CN114409772B - 一种猪繁殖与呼吸综合征病毒m蛋白的检测试剂盒及其应用 - Google Patents
一种猪繁殖与呼吸综合征病毒m蛋白的检测试剂盒及其应用 Download PDFInfo
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Abstract
本发明公开了一种猪繁殖与呼吸综合征病毒单克隆抗体,所述的单克隆抗体的重链可变区序列如SEQ ID NO.2所示,轻链可变区序列如SEQ ID NO.3所示。本发明还公开了一种用于检测猪繁殖与呼吸综合征病毒的试剂盒,所述试剂盒包括有效量的单克隆抗体和PRRSV M蛋白以及配套的检测试剂。本发明制备的单克隆抗体具有良好的特异性和敏感性,适用于制备不同的猪繁殖与呼吸综合征病毒诊断试剂,如胶体金、ELISA、化学发光等检测试剂盒。本发明的试剂盒具有良好的灵敏度和特异性,适用于PRRSV的筛查和疫苗的免疫评价。
Description
技术领域
本发明属于病毒疫病诊断技术领域,具体涉及一种猪繁殖与呼吸综合征病毒M蛋白的检测试剂盒及其应用。
背景技术
猪繁殖和呼吸障碍综合症(Porcine Reproductive and Respiratory Syndrome,简称PRRS),俗称蓝耳病(Blue Ear Disease),是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种严重危害养猪业的传染病。PRRS可导致母猪出现繁殖障碍及仔猪出现严重呼吸道疾病,是一种严重影响经济效益的猪传染病。
PRRSV为动脉炎病毒属的成员,是一种有囊膜的单股正链RNA病毒,大小约15kb,病毒粒子呈球型,直径为55~60nm。病毒有2个血清型,即美洲型和欧洲型,我国分离到的毒株为美洲型。病毒对酸、碱都较敏感,尤其很不耐碱,一般的消毒剂对其都有作用,但在空气中可以保持3周左右的感染力。PRRSV的RNA含有9个开放阅读框(ORF),分别为1a、1b、2a、2b、3、4、5、6、7。PRRSV的主要结构蛋白包括GP5蛋白(囊膜糖蛋白)、M蛋白(基质蛋白)、N蛋白(核衣壳蛋白)。因此,GP5蛋白和M蛋白是PRRSV诊断的靶标蛋白。本发明主要针对PRRSV M蛋白建立了一套诊断试剂盒,以用于PRRSV抗体的检测。
发明内容
为了弥补现有技术的不足,本发明的目的之一,提供一种PRRSV M蛋白的单克隆抗体及其制备方法;本发明的目的之二,提供了一种使用本发明制备的PRRSV M蛋白和单克隆抗体制备猪繁殖与呼吸综合征病毒抗体检测试剂盒。
因此,本发明一方面公开了一种猪繁殖与呼吸综合征病毒单克隆抗体,所述的单克隆抗体能特异性结合PRRSV M蛋白,所述的单克隆抗体的重链可变区序列如SEQ ID NO.2所示,所述的单克隆抗体的轻链可变区序列如SEQ ID NO.3所示。
优选地,本发明所述的单克隆抗体重链恒定区为lgG1型,轻链恒定区为Kappa型。
优选地,本发明所述的单克隆抗体识别的抗原表位位于PRRSV M蛋白的aa1-aa14,所述的PRRSV M蛋白的aa1-aa14的氨基酸序列为MGSSIDDFCNDSTA。
另一方面,本发明还提供了一种制备所述单克隆抗体的方法,所述方法包括以下步骤:1)PRRSV M蛋白的制备;2)特异性表达抗PRRSV M蛋白单克隆抗体的杂交瘤细胞的制备;3)使用步骤2)制备的杂交瘤细胞制备小鼠腹水,从腹水中纯化抗PRRSV M蛋白单克隆抗体。
优选地,本发明所述的步骤1)中制备PRRSV M蛋白时,表达M蛋白的核苷酸序列如SEQ ID NO.1所示。
再一方面,本发明还提供了一种用于检测猪繁殖与呼吸综合征病毒抗体的试剂盒,所述试剂盒包括有效量的所述单克隆抗体和PRRSV M蛋白;以及配套的检测试剂。
优选地,本发明所述试剂盒为阻断ELISA抗体检测试剂盒,所述的PRRSV M蛋白的包被浓度为1μg/ml。
优选地,本发明所述的试剂盒还包括样品稀释液、25×浓缩洗涤液、底物溶液、终止液、阳性对照和阴性对照。
再一方面,本发明还提供了一种所述的抗PRRSV M蛋白的单克隆抗体在猪繁殖与呼吸综合征病毒抗体诊断试剂中的应用。
本发明制备的PRRSV M蛋白适用于制备不同的猪繁殖与呼吸综合征病毒诊断试剂,如胶体金、化学发光检测试剂盒等。本发明制备的抗PRRSV M蛋白的单克隆抗体具有良好的特异性和敏感性,适用于制备不同的猪繁殖与呼吸综合征病毒诊断试剂,如胶体金、ELISA、化学发光等检测试剂盒。
本发明所提供的猪繁殖与呼吸综合征病毒抗体检测试剂盒适用于猪血清中猪繁殖与呼吸综合征病毒抗体的检测,其特异性强、灵敏度高、稳定性好、检测速度快,可用于猪繁殖与呼吸综合征病毒的筛查和疫苗的免疫评价等。
附图说明
图1PRRSV M蛋白纯化后SDS-PAGE图,1是纯化后的PRRSV M蛋白。
图2单克隆抗体纯化后SDS-PAGE图,1是纯化后的单克隆抗体。
图3BCA试剂盒标准曲线图。
图4交叉反应性鉴定结果,其中M为Marker,1-5分别为PCV2、CSFV、PRRSV、PRV、PEDV/TGEV疫苗样品,6是PRRSV M蛋白。
图5PRRSV M蛋白跨膜区预测结果。其中Inside表示胞内区,inside数值越大,表示该氨基酸位于胞内区的可能性越大;Outside表示胞外区,outside数值越大,表示该氨基酸位于胞外区的可能性越大;Transmembrane表示跨膜区,Transmembrane数值越大,表示该氨基酸在跨膜区的可能性越大。
图6单克隆抗体识别抗原表位ELISA检测结果图。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1:猪繁殖与呼吸综合征病毒M蛋白的制备
将PRRSV M基因(GenBank:MN913537.1)的完整编码序列(CDR)进行密码子优化,将密码子优化后的PRRSV M蛋白的核苷酸序列(如SEQ ID NO.1所示)亚克隆到pET-30a质粒中,构建原核表达载体pET-30a-M,将重组克隆pET-30a-M转化到大肠杆菌E.coli BL21(DE3),挑取单菌落于含有卡那霉素(终浓度为50μg/ml)的LB培养基中,培养至对数生长期(OD600为0.6~1.0)时,加入IPTG转入16℃,诱导16h,以表达目的蛋白。采用镍柱纯化方法进行纯化,用350mM的咪唑洗脱目的蛋白,再用10kD超滤管浓缩洗脱液,洗脱液透析换液至PBS中,取20μl PRRSV M蛋白使用SDS-PAGE蛋白凝胶进行检验,在约19.9kDa左右有一条清晰条带,纯度大于等于90%(图1)。使用BCA试剂盒检测蛋白含量,蛋白浓度为1.45mg/ml。
实施例2:抗PRRSV M蛋白单克隆抗体的制备
将实施例1制备的PRRSV M蛋白作为抗原,免疫BALB/c小鼠。PRRSV M蛋白与弗氏完全佐剂等体积乳化后,肌肉多点注射6~8周龄BALB/c雌性小鼠,每只100μg。两周后,改用不完全弗氏佐剂乳化,肌肉多点注射免疫,100μg/只,间隔两周后,重复一次,第三次免疫后,测定抗体效价。效价高于1:105的小鼠,采集小鼠脾脏研磨并分离脾细胞,使用PEG法将其与骨髓瘤Sp2/0细胞株融合,将融合后的细胞用含有HAT的20%新生牛血清的完全RPMI 1640培养基重悬,然后均匀铺于96孔板内,每孔100μl,37℃,5%CO2培养。15天后改用含HT的20%犊牛血清的完全RPMI 1640培养基培养。取培养上清,用PRRSV M蛋白(100ng/孔包被)包被的ELISA方法进行阳性克隆的检测。采用有限稀释法,将筛选出的抗体效价高、形态良好的细胞株进行克隆化培养,直至获得单克隆,扩大培养并保存杂交瘤细胞。
实施例3:抗PRRSV M蛋白单克隆抗体的纯化
取6~8周龄BALB/c雌性小鼠经腹腔注射降植烷,0.5ml/只,7日后每只小鼠注射杂交瘤细胞1×106个,7~10日后,抽取小鼠腹水,4℃8000rpm离心30min,收集上清液。使用辛酸-硫酸铵沉淀法纯化单克隆抗体,简述如下:取腹水上清10ml,加入40ml醋酸盐缓冲液(0.06mol/L,pH值4.5),混合均匀后在室温加入330μl辛酸,室温混合30min后,4℃静置2小时,4℃10000rpm离心30min,收集上清,在冰浴下加入相同体积预冷的饱和硫酸铵溶液,4℃静置14±2小时,4℃5000rpm离心30min,收集沉淀。用5ml PBS溶解沉淀后用PBS透析过夜。透析好的单克隆抗体用0.22μm滤膜过滤除菌,分装,0.1ml/管。取20μl单克隆抗体使用SDS-PAGE蛋白凝胶进行检验,有两条清晰条带,纯度大于等于90%(图2),使用BCA试剂盒(图3)对单克隆抗体进行浓度检测,检测结果为1.36mg/ml。
实施例4:单克隆抗体的检测
反应性测定:用间接ELISA方法(PRRSV M蛋白的包被量为100ng/孔)测定单克隆抗体的ELISA效价,单克隆抗体(起始浓度10μg/ml)按1:10的比例倍比稀释,一直稀释到1:107,结果为1:106(P/N值大于2.1时为阳性,其中P为样品OD450nm值,N为空白对照OD450nm值)。说明单克隆抗体的效价很好。
交叉反应性鉴定:从市场上面购买PCV2亚单位疫苗、CSFV活疫苗、PRRSV活疫苗、PRV活疫苗、PEDV/TGEV活疫苗,按照说明书稀释后,提取总蛋白做werstern blot检测(单克隆抗体1:1000倍稀释,HRP标记羊抗鼠IgG二抗1:10000倍稀释)。结果(图4)显示,该单抗均无特异性反应,说明该单抗具有良好的特异性。
亚类测定:用小鼠单克隆抗体亚型鉴定试剂盒(Sigma,Mouse MonoclonalAntibody Isotyping Kit)对单抗的亚型进行鉴定,鉴定结果显示单克隆抗体重链恒定区为lgG1型,轻链恒定区为Kappa型。
实施例5:单克隆抗体序列测定
使用Trizol试剂(Thermo公司),按照说明书提取杂交瘤细胞的总RNA。取2.5μg总RNA,加入1.0μl oligo(dT)(10μM),加入1μl dNTPs(10mM),用DECP水使总体积达到13μl,混合均匀,65℃孵育5min后置冰上1min,然后加入4.0μlRT buffer(5×),1.0μl DTT(100mM),1.0μl Ribonuclease Inhibitor及1.0μl逆转录酶(takara公司),50℃反应10min,80℃孵育10min以终止反应,获得的cDNA保存于-20℃。设计特异性的巢式PCR引物,该扩增反应中所使用的引物序列与抗体可变区第一框架区和恒定区互补,采用常规PCR方法扩增目的基因。其中,引物序列的设计按照文献(CN 111393525 B)进行。经过测序,抗体的重链可变区和轻链可变区的序列如SEQ ID NO.2、SEQ ID NO.3所示。单克隆抗体的可变区氨基酸序列信息见表1所示。
表1单克隆抗体氨基酸序列信息
实施例6:单克隆抗体识别抗原表位的确定
为研究单克隆抗体的识别区域,将表达的PRRSV M蛋白的跨膜区进行预测,结果(图5)所示。根据预测的结果,设计合成7段多肽,覆盖整个PRRSV M蛋白。多肽段氨基酸序列如表2所示。
表2 PRRSV M蛋白的跨膜区预测结果
用包被缓冲液(0.05M pH9.6碳酸钠溶液)稀释多肽,包被酶标板,4℃过夜;PBST洗板3次;用5%脱脂奶封闭,37℃孵育2h;以本发明获得的单克隆抗体作为一抗进行间接ELISA,37℃孵育1h;洗板后,加入HRP标记羊抗鼠IgG,37℃孵育30min;洗板后加入TMB底物显色液,37℃孵育10min后终止显色,用酶标仪读数。结果(图6)显示筛选到的单克隆抗体可与多肽P1(MGSSIDDFCNDSTA)发生特异性反应。
实施例7:PRRSV抗体的检测(阻断ELISA法)
使用实施例1制备的PRRSV M蛋白和实施例2、3制备的单克隆抗体建立猪繁殖与呼吸综合征病毒阻断ELISA抗体检测方法,以用于猪繁殖与呼吸综合征病毒的血清抗体检测。
1试剂盒的制备
1.1酶标板的制备:将PRRSV M蛋白用包被缓冲液(0.05M pH9.6碳酸钠溶液)稀释到1μg/ml,加入酶标板,100μl/孔,4℃过夜;取出,弃去孔内液体,用洗涤液洗涤3次,拍干;加入封闭液(含1%BSA的PBST溶液),200μl/孔,37℃孵育2小时;取出,弃去孔内液体,拍干,真空包装;
1.2阳性对照:使用购买的商品化疫苗(购自勃林格)免疫健康易感的28日龄仔猪(ASFV、CSFV、FMDV、PRV、PCV2、PRRSV、TGEV抗体抗原阴性,使用商品化试剂盒对其抗原和抗体进行检测),在一免14日和28日分别进行二免和三免,2ml/头/次,三免后7日采血,用间接ELISA方法进行检测,选择效价高于1:10000的猪用于血清制备。对满足条件的实验猪进行颈动脉放血,3000r/min离心10分钟,取上清,加入终浓度为0.1%的ProClin 300,混匀后0.22μm滤膜过滤除菌。定量分装,1ml/管,即为阳性对照。
1.3阴性对照:筛选健康易感的28日龄仔猪(ASFV、CSFV、FMDV、PRV、PCV2、PRRSV、TGEV抗体抗原阴性,使用商品化试剂盒对其抗原和抗体进行检测),进行颈动脉放血,3000r/min离心10分钟,取上清,加入终浓度为0.1%的ProClin 300,混匀后0.22μm滤膜过滤除菌。定量分装,1ml/管,即为阴性对照。
1.4样品稀释液的制备:含终浓度为1%的BSA、终浓度为0.1%的ProClin 300的1×PBST缓冲液,混合均匀,0.22μm滤膜过滤除菌,定量分装。
1.5 25×浓缩洗涤液的制备:为25×PBST溶液中加入终浓度为0.1%的ProClin300,混合均匀,0.22μm滤膜过滤除菌,定量分装。
1.6酶标抗体的制备:为使用样品稀释液稀释1万倍(使用本方法制备的酶标板,用直接ELISA检测HRP标记的单克隆抗体,OD450nm值大于1.1时的最大稀释倍数)的HRP标记的单克隆抗体。其中HRP标记参见HRP偶联试剂盒(ab102890)的说明书制备。
1.7底物溶液的制备:为北京索莱宝生物科技有限公司的单组份TMB显色液,定量分装。
1.8终止液的制备:为2M H2SO4。
1.9试剂盒的组装:按下表组装试剂盒。
名称 | 数量 |
抗原包被板 | 2块板(192份/盒) |
阳性对照 | 1mL/管 |
阴性对照 | 1mL/管 |
样品稀释液 | 12mL/瓶 |
25×浓缩洗涤液 | 30mL/瓶 |
酶标抗体 | 25mL/瓶 |
底物溶液 | 25mL/瓶 |
终止液 | 12mL/瓶 |
说明书 | 1份 |
2试剂盒的检测
2.1加样:在每孔中加入50μl样本稀释液,再在对应孔中加入50μl阳性对照、阴性对照和待检血清,振荡混匀。置37℃孵育60分钟。
2.2洗涤:孵育结束后,甩去孔内液体,加入洗涤液,300μl/孔,洗涤3~5次,拍干。
2.3酶标抗体孵育:加入酶标抗体,100μl/孔,置37℃孵育30分钟。
2.4洗涤:方法同2.2。
2.5显色:加入底物溶液,100μl/孔,置37℃避光孵育10分钟。
2.6终止:加入终止液,50μl/孔,轻微震荡混合均匀。
2.7读数:加入终止液后,立即将包被板置于酶标仪中,读取OD450nm值。
2.8S/N值计算按照以下计算公式,计算S/N值。
2.9判定
2.9.1试验成立条件:阴性对照孔每孔OD450nm读数应大于0.7且各孔间最大差值应<0.3,阳性对照孔每孔OD450nm读数应<0.3。
2.9.2当S/N值大于0.6时判为阴性;当S/N值小于等于0.6时判为阳性。
3试剂盒性能验证:按照上述方法连续生产了3批试剂盒,批号分别为211101、211102、211103,使用这三批试剂盒进行性能验证。
3.1敏感性验证:三批试剂盒对制备的阳性对照进行梯度检测,最低检测梯度能达到1600倍,说明该试剂盒的敏感性很好。具体数据见表3。
表3敏感性检测结果(S/N值)
3.2特异性验证:三批试剂盒对7份猪源特异性质控血清进行检测,结果均为阴性,说明本试剂盒的特异性很好。具体数据见表4。
表4特异性检测结果
3.3重复性验证:三批试剂盒对一份阳性血清和一份阴性血清的批间和批内的变异系数均小于10%,说明本试剂盒的重复性很好。具体见表5和表6。
表5重复性检测结果(批内)
表6重复性检测结果(批间)
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明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,都包含在本发明的保护范围之内。
序列表
<110> 梁荣乾
<120> 一种猪繁殖与呼吸综合征病毒M蛋白的检测试剂盒及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 522
<212> DNA
<213> 密码子优化后的PRRSV M蛋白的核苷酸序列(Artificial Sequence)
<400> 1
atgggttctt ccatcgatga tttctgcaac gactccaccg ctgcccagaa agtactgctg 60
gctttcagca tcacctatac cccgatcatg atctacgccc tgaaagtttc tcgtggtcgt 120
ctgctgggtc tgctgcacct gctgatcttc ctgaactgca ccttcacctt tggttacatg 180
acctttgtac acttccagtc taccaacaaa gtggcgctga ccatgggcgc tgtcgttgca 240
ctgctgtggg gtgtttacag cgccatggaa acctggcgtt tcatcacttc tcgttgccgt 300
ctgtgtctgc tgggtcgcaa atacatcctg gcaccggcgc accacgttga atctgctgcg 360
ggtttccacc ctatcaccgc gagcgacaac catgccttcg tcgtacgtcg cccgggttcc 420
actaccgtta acggtactct ggttccgggt ctgaaatccc tggtactggg cggtcgtcgt 480
gcagtgaaac gtggtgtagt taacctggtt aaatacgcga aa 522
<210> 2
<211> 369
<212> DNA
<213> 抗PRRSV M蛋白单克隆抗体重链可变区序列(Artificial Sequence)
<400> 2
gaaaccctgg tggaaagcgg cgatctgcag atgaacagcc tgaaaagcga agatagctgc 60
gcggcgagcg gctttacctt tagccattat ggcagctggg tgcgccagac cccggataaa 120
cgcctggaat gggtggcgac cattggcagc cgcggcacct atacccatta tccggatagc 180
gtgaaaggcc gctttaccat tagccgcgat aacgataaaa acgcgctgta tctggtgaaa 240
ccgggcggca gcctgaaact gaccgcgatg tattattgcg cgcgccgcag cgaattttat 300
ctggtgaaac cgagcaacaa cggcggcagc ctgaaactgg gccagggcac caccgtgacc 360
gtgagcagc 369
<210> 3
<211> 327
<212> DNA
<213> 抗PRRSV M蛋白单克隆抗体轻链可变区序列(Artificial Sequence)
<400> 3
gatattctga cccagagccc ggcgagcctg gcggtgagcc tgaccagcgt gggcgatcgc 60
gtgagcatta cctgcaaagc gagccagaac gtgggcaccg cggtgcagaa accgggccag 120
ccgccgaaac tgatttatgc gattagcaac cgcggcagcg gcgtgccggc gcgctttagc 180
ggcagcggca gcggcaccga ttttagcctg aacattcatc cggtggaaga agatgatccg 240
gcgatgtatt tttgccagca gaccagcggc accgatacca aagaagtgcc gtggaccttt 300
ggcgcgggca ccaaactgga aattaaa 327
Claims (7)
1.一种猪繁殖与呼吸综合征病毒单克隆抗体,所述的单克隆抗体能特异性结合PRRSVM蛋白,其特征在于,所述的单克隆抗体的重链可变区序列如SEQ ID NO.2所示,所述的单克隆抗体的轻链可变区序列如SEQ ID NO.3所示。
2.根据权利要求1所述的单克隆抗体,其特征在于,所述的单克隆抗体重链恒定区为lgG1型,轻链恒定区为Kappa型。
3.根据权利要求1所述的单克隆抗体,其特征在于,所述的单克隆抗体识别的抗原表位位于PRRSV M蛋白的aa1-aa14,所述的PRRSV M蛋白的aa1-aa14的氨基酸序列为MGSSIDDFCNDSTA。
4.一种用于检测猪繁殖与呼吸综合征病毒抗体的试剂盒,其特征在于,所述试剂盒包括有效量的权利要求1所述的单克隆抗体和PRRSV M蛋白;以及配套的检测试剂。
5.根据权利要求4所述的试剂盒,其特征在于,所述试剂盒为阻断ELISA抗体检测试剂盒,所述的PRRSV M蛋白的包被浓度为1µg/ml。
6.根据权利要求4所述的试剂盒,其特征在于,所述的试剂盒还包括样品稀释液、25×浓缩洗涤液、底物溶液、终止液、阳性对照和阴性对照。
7.一种如权利要求1所述的抗PRRSV M蛋白的单克隆抗体在猪繁殖与呼吸综合征病毒抗体诊断试剂中的应用。
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