CN116731128A - 一种副猪嗜血杆菌基因II型Omp P2蛋白的特异性B细胞线性表位及其应用 - Google Patents
一种副猪嗜血杆菌基因II型Omp P2蛋白的特异性B细胞线性表位及其应用 Download PDFInfo
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Abstract
本发明公开了一种副猪嗜血杆菌基因II型OmpP2蛋白的特异性B细胞线性表位,该线性表位序列为TYKVDESITVNNTQGTFKYSAPQE,处于成熟基因II型OmpP2蛋白序列的第129‑152氨基酸,处于基因II型比基因I型多出来的Loop环中。本发明的副猪嗜血杆菌基因II型OmpP2蛋白的B细胞线性表位,为基因II型的基因型特异性B细胞线性表位,实现猪血清中基因II型副猪嗜血杆菌抗体的特异性检测,并可以初步筛选无毒或者低毒副猪嗜血杆菌的感染,有助于副猪嗜血杆菌病的准确诊断。另外,疫苗株中该段序列突变或缺失获得的突变OmpP2蛋白在免疫猪之后,无法诱导产生抗该表位的抗体,可以达到区分无毒寄生株与疫苗株的鉴别诊断。
Description
技术领域
本发明涉及免疫学领域,特别是涉及一种副猪嗜血杆菌基因II型OmpP2蛋白的特异性B细胞线性表位以及该线性表位的应用。
背景技术
副猪嗜血杆菌(Haemophilus parasuis,Hps)是猪上呼吸道的常在菌,当宿主免疫力下降时,强毒力的Hps会突破粘膜屏障侵入机体引起以肺炎、纤维性多发性浆膜炎、关节炎、脑膜炎等炎症为特征的病。而且Hps是引起猪呼吸系统疾病的主要病原菌,参与了中国大约28.54%的猪呼吸系统疾病。Hps主要威胁保育猪,发病率为10-15%,死亡率可达到50%;在和PRRSV、PCV等病毒混合感染后发病率和死亡率会进一步上升。所以致病性Hps感染已成为保育猪死亡的主要原因之一,给全球生猪养殖业造成了巨大的经济损失。
Hps菌株中既有正常寄生菌,也有致病病原体,不同Hps菌株的毒力差别较大。区别呼吸道无毒株和强毒株对防控和诊断该疾病就显得至关重要了。目前对Hps菌株毒力的划分一般只是根据血清型简单划分,但随着临床分离菌株数量的增加,同一血清型的Hps菌株表现出不同毒力的现象越来越多;并且强毒力血清型菌株从健康猪上呼吸道分离的几率与患病猪的实质器官中分离的概率没有明显区别,说明根据血清型划分Hps菌株毒力的方法并不完善,Hps中存在血清型之外的毒力决定因子,而这些毒力因子的检测是区分正常寄生菌和致病菌最直接的方法。
外膜蛋白P2(OmpP2)是Hps中丰度最高的外膜蛋白,属膜孔蛋白家族,是Hps主要的毒力因子之一。首先OmpP2蛋白是细菌主要的膜结构蛋白和营养物质的流通通道,它的缺失会导致细菌生长繁殖能力出现明显下降。其次,OmpP2蛋白还介导细菌对宿主细胞粘附和入侵的过程,并参与抵抗血清的杀伤,帮助细菌逃避宿主的免疫清除。另外,OmpP2蛋白还具有细胞毒性,并能诱导细胞分泌促炎症细胞因子,参与炎症反应。
另外,OmpP2蛋白还是Hps中的优势抗原。不管是免疫小鼠还是猪都能够迅速产生高效价的特异性抗体,并通过抗体介导补体依赖的细胞毒作用有效的杀伤细菌,抑制细菌在宿主体内生长。这说明OmpP2中可能存在中和抗体识别表位,而OmpP2的B淋巴细胞表位不仅与Hps的致病性有关,还有可能与抗Hps感染的保护性免疫也有关系。
OmpP2蛋白存在两种明显不同的蛋白结构,分为基因I型和II型。对比国内外不同血清型的代表株发现,所有强毒株和中等毒株Hps的OmpP2蛋白基因都属于基因I型,而弱毒株或者非致病株都属于基因型II型。而且大部分来自患病猪的分离株OmpP2蛋白都属于基因I型,而大部分健康猪分离株的OmpP2蛋白都属于基因II型。动物和细胞毒力实验也证实基因I型的OmpP2蛋白具有更高的细胞毒性和血清抵抗力,说明OmpP2蛋白的结构与细菌毒力直接相关。因此可通过建立两基因型的通用表位及差异表位的多肽ELISA诊断方法及其诊断试剂对OmpP2蛋白的结构进行鉴定,实现致病性菌株和非致病性菌株的区分。
发明内容
本发明的一个目的在于,提供一种副猪嗜血杆菌基因II型OmpP2蛋白的特异性B细胞线性表位,该线性表位序列为TYKVDESITVNNTQGTFKYSAPQE,处于成熟基因II型OmpP2蛋白序列的第129-152氨基酸,处于基因II型比基因I型多出来的Loop环中。
本发明的另一个目的在于提供上述副猪嗜血杆菌基因II型OmpP2蛋白的特异性B细胞线性表位在制备副猪嗜血杆菌病的诊断试剂或防治副猪嗜血杆菌病的试剂或药物中的应用。
本发明所采取的技术方案是:
1)重组表达基因I型和II型的OmpP2蛋白,并进行纯化;
2)对不同基因型重组蛋白进行乳化,制备成抗原;
3)分别免疫小鼠,分别制备两基因型的鼠阳性血清;
4)合成基因II型蛋白中插入片段组成的多肽,并以这些多肽为抗原进行包被,以阳性鼠血清为一抗,进行Peptide-ELISA检测,以筛选OmpP2蛋白中基因II型特异性B细胞线性表位。
本发明的有益效果是:
本发明的副猪嗜血杆菌基因II型OmpP2蛋白的B细胞线性表位,为基因II型的基因型特异性B细胞线性表位,疫苗株中该段序列突变或缺失后获得的突变OmpP2蛋白在免疫猪之后,无法诱导产生抗该表位的抗体,从而达到自然感染与疫苗接种免疫的鉴别诊断,可为新型分子标记疫苗的研制提供依据。
本发明的副猪嗜血杆菌OmpP2蛋白基因II型的基因型特异性B细胞线性表位,包被酶标板之后可用于检测猪血清中的抗副猪嗜血杆菌基因II型OmpP2蛋白抗体。由此建立Peptide-ELISA检测方法,可以特异性检测基因II型副猪嗜血杆菌的感染引起的特异性抗体,实现基因II型副猪嗜血杆菌的特异性检测,并且能够初步判定所感染副猪嗜血杆菌为非致病株或弱毒株。
附图说明
图1是基因II型重组omp2蛋白(rP2-II)包涵体洗涤、复性及浓缩的电泳结果;1:大量诱导表达后的破碎上清;2:大量诱导表达后的破碎沉淀;3:洗涤后包涵体;4:包涵体溶解后上清;5:包涵体溶解后沉淀;6:蛋白复性后上清;7:复性蛋白液浓缩后沉淀;8:复性蛋白液浓缩后上清;M:蛋白marker,条带大小由大到小分别为97.2、66.4、44.3、29、20.1、14.3KDa。
图2是OmpP2重组蛋白复性及浓缩后的WB检测结果结果;1:基因I型重组蛋白;2:基因II型重组蛋白;3:未诱导菌体沉淀;M:预染蛋白分子质量标准品,从上到下分子量大小为72、55、43、34、26KDa。
图3是鼠阳性血清(rP2-II免疫)中抗rP2-II、rP2-I2抗体的效价测定。
图4是Pt22及Pt23多肽与鼠阳性血清的Peptide-ELISA结果。
图5是成熟基因II型OmpP2蛋白中第129-152位氨基酸序列多重比对结果;DT3、ZS7为本实验室分离保存的副猪嗜血杆菌菌株,Hs-DY01~Hs-DY15依次为中国来源副猪嗜血杆菌1~15血清型的代表株;No.4等其它菌株为国际上公认副猪嗜血杆菌各血清型的代表株;毒力一栏中“++”:强毒性,可引起明显病变及实验动物死亡;“+”:中等毒性,可引起明显病变但不致死;“±”:弱毒性,引起的症状轻微,呈一过性感染;“-”:无毒性,不能引起明显症状。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
副猪嗜血杆菌重组外膜蛋白OmpP2的制备
1)表达载体构建
设计一对引物用于重组表达全长的基因II型成熟蛋白(第20位氨基酸开始)。引物序列为:opm2/F:CATGCCATGGTAACAGTTTATGAAAATGAAGGT;和opm2/R:CCGCTCGAGCCATAATACACGTAAACC,该引物由生工生物工程(上海)股份有限公司(下简称生工)合成。扩增后的序列测序正确后进行Nco I和Xho I双酶切,然后与表达载体PET-28a连接,构建原核表达载体PET-28a-OmpP2,这样就能够获得N端融合有6×His标签的重组OmpP2蛋白(截除信号肽的成熟蛋白)。
2)重组蛋白的诱导表达
使用上述引物构建了4个OmpP2蛋白的原核表达载体,其中1个为基因II型OmpP2蛋白表达载体,3个为基因I型OmpP2蛋白表达载体。构建成功的表达载体转化入大肠杆菌感受态BL21(DE3,货号B528414,生工),随后将重组菌摇至菌液OD600为0.8时,加入IPTG(货号A100487,生工)至终浓度0.5mM诱导目的蛋白表达。4个OmpP2蛋白原核表达载体均有目的蛋白表达,分别标记为rP2-I1、rP2-I2、rP2-I3、rP2-II。经过鉴定和多次优化,4个重组蛋白都是以包涵体的形式存在。
3)包涵体洗涤、复性和浓缩
随后对基因II型的rP2-II蛋白的包涵体进行洗涤。首先使用30mL包涵体洗涤缓冲液(0.5%TritonX-100,50mM Tris-HCl PH8.0,300mM NaCl,2mM EDTA,2M尿素)超声破碎仪超声洗涤3次。然后用30mL包涵体重悬缓冲液(50mM Tris-HCl PH8.0,300mM Na Cl,2mMEDTA)超声洗涤1次。洗涤后用包涵体溶解缓冲液(8M尿素,10%甘油,50mM Tris-HClPH8.0,150mM NaCl,2mM EDTA)溶解,每30mg包涵体加入1mL包涵体溶解缓冲液,并在4℃搅拌下过夜溶解,收集溶解上清即为洗涤后包涵体。
然后采用尿素梯度透析的方法对rP2-II蛋白的溶解后包涵体进行复性,具体步骤如下:以1:25的比例将包涵体溶解液加入预冷的6M尿素的复性液①(6M尿素,10%甘油,50mM Tris-HCl PH8.0,150mM NaCl,2mM EDTA,0.1%吐温-20)中,混匀后装入10KDa截留的透析袋内;将透析袋放入含4M尿素的复性液②(4M尿素,10%甘油,50mM Tris-HCl PH8.0,150mM NaCl,2mM EDTA)中,4℃低速搅拌透析8h;然后将透析袋转移至含2M尿素的复性液③(2M尿素,10%甘油,50mM Tris-HCl PH8.0,150mM NaCl,2mM EDTA)中,4℃低速搅拌透析8h;随后将透析袋放入分子筛缓冲液(20mM Tris-HCl PH8.0,50mM NaCl)中透析8h;最后收集透析袋内上清进行蛋白浓缩。浓缩时选用10KDa截留的Millipore超滤离心管(货号UFC9010,默克生命科学技术有限公司),将复性液浓缩至1-1.5mL,即为复性及浓缩后的rP2-II蛋白。
4)重组蛋白的鉴定
对复性和浓缩后的rP2-II蛋白进行SDS-PAGE检测,结果如图1中2泳道所示,大量外源蛋白以包涵体的形式集中在破碎后的沉淀中,且蛋白含量明显高于菌体蛋白量。经过洗涤和溶解后,包涵体的纯度明显上升,达到为66.1%。rP2-II蛋白的复性液浓缩至为1.4mL,发现浓缩液中都含有大量目的蛋白沉淀(图1中7泳道),浓缩后上清中只含有小部分目的蛋白。经检测和计算,rP2-II蛋白浓缩上清的蛋白含量为1.95mg/mL,纯度(由BioRadGel Doc XR+IMAGELAB的条带分析功能自动计算)为88.4%,蛋白复性率为34.4%;均达到免疫动物对抗原纯度和浓度要求。
再次用上述方法对基因I型的rP2-I2、rP2-I3、rP2-II蛋白的包涵体进行洗涤、复性和浓缩(即纯化)。然后用抗HIS标签抗体(货号D191001,生工)对纯化的两基因型目的蛋白进行WB检测,结果如图2所示,抗HIS标签抗体能够特异的和目的蛋白结合,而未诱导菌体破碎沉淀中并没有对应蛋白条带。rP2-II蛋白分子量大小在44.3KDa左右,稍高于基因I型。
制备重组外膜蛋白OmpP2的鼠阳性血清
1)选择6只6周龄的纯系雌性BALB/c小鼠(购自广东省医学实验动物中心),分为2组(3只/组),分别用浓缩的重组rP2-I2和rP2-II蛋白作为抗原免疫各组小鼠。将两基因型重组蛋白用PBS稀释至终浓度为0.5mg/mL,并与完全福氏佐剂(CFA,货号F5881,默克生命科学技术有限公司)等体积混匀乳化后,小鼠腹腔皮下多点注射,进行初次免疫;每只小鼠总共注射200μL乳化悬液,即每只小鼠100μg抗原。
2)初次免疫后2周,将重组rP2-I2和rP2-II蛋白分别与非完全福氏佐剂(IFA,货号F5506,默克生命科学技术有限公司)乳化后,同样腹部皮下多点注射;进行常规免疫,每只小鼠注射50μg抗原蛋白。
3)2周后,取少许小鼠尾静脉血,离心获得血清。用免疫用的重组rP2-I2和rP2-II蛋白包被,用间接ELISA检测血清中的抗体效价,效价高于1:12800时即可进行融合前加强免疫。
4)融合前3天分别对两组中效价最高的小鼠直接腹腔注射含50μg对应重组蛋白的PBS缓冲液加强免疫,注意这次抗原无需乳化。
5)加强免疫后第3天,眼角取血后断颈处死BALB/c小鼠,血液37℃静止1h后,5000rpm/min离心5min,取上清冻存于-20℃冰箱备用。
当然,也可以使用重组蛋白免疫其他实验动物,取得相应动物的阳性血清。
鼠阳性血清的鉴定及效价测定
分别以重组的OmpP2蛋白rP2-I2、rP2-II为抗原包被酶标板,包被浓度为5μg/mL,每孔100μL 4℃包被过夜;第二天PBST洗涤3次后,每孔加入200μL含4% BSA的PBS,37℃培养箱封闭2小时;随后PBST洗涤3次,每孔加入100μL抗体稀释液(含1% BSA的PBST)稀释后的小鼠血清(稀释倍数为1:200-1:102800),作为一抗37℃孵育1小时;然后PBST洗涤5次,加入1:10000抗体稀释液稀释的HRP标记兔抗鼠IgG为二抗(货号D110097,生工),每孔100μL,37℃孵育45min;PBST洗涤5次后,加入100μL先配置的TMB显色液(货号C520026,生工),每孔100μl,37℃避光显色20min,最后每孔加入50μl 2M H2SO4终止反应。使用酶标仪测量450nm波长下每孔的光密度(OD)。以非洲猪瘟病毒P30蛋白的阳性鼠血清为阴性对照,阳性的判定标准为:以阴性对照OD450nm值的2.1倍为阈值,各待测孔OD450nm值(S)与阈值(CO)对比,计算S/CO。以S/CO值大于1的为阳性。结果如图3所示,rP2-II免疫小鼠后获得的血清能够与rP2-I2和rP2-II都产生阳性反应,效价分别是1:6400、1:12800,抗rP2-II的总抗体效价稍高一些,说明rP2-II能够诱导一些基因II型蛋白的型特异性抗体。
重叠多肽与鼠阳性血清的Peptide-ELISA
根据rP2-II的测序获得的蛋白序列为基础,预测蛋白的结构,针对基因II型OmpP2蛋白特有的插入序列设计重叠多肽,序列分别是Pt22:TYKVDESITVNNTQGTFKYSAPQE(SEQ IDNO:1),位于成熟基因II型蛋白的129-152氨基酸;Pt23:GTFKYSAPQEGFDILT QSSDSA(SEQ IDNO:2),位于成熟基因II型蛋白的143-164氨基酸;两多肽前后之间重叠10个氨基酸。以两多肽作为抗原进行包被,每条多肽包被浓度均为5μg/mL,以稀释好的rP2-II、rP2-I2免疫后阳性鼠血清作为一抗进行Peptide-ELISA检测。具体步骤与重组蛋白的间接ELISA相似,结果如图4A显示,rP2-II免疫后小鼠中存在抗Pt22抗体,效价为1:200,但不存在抗Pt23多肽的抗体;另外图4B显示,基因I型的rP2-I2免疫后小鼠的阳性血清不存在抗Pt22和Pt23的抗体。说明Pt22含有基因II型OmpP2的B细胞线性表位,是基因II型蛋白的型特异性表位;而且这个表位的关键氨基酸只存在于Pt22中,不在与Pt23的10个重叠氨基酸里面,在成熟基因II型OmpP2蛋白的第129-142氨基酸序列中。
基因型特异性表位序列保守性分析
为确定成熟基因II型OmpP2蛋白第129-152位氨基酸的保守情况,我们对rP2-I1、rP2-I2、rP2-I3、rP2-II 4个重组蛋白和DT3、ZS7及国内外各血清型代表株的OmpP2蛋白序列进行多重比对,结果如图5所示,显示所有基因I型OmpP2蛋白中这一段序列都是缺失的,而在基因II型蛋白中这一段序列虽然在不同菌株之间具有多态性,但有一定的相似性,所以可以推测成熟基因II型OmpP2蛋白该表位区域的表位序列不仅仅是Pt22,其它序列相似序列也有可能是基因II型特异性的B细胞线性表位,包括但不限于图上的TYKVNKPITVNNQQGTFKYSAPQE(SEQ ID NO:3),TYKVNESITVDNKRGTFKYSAPQE(SEQ ID NO:4),TYEVEEPITVNNTQGTSQGTFKYSAPQE(SEQ ID NO:5),TYKVDKPITGNNRQGTL YSAPQK(SEQ ID NO:6),TYEVEESITVDNKQGTFKYSAAQE(SEQ ID NO:7)等。再结合图4中代表株致病性的分布,可以看出基因II型的菌株(10/10)都为弱毒株或者无毒株,所以基因II型OmpP2蛋白第129-152位氨基酸代表的表位不仅可以用来区分基因型,也可以用来区分感染菌株的致病性。
表位多肽检测猪血清样品
将Pt22表位多肽分别作为抗原,5μg/mL包被过夜;抗副猪嗜血杆菌抗体阳性的猪血清为一抗,HRP标记兔抗猪IgG抗体为二抗进行ELISA检测。其中阳性猪血清58份,都是商品化副猪嗜血杆菌抗体ELISA试剂盒(购自深圳子科生物科技有限公司)检测为阳性的血清,检测时以1:400稀释作为一抗与表位多肽进行孵育。结果如表1所示,58份商品化试剂盒检测阳性样品中,Pt22表位多肽检出阳性样品8份,阳性检出率为13.79%,阳性率较低。推测Pt22只存在于一小部分的基因II型副猪嗜血杆菌中,这和图5代表株中也只1条序列与Pt22相同的情况是相符的。也说明基因II型OmpP2蛋白第129-152位氨基酸内表位的关键氨基酸存在于该序列中的突变氨基酸,比如第133位的D、N、E,第135位的S、P,第141位的T、Q、R、K等等。
表1.副猪嗜血杆菌病猪阳性血清检测
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,则本发明也意图包含这些改动和变形。
Claims (2)
1.一种副猪嗜血杆菌基因II型OmpP2蛋白的特异性B细胞线性表位,其特征在于:所述线性表位序列为TYKVDESITVNNTQGTFKYSAPQE,处于成熟基因II型OmpP2蛋白序列的第129-152氨基酸。
2.权利要求1所述的副猪嗜血杆菌基因II型OmpP2蛋白的特异性B细胞线性表位在制备副猪嗜血杆菌病的诊断试剂或防治副猪嗜血杆菌病的试剂或药物中的应用。
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