRRSV病毒上存在保守的抗原表位可刺激机体产生广谱中和抗体,例如Eric Nelson对来自一个群体的5个血清样品试验使用多个1型和2型PRRSV分离株进行检测,血清与90%的2型PRRSV毒株发生反应,滴度为16-56;并意外发现可与90%的1型PRRSV发生反应,滴度大于80(陈错等,《猪业科学》,2017,34(4):25-26)。现有技术中也出现了具有较高中和活性的单克隆抗体,例如刘梦莹等(《中国预防兽医学报》,2019,41(6):641-644)制备的MAb LM26仅针对PRRSV SD53株具有中和活性,中和效价最高为1:50;B. Pirzadeh等(Journal of General Virology (1997), 78, 1867–1873)所制备的针对GP5的单克隆抗体对于美洲型VR-2332具有中和活性,中和效价为1:32-1:128,但是其对于欧洲型(Lelystad原型)不具备中和活性。但是,现有研究中尚未报道具有广谱中和的单克隆抗体及其应用。本发明鉴定出一株PRRSV特异性单克隆抗体,其所识别的保守表位广泛存在于不同基因型的PRRSV毒株,并且该抗体可在体外实验上中和不同PRRSV毒株对易感细胞的感染。
发明内容
针对现有技术中缺乏PRRSV广谱中和抗体的问题,本发明鉴定出一株PRRSV广谱中和抗体MAB-GP3,对于PRRSV-I型和PRRSV-II型病毒均具有较高的中和活性,可以用于PRRSV的检测和猪繁殖与呼吸综合征的治疗药物的开发。
为解决以上技术问题,本发明采用如下技术手段:
一种PRRSV广谱中和单克隆抗体MAB-GP3,包括重链和轻链,其特征在于:
所述重链的氨基酸序列如SEQ ID No:1所示;
所述轻链的氨基酸序列如SEQ ID No:2所示。
本发明还请求保护所述PRRSV广谱中和单克隆抗体MAB-GP3的编码DNA,包括重链编码DNA和轻链编码DNA,其特征在于:
所述重链的编码DNA序列如SEQ ID No:3所示;
所述轻链的编码DNA序列如SEQ ID No:4所示。
所述单克隆抗体MAB-GP3为IgG2b亚型。
本发明还请求保护所述PRRSV广谱中和单克隆抗体MAB-GP3在制备PRRSV检测试剂盒中的应用,优选的,所述检测试剂盒为ELISA检测试剂盒或者试纸条,所述试纸条为胶体金试纸条。
本发明还请求保护由单克隆抗体MAB-GP3重链和轻链在抗体制备中的应用,所述抗体为单克隆抗体,基因工程抗体;其中,所述基因工程抗体包括单链抗体、嵌合单克隆抗体、猪源单克隆抗体等。
基于以上技术方案,本发明具有如下技术效果:
本发明采用PRRSV病毒液-SD16作为免疫原,免疫Balb/c小鼠。经细胞融合,病毒感染Marc145细胞筛选克隆,获得高效分泌单克隆抗体的阳性杂交瘤细胞系,获得鼠单克隆抗体MAB-GP3。用ELISA技术测定该单克隆抗体MAB-GP3的亚型为IgG2b型单克隆抗体。PRRSV-I型和PRRSV-II型病毒感染Marc145细胞,利用IFA技术用该单克隆抗体进行检测,证明单克隆抗体MAB-GP3对PRRSV-I和PRRSV-II型病毒具有广谱反应性。随后用其进行病毒中和实验,利用Western blot技术和qPCR技术证明该单克隆抗体对PRRSV-I和PRRSV-II型病毒都具有中和活性。
附图说明:
图1:单克隆抗体MAB-GP3病毒中和实验结果图:图1-A为Western blot检测结果,其中SD16表示PRRSV-SD16病毒;Isotype Control为同型对照组,即未感染PRRSV的小鼠IgM;N表示基于PRRSV-N蛋白检测表征的病毒繁殖水平;α-Tublin为α微管蛋白,作为Western blot内参。图1-B为基于荧光定量PCR检测PRRSV-N基因mRNA表达水平的检测结果。
图2:单克隆抗体MAB-GP3广谱反应活性测定结果图。
图3:单克隆抗体MAB-GP3广谱中和活性测定结果图。
图4:单克隆抗体MAB-GP3腹水中和活性的检测结果图:图4-A,单克隆抗体MAB-GP3腹水针对PRRSV-SD16病毒(PRRSV-II型)的中和效价;图4-B,单克隆抗体MAB-GP3腹水针对PRRSV-GZ11病毒(PRRSV-I型)的中和效价。
具体实施方式:
下面列出具体实施方案对本发明做进一步阐述,以使本领域技术人员可以更清楚得得知本发明的技术方案,并非对本发明的限制。
实施例1:单克隆抗体MAB-GP3的制备和鉴定
1. 杂交瘤细胞系的建立
将PRRSV病毒:SD16(由西北农林科技大学动物医学院免疫生物学实验室提供)作为免疫原,用弗氏完全佐剂(Sigma公司)1:1混合乳化,免疫6周龄雌性Balb/c小鼠(由西安交通大学提供),腹部皮下注射,剂量为3×10^7 PFU/只,每14天加强免疫一次。第三次免疫后7天尾静脉采血用IFA检测小鼠血清中抗免疫原的抗体效价,效价最好的的小鼠以尾静脉注射冲击免疫,免疫原用生理盐水1:1混匀,剂量为3×10^7 PFU/只
2. 细胞融合
2.1无菌获取免疫后小鼠的脾细胞,以扩大培养好的小鼠骨髓瘤细胞(SP2/0),两者比例5:1,将50ml离心管中脾细胞悬液和瘤细胞悬液1000rpm离心10min。
离心后分别弃尽上清,各添加10ml不完全1640培养基,用吸管重新悬浮细胞,用吸管将10ml脾细胞悬液添加到10ml瘤细胞悬液中,充分混匀。1000rpm离心10min。离心完成后,弃上清,用手指轻轻弹击离心管底部,使细胞松散呈糊状,散在管底壁上。
准备37℃水浴烧杯,将离心管置于烧杯中,右手用吸管吸取1ml PEG1500(Roche公司),沿离心管壁,尽量接近细胞处缓缓加入PEG,右手始终不停均匀转动离心管,时间控制在60s。
轻轻摇晃,缓缓添加15ml培养基,终止融合,充分混匀,在水浴中静置5min。700rpm离心8min弃上清,添加HAT培养基10ml,补加HAT培养基至所需的量。用吸管将细胞悬液滴加在含有饲养细胞的96孔板中,每孔添加1滴,大约200μl每孔。添加完毕后,将96孔板置于37℃,5%CO2温箱中培养。
挑克隆
融合后七天,显微镜下观察,选取已经出现大小适中的细胞团的孔,细胞量大约占孔1/2。检测前一天将Mac145铺板并感染PRRSV病毒,取融合成功孔的上清进行IFA试验检测筛选,阳性孔进行亚克隆筛选,并转入24孔板扩大培养并冻存,至到筛选出阳性的单克隆抗体。
腹水诱生法大量制备单克隆抗体
提前一周给小鼠注射石蜡油致敏,一周后将筛选出的阳性对数生长期的杂交瘤细胞腹腔注射至小鼠体内,每只5×10^5个细胞。注射杂交瘤细胞7天后开始收集腹水,取出的腹水4℃理性5000g10min,吸出上清腹水收集于EP管中,-20℃保存。
单克隆抗体的纯化
5.1饱和硫酸铵粗提。按腹水:PBS:饱和硫酸铵=1:1:2的比例将腹水和PBS混合,后将饱和硫酸铵逐滴缓慢加入其中,边加边搅拌,滴加完后,放于4℃沉淀6h以上。
沉淀完成后,离心5000g10min,弃上清取沉淀,用与腹水等体积的PBS重悬,透析2次,中间隔12h换一次透析液,以除去样品中残留的硫酸铵。
透析完后,将粗提IgG样品10000r/min离心5min,收上清,与Protein G Resin(Transgene)混匀,根据需要取适量加入纯化柱中,甘氨酸洗脱目的抗体,收集,再次透析2次,得最终的目的抗体。纯化的抗体保存在-20℃。
单克隆抗体的亚型鉴定和测序
6.1 亚型鉴定
用亚型测定试剂盒(Sigma公司)进行测定,测得其亚型为IgG2b。
单克隆抗体序列测定
(1)总RNA提取
取10^6个细胞,Trizol裂解,加入氯仿分层获取RNA,异丙醇沉淀后用乙醇洗涤,干燥,用DEPC水溶解RNA。
(2)反转录PCR
取500ngRNA,加入Oligo(dT),Random,dNTP及5×RT buffer,反转录酶(Takara公司),37℃25min,85℃5sec,4℃终止反应获得cDNA,之后进行PCR扩增。
(3)测序和分析
将扩增得到的重链和轻链克隆至pMD18-T,送至公司测序,使用IMGT/V-QUEST数据库进行数据比对分析。经测序,
所述重链的编码DNA序列如SEQ ID No:3所示;
所述轻链的编码DNA序列如SEQ ID No:4所示。
根据密码子编码规则,可知:
所述重链的氨基酸序列如SEQ ID No:1所示;
所述轻链的氨基酸序列如SEQ ID No:2所示。
实施例2. 单克隆抗体MAB-GP3的中和活性测定
1 中和活性测定
利用该单克隆抗体进行病毒中和实验,以检测其中和活性。用PAM细胞铺至24孔板,分别接入0.01MOI不同型的PRRSV病毒,每种病毒分别和100μg/ml,200μg/ml,300μg/ml,400μg/ml的抗体,37℃孵育1h之后换液,36h后进行Western blot及qPCR检测,确定其具有中和活性。具体结果参见图1。
基于图1的结果可知,使用PRRSV在宿主体内的天然靶细胞肺泡巨噬细胞(PAMs)在体外培养,使用纯化的MAB-GP3单抗按照100μg,200μg,300μg,400μg/mL的剂量与0.01MOI剂量的PRRSV-SD16病毒在37℃培养1小时,接种PAMs,病毒接种24小时后收集样品,分别通过westernblot和荧光定量PCR检测PRRSV-N基因在蛋白和mRNA水平的表达,显示MAB-GP3单抗对PRRSV病毒的抑制作用呈现出剂量依赖型增加。
单克隆抗体MAB-GP3广谱反应活性测定
分别用PRRSV-1和PRRSV-2型病毒感染Marc145细胞,用该单抗作为一抗进行免疫荧光试验检测。具体检测结果参见图2。
基于图2的结果可知,使用PRRSV-2型病毒经典毒株(VR2332,CH1a),高致病PRRSV-2毒株(GD-HD,JXA1),以及PRRSV-2经典毒株突变株VR2385,以及PRRSV-2新型突变毒株NADC30,和PRRSV-1型毒株(1型)感染MACR-145细胞,24小时后使用4%多聚甲醛溶液固定细胞,含有0.5% TritonX100的PBS对固定细胞进行破膜处理,使用单克隆抗体MAB-GP3(红色荧光通道)和PRRSV多克隆猪阳性血清(绿色通道,阳性对照)对细胞进行免疫荧光共染色后可发现单抗MAB-GP3可识别多种PRRSV病毒所感染的MARC-145细胞,具有广谱反应性。
单克隆抗体MAB-GP3广谱中和活性测定
挑选目前在国内广泛流行的代表性PRRSV毒株,如高致病PRRSV(JXA1,GD),经典PRRSV毒株(VR2332),以及不同基因型PRRSV-1型毒株GE11,以200ug/mL的剂量的抗体剂量与病毒在37℃孵育后感染PAMs细胞,荧光定量PCR检测PRRSV-N基因表达,发现MAB-GP3可广谱抑制不同PRRSV毒株的感染,结果见图3。
实施例3. 单克隆抗体MAB-GP3腹水的制备和中和活性的检测
1. 腹水诱生法大量制备单克隆抗体
提前一周给小鼠注射石蜡油致敏,一周后将筛选出的阳性对数生长期的杂交瘤细胞腹腔注射至小鼠体内,每只5×10^5个细胞。注射杂交瘤细胞7天后开始收集腹水,取出的腹水4℃理性5000g10min,吸出上清腹水收集于EP管中,-20℃保存。
2. 单克隆抗体MAB-GP3腹水中和活性的检测
利用含有该单克隆抗体的腹水通过4倍稀释法,进行病毒中和实验,以检测其具有中和活性的最高效价。用PAM细胞铺至24孔板,分别接入0.01MOI PRRSV-SD16病毒(PRRSV-II型)或者PRRSV-GZ11(PRRSV-I型毒),将病毒分别和使用无菌PBS进行1:4倍,1:16倍,1:64倍,1:256倍,1:1024倍,1:4096倍稀释的的小鼠腹水,37℃孵育1h之后换液,36h后进行qPCR检测PRRSV-N蛋白的mRNA表达水平,以实现50%N蛋白mRNA抑制的最大稀释度,确定为腹水具有中和病毒感染活性的最大稀释倍数。
基于图4的结果可知,其中图4-A展示了使用MAB-GP3单抗所制备的腹水进行倍比稀释,与PRRSV-SD16毒株进行孵育后,检测被感染细胞内PRRSV-N基因的mRNA水平,以实现50%抑制的稀释度为最高中和效价,检测可知MAB-GP3腹水针对PRRSV病毒的中和效价可达1:256。其中图4-B展示了单克隆抗体MAB-GP3腹水针对PRRSV-GZ11病毒(PRRSV-I型)的中和效价,经检测可知单克隆抗体MAB-GP3针对PRRSV-GZ11病毒的中和效价可达1:64。
序列表
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Claims (6)
1.一种PRRSV广谱中和单克隆抗体MAB-GP3,包括重链和轻链,其特征在于:
所述重链的氨基酸序列如SEQ ID No:1所示;
所述轻链的氨基酸序列如SEQ ID No:2所示。
2.编码权利要求1所述的PRRSV广谱中和单克隆抗体MAB-GP3的DNA,包括重链的编码DNA和轻链的编码DNA,其特征在于:
所述重链的编码DNA序列如SEQ ID No:3所示;
所述轻链的编码DNA序列如SEQ ID No:4所示。
3.根据权利要求1所述的PRRSV广谱中和单克隆抗体MAB-GP3,其特征在于,所述单克隆抗体MAB-GP3为IgG2b亚型。
4.根据权利要求1所述的PRRSV广谱中和单克隆抗体MAB-GP3在制备PRRSV检测试剂盒中的应用,所述检测试剂盒为ELISA检测试剂盒。
5.根据权利要求1所述的PRRSV广谱中和单克隆抗体MAB-GP3的重链和轻链在抗体制备中的应用,所述抗体为单克隆抗体或基因工程抗体。
6.根据权利要求5所述的PRRSV广谱中和单克隆抗体MAB-GP3的重链和轻链在抗体制备中的应用,其中,所述基因工程抗体为单链抗体、嵌合单克隆抗体或猪源单克隆抗体。
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