CN109554375A - 美洲型prrsv n蛋白抗原表位及其应用 - Google Patents
美洲型prrsv n蛋白抗原表位及其应用 Download PDFInfo
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- CN109554375A CN109554375A CN201811527186.0A CN201811527186A CN109554375A CN 109554375 A CN109554375 A CN 109554375A CN 201811527186 A CN201811527186 A CN 201811527186A CN 109554375 A CN109554375 A CN 109554375A
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Abstract
本发明公开了一种美洲型PRRSV N蛋白抗原表位及其应用,旨在解决对PRRSV N蛋白细胞表位研究不足的技术问题。本发明设计一种短肽,其氨基酸序列如SEQ NO.1所示氨基酸序列;或由SEQ ID NO.1所示的氨基酸序列衍生的具有同等功能的氨基酸序列。筛选一种美洲型PRRSV N蛋白抗原表位,含所述短肽的氨基酸序列。将所述短肽或所述的美洲型PRRSV N蛋白抗原表位在制备预防和/或治疗美洲型PRRSV抗体中应用。本发明提供一种N蛋白新抗原表位,完成了对该表位的鉴定,完善了N蛋白的抗原表位图谱,为美洲型PRRSV交叉保护性表位疫苗的研究打下基础,对PRRSV美洲型、欧洲型的区分检测具有重要意义。
Description
技术领域
本发明涉及细胞免疫技术领域,具体涉及一种美洲型PRRSV N蛋白抗原表位及其应用。
背景技术
猪繁殖与呼吸综合征(Porcine Reproduetive and Respiratory Syndrome,PRRS)是目前世界养猪业危害最为严重的病毒性疾病。感染PRRSV 后母猪以生殖障碍为特征,主要表现为早产、晚期流产、死胎、木乃伊胎、发热、厌食等,感染的仔猪表现为呼吸道症状,也有神经症状。在发病过程中会出现两耳皮肤发绀发紫,故又称为“蓝耳病”。接触感染、精液传播、空气传播是PRRSV的主要传播途径,鸟类、鼠类、人类等运输工其都可能成为本病的媒介。猪繁殖与呼吸综合征病毒(PRRSV)是引起该病的致病因子。该病毒有两种主要的血清型类型,分别是基因I型(欧洲型)和II型(美洲型)。欧洲型以 Lelystad 株(LV 株)为代表,美洲型以 VR2332 株为代表。感染PRRSV的猪和感染后康复的猪都能排毒,有研究报道能分别从感染后210天的血清、42天的唾液、43~92天的精液、38天的粪便、21天的鼻腔液、28天的尿液、157天的口腔刮脱物中分离到病毒。
表位是病毒结构蛋白的重要抗原元件,一般为2~18氨基酸序列,在诱导和细胞介导的病毒免疫反应中发挥重要作用。根据它们各自的受体不同,表位可以分类为B细胞表位和T细胞表位,同时根据结构不同,表位又分为线性表位和构象表位。
表位的研究对于完善病毒蛋白结构及病毒与宿主作用关系的研究意义重大,同时对病毒的检测,表位疫苗的制备提供材料基础应用价值巨大。
国内外对于PRRSV N蛋白表位的研究并不是非常完善,Meulenberg等人用肽扫描技术对PRRSV I 型N蛋白表位进行分析,得到2~12,25~30,40~463段B细胞表位;多年后,Plagemann等人用基于肽扫描方法对PRRSV II 型N蛋白表位进行分析,得到23~33,31~50,43~563段B细胞表位;T.Q.An等用噬菌体展示技术结合生物套选法对PRRSV II 型N蛋白表位进行分析,获得表位78~87。选择合适的靶抗原研制新型PRRSV疫苗是免疫预防PRRS的研究重点。
然而,如何获得合适的疫苗靶抗原及其表位研制新型PRRSV表位疫苗或药物则一直是本领域技术人员所努力解决的技术难题。
发明内容
本发明要解决的技术问题是提供一种美洲型PRRSV N蛋白抗原表位及其应用,以解决对PRRSV N蛋白细胞表位研究不足的技术问题。
为解决上述技术问题,本发明采用如下技术方案:
设计一种短肽,其氨基酸序列为
(1)如SEQ NO.1所示氨基酸序列;或
(2)由SEQ ID NO.1所示的氨基酸序列衍生的具有同等功能的氨基酸序列。
筛选一种美洲型PRRSV N蛋白抗原表位,具有所述短肽的氨基酸序列。
优选的,所述表位是一种位于N蛋白柔性区的B细胞表位,该区域为PRRSV N 蛋白RNA的结合区。
优选的,所述表位为构象性表位。
优选的,所述表位为含Alpha、Beta、Turn、coil结构中的至少一种,富含正电荷,亲水性强,表面可及性强,抗原指数高。
优选的,所述表位在美洲型各毒株之间保守性高,在欧洲型毒株之间保守性低。
将所述短肽或所述的美洲型PRRSV N蛋白抗原表位在制备预防和/或治疗美洲型PRRSV抗体中应用。
设计一种含有所述短肽或/和该短肽的抗体的检测美洲型PRRSV的试剂盒或/和疫苗。
与现有技术相比,本发明的有益技术效果在于:
1.本发明研究筛选出了一种N蛋白新抗原表位,完成了对该表位的鉴定,完善了N蛋白的抗原表位图谱。
2.本发明的美洲型PRRSV N蛋白表位为首次发现,在美洲型PRRSV各毒株间保守性较高,与欧洲型差异较大,对PRRSV美洲型、欧洲型的区分检测具有重要意义。
3.本发明的美洲型PRRSV N蛋白表位有助于N蛋白结构的研究,为美洲型PRRSV交叉保护性表位疫苗的研究打下良好的技术基础,具有良好的应用前景。
附图说明
图1为pET-28a-N重组载体构建后PCR鉴定结果图;
图中,M为DNA分子质量标准;1为ORF7基因;
图2为PRRSV N蛋白表达及纯化SDS-PAGE鉴定结果图;
图中,M为蛋白分子质量标准;方框标出为N蛋白;
图3为纯化后N蛋白与PRRSV阳性猪血清反应western-blot鉴定结果图;
图中,M为蛋白预染marker;方框标出为阳性猪血清所识别的N蛋白;
图4为单抗与PRRSV JX-A1株结合能力的IPMA法检测结果图;
图5为单抗与PRRSV JX-A1及BJ-4株结合能力的IFA法检测结果图;
图6为N蛋白重叠多肽的位点图;
图中,分别合成6段多肽,命名P1~P6;
图7为N蛋白B细胞表位的间接ELISA鉴定结果图;
图中,P1~P6为包被的合成短肽;NC为包被的原核表达无关蛋白,作为阴性对照;
图8为N蛋白B细胞表位的Dot-ELISA鉴定结果图;
图中,P1~P6为合成的短肽,其中P1为经PBS稀释的短肽MPNNNGKQQKKKKGN(1~15aa);PC为经PBS稀释的N蛋白,作为阳性对照;NC为经PBS稀释的原核表达无关蛋白,作为阴性对照;
图9为抗原表位MPNNNGKQQKKKKGN(1~15aa)的SDS-PAGE及Western-blot结果图;
图中,1为经SDS-PAGE处理的抗原表位MPNNNGKQQKKKKGN(1~15aa);M为蛋白预染marker;2为抗原表位MPNNNGKQQKKKKGN(1~15aa);
图10为PRRSV N蛋白结构及抗原表位的生物信息学预测结果图;
图11为抗原表位MPNNNGKQQKKKKGN(1~15aa)在不同毒株间的保守性生物信息学软件分析结果图。
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;所涉及的试剂或产品如无特别说明,均为市售常规试剂或产品;所涉及的试验方法,如无特别说明,均为常规方法。
实施例一:免疫原的选择和制备
1.免疫原的选择
发明人经长期、大量研究、实践、试验发现:
首先,在PRRSV基因组所编码的蛋白中,由ORF7编码的核衣壳蛋白即N蛋白高度保守;
其次,在机体中由N蛋白激发的免疫反应性远远高于其他蛋白,从而容易获得高亲和力的单抗;
再次,PRRSV感染机体后N蛋白含量最高,所占结构蛋白总量的40%~60%,是PRRSV抗原检测的优势靶标。
基于以上三点N蛋白成为首选靶标蛋白。
2.免疫原的制备
N蛋白的核苷酸序列在Genebank上的登录号为:EF112445。
(1)构建表达N蛋白的pET-28a-N重组载体,构建后PCR鉴定结果如图1所示;
(2)将重组表达载体pET-28a-N电转入感受态细胞E.coliBL21(DE3)中,获得阳性重组大肠杆菌BL21(DE3);
(3)将阳性重组大肠杆菌BL21(DE3)接种于LB培养基中,在IPTG浓度为 1mM的条件下,16℃诱导表达10小时;
(4)收集样品进行超声破碎及Ni柱纯;
(5)将纯化后蛋白经SDS-PAGE电泳和Western blot检测。
鉴定结果显示:
如图2所示,纯化后获得的N蛋白纯度较高;如图3所示,纯化后获得的N蛋白在Westernblot检测中显示出与PRRSV阳性猪血清有较高的结合能力。
实施例二:单克隆抗体的制备及鉴定
1. 动物免疫
(1)将免疫原N蛋白加入弗氏完全佐剂首次免疫,乳化制成免疫抗原;
(2)通过背部皮下多点注射的方法,免疫4~8周龄的雌性BALB/c小鼠3只,免疫剂量50μg/只;
(3)每隔3周用弗氏不完全佐剂与免疫抗原乳化后以相同的方法和剂量对BALB/c小鼠进行加强免疫;
(4)四免后,细胞融合前3~4天,通过尾静脉注射的方法,用不含佐剂的免疫原对BALB/c小鼠进行超强免疫,免疫剂量是100 μg/只;
(5)多抗血清效价和敏感性测定:
最后一次加强免疫后一周,分别对3只小鼠进行剪尾采血,然后通过间接ELISA法和间接竞争ELISA法分别对3只小鼠多抗血清的效价和敏感性进行测定。选择免疫效果最好的免疫小鼠进行细胞融合。
2. 细胞融合及单克隆抗体制备
(1)采用聚乙二醇的方法,将免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0按细胞数量8:1的比例进行细胞融合,融合后的细胞用HAT选择培养基进行筛选;
(2)分装在铺有饲养细胞的96孔细胞培养板,置37℃、5% CO2培养箱内培养,于融合后第五天每孔补加培养基50 μl,12天后,以N蛋白或多肽作为包被抗原,通过间接ELISA法对杂交瘤细胞上清测OD值,作为阳性杂交瘤的初筛;
(3)通过有限稀释法对阳性杂交瘤细胞进行3~4次亚克隆筛选,最终获得了能稳定分泌抗N蛋白单克隆抗体的杂交瘤细胞株(1G4)。
3. 免疫过氧化物酶单层细胞试验(IPMA)
IPMA操作过程:
Marc145细胞铺96孔细胞板,待细胞汇合度长至70%左右接毒(PRRSV 强毒株JX-A1)。接毒后1h补加2%胎牛血清的DMEM培养基;48h后固定细胞:弃去96孔板中的培养基,PBST洗2~3次,50uL/孔加-20℃预冷的固定液(乙醇/0.3%双氧水的甲醇),室温静置10~15min;弃去固定液,PBST洗3次,200 μL/孔加脱脂奶,四度过夜封闭;弃去封闭液,PBST洗3次,50 μL /孔,分别加入单抗1G4或PBS(作为阴性对照NC),37℃,1h;弃去一抗,PBST洗5~6次,拍干,50μL /孔加一定倍数稀释的对应二抗(HRP-羊抗鼠酶标二抗),37℃,1h;弃去二抗,ddw洗5~6次,拍干,50 μL /孔,显色10min。(AEC显色液:每1ml ddw依次加A液1滴,B液2滴,C液1滴)。
如图4所示,经IPMA筛选,1G4能与PRRSV结合。
4. 间接免疫荧光试验(IFA)
IFA操作过程:
Marc145细胞铺96孔细胞板,待细胞汇合度长至70%左右接毒(PRRSV 强毒株JX-A1、BJ-4)。接毒后1h补加2%胎牛血清的DMEM培养基;48h后固定细胞:弃去96孔板中的培养基,PBST洗2~3次,50 μL/孔加-20℃预冷的固定液(乙醇/0.3%双氧水的甲醇),室温静置10~15min;弃去固定液,PBST洗3次,200 μL/孔加脱脂奶,四度过夜封闭;弃去封闭液,PBST洗3次,50 μL /孔加一定稀释度的单抗1G4或PBS(作为阴性对照NC),37℃,1h;弃去一抗,PBST洗5~6次,拍干,50 μL /孔加一定倍数稀释的对应二抗(FITC-羊抗鼠酶标二抗),37℃,1h;弃去二抗,PBST洗5~6次,于荧光显微镜下观察荧光。
结果如图5所示单抗IG4与美洲型PRRSV不同毒株反应均良好。
5. 体内诱生腹水法制备单抗
选择经产的雌性Balb/c小鼠,腹腔内注射500μl灭菌石蜡,一周后,再次腹腔内注射获得的单克隆杂交瘤细胞,注射量为2×105个细胞,再过一周后,待小鼠腹部膨大后抽取腹水,离心后取上清,用辛酸硫酸铵法对腹水进行纯化。
6. 单抗腹水的鉴定
(1)间接ELISA测定IPMA阳性单抗上清与N蛋白在不同孵育时间的效价均达到105;
(2)亚型鉴定:用亚型鉴定试剂盒对单抗的亚型进行鉴定,鉴定结果显示1G4单克隆抗体属于lgG1,轻链型为Kappa 型。
7. 稳定性鉴定
将所建立的单克隆杂交瘤细胞株连续培养3个月并反复液氮冻存复苏,从而鉴定杂交瘤细胞的稳定性。
结果显示单克隆杂交瘤细胞株稳定性良好。
实施例三:抗体的纯化
辛酸-硫酸铵法进行抗体纯化,操作方法如下:
(1)取出冻存的单抗腹水或抗血清,6000 r/min离心30min,获得上清液,用醋酸钠缓冲液(0.06mol/L pH4.8)稀释5倍。
(2)用NaOH(5mol/L)调节pH为4.5,室温缓慢搅拌0.5 h,加正辛酸,最终浓度为25μL/mL。
(3)4℃下6000 r/min离心30min,保留上清。
(4)用中速滤纸过滤得到的上清,并加入10×PBS缓冲液(pH7.4),体积为1/10,混合均匀后调节pH为7.4。
(5)在4℃条件下向混合液中添加固体硫酸铵,添加比例为0.2778 g/ mL,可多次添加,30 min内添加完毕。
(6)6000 r/min离心20min,扔掉上清液。添加起始腹水体积1/3的PBS(pH7.2)进行重悬,4℃下用PBS缓冲液透析24 h,期间换液3~4次,收集mAb,分装,-20℃保存。
实施例四:PRRSV N蛋白抗原表位的制备及鉴定
1. PRRSV N蛋白抗原表位的制备
如图6所示,采用肽扫描法设计合成6段多肽,每段多肽5个氨基酸重叠,覆盖整个N蛋白。多肽段氨基酸序列如表1所示。
表1多肽段氨基酸序列
。
2. 间接ELISA筛选能与抗体结合的短肽
多肽用CBS缓冲液稀释包被ELISA板,37℃ 3h;PBST洗板3~5次;用5%脱脂奶封闭,37℃3h;所述单抗作为一抗进行间接ELISA,37℃孵育1h;洗板后,加入HRP羊抗鼠酶标二抗,37℃孵育1h;洗板后加入底物显色液,3min后终止显色,用酶标仪读数。
如图7所示,结果显示单抗1G4可以特异性识别表位1~15aa,其OD450读数为1.78。
3. Dot-ELISA筛选能与抗体结合的短肽
多肽、N蛋白(PC)、原核表达无关蛋白(NC)用PBS缓冲液稀释,取2ug在NC膜上点样,完全风干后,用5%脱脂奶封闭,4℃过夜;用所述抗体孵育,37℃ 1h;PBST洗涤3~5次,HRP酶标二抗(1:1000稀释),37℃孵育1h;加入AEC底物显色液,室温显色3min后用去离子水终止显色。
如图8所示,结果显示单抗1G4可以特异性识别表位1~15aa及N蛋白(PC)。
4. Western-blot鉴定N蛋白B细胞表位
将短肽P1经SDS-PAGE处理后,电转到NC膜上(电压15V,30min),将NC膜至于5%脱脂奶的封闭液里,4℃过夜;次日PBST洗膜3次,加入单抗为一抗室温孵育1h;洗膜3~5次,加入HRP羊抗鼠酶标二抗,室温孵育1h;ddw洗膜5次,AEC底物显色液显色。
由于经SDS-PAGE处理后的蛋白样品为变性了的线性蛋白质(如图9,泳道1所示),若结果为阳性证明所得表位为线性表位,反之为构象表位。
如图9所示,泳道2显示短肽1~15aa在此试验中并未获得阳性结果,加之该短肽在Dot-ELISA及间接ELISA试验中均为阳性,该表位为构象表位。
5. 分析所得表位的结构及在各毒株之间的保守性
(1)利用生物信息学软件DNA-STAR,完成对美洲型PRRSV N蛋白二级结构、亲水性、疏水性、表面可及性、抗原性等特性的分析。
如图10所示,该表位位于N蛋白柔性区的B细胞表位,含有丰富的Alpha、Beta、Turn、coil结构,该区域富含正电荷,亲水性强,表面可及性强,抗原指数高。
(2)利用生物信息学软件ClustalX2.0,对该表位1~15aa进行不同毒株间的同源比对分析(所用毒株见表2),比较该表位所在区域在不同序列间的保守性。
表2 毒株信息
。
如图11所示,利用生物信息学软件ClustalX2.0比对结果显示该表位在美洲型PRRSV各毒株之间保守性较高,与欧洲型序列差异较大。
上面结合附图和实施例对本发明作了详细的说明,但是,所属技术领域的技术人员能够理解,在不脱离本发明宗旨的前提下,还可以对上述实施例中的各个具体参数进行变更,形成多个具体的实施例,均为本发明的常见变化范围,在此不再一一详述。
SEQUENCE LISTING
<110> 郑州大学
<120> 美洲型PRRSV N蛋白抗原表位及其应用
<130> 2018
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Claims (9)
1.一种短肽,其氨基酸序列为
(1)如SEQ NO.1所示氨基酸序列;或
(2)由SEQ ID NO.1所示的氨基酸序列衍生的具有同等功能的氨基酸序列。
2.一种美洲型PRRSV N蛋白抗原表位,具有权利要求1所述短肽的氨基酸序列。
3.根据权利要求2所述的美洲型PRRSV N蛋白抗原表位,其特征在于, 所述表位为一种位于N蛋白柔性区的B细胞表位。
4.根据权利要求2所述的美洲型PRRSV N蛋白抗原表位,其特征在于, 所述表位为构象性表位。
5.根据权利要求2所述的美洲型PRRSV N蛋白抗原表位,其特征在于, 所述表位为含Alpha、Beta、Turn、coil结构中的至少一种。
6.根据权利要求2所述的美洲型PRRSV N蛋白抗原表位,其特征在于, 所述表位在美洲型各毒株之间保守性高,在欧洲型毒株之间保守性低。
7.权利要求1所述短肽或权利要求2所述的美洲型PRRSV N蛋白抗原表位在制备预防和/或治疗美洲型PRRSV抗体或药物中的应用。
8.一种检测美洲型PRRSV的试剂盒,含有权利要求1所述短肽或/和该短肽的抗体。
9.一种预防美洲型PRRSV的疫苗,含有权利要求1所述短肽或/和该短肽的抗体。
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