CN104650195B - Ev71病毒vp1重组抗原及其单克隆抗体与应用 - Google Patents
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Abstract
本发明公开了一组EV71病毒VP1重组抗原,是由SEQ ID No.1~SEQ ID No.11所示的11种氨基酸序列分别与柔性短肽氨基酸序列连接制得。本发明还公开了能与所述EV71病毒VP1重组抗原特异性结合的单克隆抗体,以及所述抗原在制备EV71病毒抗原或抗体检测试剂盒中的应用。本发明提供的重组抗原和单克隆抗体具有高灵敏度、高特异性等特点,是可以作为研制EV71病毒检测试剂的关键原料。
Description
技术领域
本发明涉及一组肠道病毒71型(EV71)病毒VP1重组抗原及其单克隆抗体与应用,属于分子生物学技术领域。
背景技术
手足口病(Hand foot mouth disease,HFMD)是由肠道病毒引起的一种全球性常见的急性传染病。该病多发生于5岁以下儿童,主要表现为口痛、厌食、低热、手、足、口腔等部位出现小疱疹或小溃疡,少数患儿可引起心肌炎等并发症。个别重症患儿病情发展快,导致死亡。手足口病是由肠道病毒引起的传染病,引发手足口病的肠道病毒有20多种(型)。目前国内爆发的手足口病以柯萨奇病毒A16型(Cox A16)和肠道病毒71型(EV 71)引起最为常见。Cox A16引起的手足口病一般症状较轻,容易自愈。感染EV71病症则相对较重,20%患者可能出现重症炎症及伤害中枢系统造成后遗症。故早发现早诊断可有效降低该病毒传播风险,并有助于患者得到及时治疗。因此,开发好研制有关EV71病毒检测试剂和方法显得尤为重要。
肠道病毒71型(EV 71)于1969年首次从加利福尼亚患有中枢神经系统疾病的婴儿粪便标本中分离出来。病毒基因组为单股正链RNA,全长约7.5kb,编码含2194个氨基酸的多聚蛋白。该蛋白可进一步被水解成P1、P2、P3 3个前体蛋白;其中P1蛋白可分解为VP1、VP2、VP3和VP4四个外壳蛋白。VP1、VP2、VP3暴露在病毒衣壳蛋白的表面,具有大量抗原决定簇,而其中的VP1是最主要的衣壳蛋白,具有最多的特异性中和位点,因此,VP1蛋白的编码基因序列与病毒血清具有较高的相关性,常被用于病毒的鉴定和进化分析。
高相关的EV71病毒抗原和抗体是EV71病毒检测试剂制备的关键。近年来,基因工程的发展为研制EV71病毒抗原和抗体提供了良好基础。故加强研制EV71病毒VP1特异性抗原和其单克隆抗体,对开发更加有效的早期诊断EV71的方法和试剂盒具有重要意义。
发明内容
本发明目的在于提供一组EV71病毒VP1重组抗原及其单克隆抗体与应用。
本发明所述的EV71病毒VP1重组抗原,其特征在于:所述重组抗原由SEQ ID No.1~SEQ ID No.11所示的11种氨基酸序列分别与柔性短肽氨基酸序列连接制得;其中,所述柔性短肽氨基酸序列是:GGGGS或GGGGSGGGGSGGGGS或AAY,所述11种氨基酸序列分别与柔性短肽氨基酸序列连接时,每种氨基酸序列均重复3次。
进一步的,所述重组抗原有11种,其编号为EV71 VP1 1号~EV71 VP1 11号,其中EV71 VP1 1号~EV71 VP1 11号重组抗原的核苷酸序列由SEQ ID No.12~SEQ ID No.22所示。
基于对手足口病不同疫区的EV71病毒的研究,本发明进一步综合分析了国内外有关EV71的文献,确定了EV71的VP1蛋白作为抗原靶位的主要研究对象。通过多种生物信息学数据库及生物信息学软件对VP1基因及其编码蛋白序列对比分析,考虑氨基酸序列亲水性,对VP1抗原表位预测,筛选得到上述EV71病毒11种潜在高交叉反应性的线性亲水表位氨基酸序列,合成基因重组表达,并经过实验验证具有高交叉反应,其氨基酸序列具体为:
VP1(10-20AA)SEQ ID NO1:SSIGDSVSRAL
VP1(25-40AA)SEQ ID NO2:PAPTGQNTQVSSHRLD
VP1(51-65AA)SEQ ID NO3:EIGASSNASDESMIE
VP1(105-119AA)SEQ ID NO4:GYANWDIDITGYAQM
VP1(119-133AA)SEQ ID NO5:MRRKVELFTYMRFDA
VP1(137-150AA)SEQ ID NO6:FVACTPTGEVVPQL
VP1(161-180AA)SEQ ID NO7:PKPDSRESLAWQTATNPSVF
VP1(206-224AA)SEQ ID NO8:DGYPTFGEHKQEKDLEYGA
VP1(231-245AA)SEQ ID NO9:GTFSVRTVRDLQVQV
VP1(255-269AA)SEQ ID NO10:MKHVRAWIPRPMRNQ
VP1(270-284AA)SEQ ID NO11:NYLFKANPNYAGNSI
利用基因重组方法,将上述各线性表位氨基酸序列分别加入柔性短肽并连接在一起,可以获得具有高交叉反应的融合VP1重组抗原,即EV71病毒VP1重组抗原。
本发明还提供了一组抗体,其特征在于:所述抗体是能与上述EV71病毒VP1重组抗原特异性结合的单克隆抗体。
将上述EV71病毒VP1重组抗原蛋白免疫小鼠(注射小鼠),经常规单克隆抗体制备方法,纯化分离,即可制备出上述重组抗原特异性结合的单克隆抗体。
本发明所述EV71病毒VP1重组抗原在制备EV71病毒抗原或抗体检测试剂盒中的应用。
本发明所述EV71病毒VP1特异性抗原和其单克隆抗体,可以用于开发体外检测样品中是否存在抗EV71病毒抗体的试剂盒或作为其他EV71检测产品的重要原料。或者,可以用于开发体外检测样品中是否存在EV71病毒抗原的试剂盒或作为其他EV71检测产品的重要原料。对开发更加有效的早期诊断EV71的方法和试剂盒提供了基础。
与现有技术比,本发明技术具有以下特点:
1筛选得到的VP1抗原是线性亲水表位,易于与患者血清中抗EV71病毒抗体发生特异性结合反应。运用本发明的特异性重组蛋白可用于免疫于兔、鼠等动物,制备单克隆抗体。
2本发明得到的单克隆抗体可以通过本领域技术人员已知的各种技术进行制备(如杂交瘤技术),亦可以通过小抗体技术制备Fab段。
3本发明的重组抗原蛋白和抗体是高特异性、高灵敏度的优质生物原料,均可以直接用于检测人血清中EV71病毒或抗EV71抗体,从而有利于EV71病毒感染早期筛查。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明范围。若未特别指明,实施例中所采用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
实施例1:候选抗原表位的选择
通过查找NCBI(美国国立生物技术信息中心,http://www.ncbi.nlm.nih.gov/),对EV71 3种基因型的共70条EV71序列进行分析,根据氨基酸序列的同源性,依次分筛选出11条各基因型序列保守的线性表位抗原序列。其氨基酸序列分别如表1所示。
表1 本发明筛选出的11条EV71 VP1线性抗原表位抗原序列
序号 | 氨基酸位点 | 氨基酸序列 |
SEQ ID No.1 | 10-20AA | SSIGDSVSRAL |
SEQ ID No.2 | 25-40AA | PAPTGQNTQVSSHRLD |
SEQ ID No.3 | 51-65AA | EIGASSNASDESMIE |
SEQ ID No.4 | 105-119AA | GYANWDIDITGYAQM |
SEQ ID No.5 | 119-133AA | MRRKVELFTYMRFDA |
SEQ ID No.6 | 137-150AA | FVACTPTGEVVPQL |
SEQ ID No.7 | 161-180AA | PKPDSRESLAWQTATNPSVF |
SEQ ID No.8 | 206-224AA | DGYPTFGEHKQEKDLEYGA |
SEQ ID No.9 | 231-245AA | GTFSVRTVRDLQVQV |
SEQ ID No.10 | 255-269AA | MKHVRAWIPRPMRNQ |
SEQ ID No.11 | 270-284AA | NYLFKANPNYAGNSI |
实施例2:EV71 VP1重组抗原的克隆与表达
1.EV71 VP1重组抗表达质粒的构建
1.1EV71 VP1重组抗原的合成
根据上述11条EV71 VP1抗原氨基酸序列,并利用基因重组方法,将上述各EV71VP1序列分别加入柔性短肽并连接在一起,其中,所述柔性短肽氨基酸序列是:GGGGS或GGGGSGGGGSGGGGS或AAY,所述11种氨基酸序列分别与柔性短肽氨基酸序列连接时,每种氨基酸序列均重复3次。
采用大肠杆菌偏好密码子,将氨基酸序列翻译成核苷酸序列,分别在5’端和3’端引入BamHI和Xho I两个酶切位点(见序列表),并委托上海捷瑞生物工程有限公司合成上述EV71 VP1抗原序列的基因。
1.2EV71 VP1抗原表达质粒的构建
1.2.1EV71 VP1抗原基因序列及表达载体pET28a的双酶切
取以上11条抗原合成基因产物及pET28a表达载体各30ul,分别置于1.5mlEppendorf离心管中,加入10×buffer 5ul、BamHI(10U/ul)和Xho I(10U/ul)各3ul,加入灭菌蒸馏水4ul,置37℃水浴酶切3小时。
酶切产物的琼脂糖凝胶电泳纯化和回收:合成基因及pET28a表达载体双酶切后产物按照TaKaRa DNA凝胶回收试剂盒产品说明书进行。
1.2.2连接:于灭菌Eppendorf离心管中加入上述酶切后的载体和目的基因各2ul、10×T4 DNA Ligase buffer 2ul、T4 DNA Ligase(5U/ul)1ul,加入灭菌蒸馏水至20ul,置16℃过夜。
1.2.3转化:在超净工作台中,用无菌吸头吸取100ul感受态细胞BL21(感受态细胞按照《分子克隆》(科学出版社,第三版)的方法进行)悬液于Eppendorf中,加入上述连接产物10ul,轻轻涡旋混匀,冰浴30分钟。立即转移到42℃水浴中放置2分钟,每管加入0.5ml LB培养基,37℃温箱振荡培养60分钟,吸取培养液涂布与LB培养基上(含抗生素),置37℃温箱倒置培养过夜。
1.2.4阳性重组子的筛选:各挑取上述培养过夜的平皿中的单克隆菌落,接种于5ml LB液体培养基(含抗生素)中,37℃温箱振荡培养5小时。保存各单克隆菌液并提取质粒(按照《分子克隆》(科学出版社,第三版)的方法进行)。提取后的质粒用BamHI和Xho I酶切,酶切产物用琼脂糖凝胶电泳进行鉴定。
2EV71 VP1抗原的表达与纯化
2.1表达菌株的培养:分别取上述11条抗原的阳性表达菌株单克隆菌液20ul,接种于100ml LB培养基中,37℃温箱振荡培养过夜。次日,按照1%接种比例转种于LB培养基,37℃温箱振荡培养3小时,当OD600值达到0.6时,加入异丙基硫代半乳糖苷(IPTG)诱导培养,终浓度1mmol/L,37℃温箱振荡培养5小时。将菌液合并,6000rpm离心20分钟,弃上清,收集沉淀部分。
2.2提取包涵体:将沉淀称湿重,用10倍体积的20mmol/L,pH8.0Tris缓冲液将沉淀悬起,加入溶菌酶,在室温下磁力搅拌10分钟。在冰浴中超声破碎菌体。12000rpm,离心10分钟,其上清,沉淀用1mol/L NaCl洗1次,再用TE洗2次,收集沉淀。沉淀用8M尿素(用20mmol/L,pH8.0Tris配制)溶解,加入1‰DTT,于4℃,12000rpm,离心10分钟,去沉淀取上清。
2.3纯化:将上述包涵体溶解液过S-Sepharose柱阶段梯度洗脱,用不同浓度的NaCl(用平衡器配制)洗脱,收集0.15M NaCl洗脱峰,再经Sephadex G50柱脱盐,收集第一个洗脱峰。重组多型别HVR1融合抗原用SDS-PAGE鉴定。
3EV71 VP1重组抗原活性鉴定
分别使用经过处理的实施例2制备的EV71 VP1重组抗原,用pH7.4的磷酸盐缓冲液稀释到5.0ug/ml,包被,与50例病人(已确诊)的血清混合反应。
结果表明,本发明的重组抗原可以识别患者血清中早期出现的针对EV71的特异性抗体,并与之结合,洗涤后加入羊抗人IgG-HRP(辣根过氧化物酶)酶联二抗,最后加入显色底物液,A450检测。
基于以上结果,将11种EV71 VP1重组抗原用pH7.4的磷酸盐缓冲液稀释到5.0ug/ml,包被ELISA测定板,经封闭后,对收集的90份EV71验证为阴性血清(其中包括部分HSV、腺病毒、RSV、麻疹病毒血清)进行测定。结果如表2所示。
表2 11种EV71 VP1抗原包被测定板检测血清结果
结果表明本发明11种抗原包被的酶联免疫吸附板没有假阳性出现。说明本发明重组抗原特异性良好。
实施例3:重组EV71 VP1抗原蛋白免疫小鼠制备单克隆抗体
1免疫抗原的制备:11种EV71 VP1抗原制备方法按照实施例2方法进行。
2小鼠免疫:采用重组VP1蛋白进行BALB/c小鼠免疫。免疫程序:首次免疫取重组VP1蛋白与弗氏完全佐剂混合乳化,免疫BALB/c小鼠,50ug/只,腹腔注射;间隔14天后进行第二次免疫:取重组VP1蛋白与弗氏不完全佐剂混合乳化,免疫BALB/c小鼠,50ug/只,腹腔注射;7天后采血,采用间接ELISA法测定EV71抗体活性。对检测抗体效价最高的小鼠不加佐剂,用重组VP1蛋白进行静脉注射加强免疫。
3细胞融合、细胞筛选与培养、制备腹水、抗体纯化均采用本领域技术人员所熟知的常规手段。
通过以上实验,本发明获得单克隆抗体共12个。分别将12个经纯化的单克隆抗体,采用经典过碘酸钠法进行HRP标记,标记后的抗体加等量甘油后-70℃保存。
利用实施例3获得的12个单克隆抗体和12个HRP标记的单克隆抗体采用双抗体夹心法进行两两配对组合检测EV71病毒抗原,得到A(6,5-HRP)、B(5,7-HRP)、C(7,8-HRP),3组有效的组合,分别测得OD450>3.0。结果如表3。
表3 单克隆抗体的优化组合
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
1-HRP | - | 1.523 | 1.231 | 1.221 | 1.869 | 1.601 | 1.542 | 1.467 | 1.467 | 1.369 | 1.699 | 0.986 |
2-HRP | 1.563 | - | 1.645 | 1.841 | 1.444 | 1.356 | 1.006 | 1.698 | 1.256 | 1.055 | 1.536 | 1.322 |
3-HRP | 1.869 | 1.321 | - | 1.599 | 1.578 | 1.723 | 1.548 | 1.006 | 1.875 | 1.469 | 1.875 | 1.648 |
4-HRP | 1.562 | 1.254 | 1.232 | - | 1.337 | 1.226 | 1.201 | 1.745 | 1.528 | 1.415 | 2.046 | 1.250 |
5-HRP | 1.632 | 1.369 | 1.482 | 1.674 | - | 3.001 | 1.864 | 1.457 | 1.268 | 1.758 | 1.465 | 1.485 |
6-HRP | 1.445 | 1.325 | 1.474 | 1.685 | 1.876 | - | 2.036 | 1.355 | 1.394 | 1.694 | 1.375 | 1.852 |
7-HRP | 1.694 | 1.445 | 1.536 | 1.750 | 3.213 | 1.764 | - | 1.804 | 1.955 | 1.399 | 1.910 | 1.733 |
8-HRP | 1.211 | 1.120 | 1.587 | 1.624 | 1.725 | 2.511 | 3.102 | - | 1.376 | 1.487 | 1.935 | 1.629 |
9-HRP | 1.645 | 1.845 | 1.455 | 1.620 | 1.625 | 2.134 | 2.001 | 1.746 | - | 1.647 | 1.385 | 1.641 |
10-HRP | 1.564 | 1.631 | 1.654 | 1.456 | 2.663 | 1.036 | 1.874 | 1.365 | 1.846 | - | 1.123 | 1.541 |
11-HRP | 1.785 | 1.666 | 1.674 | 1.751 | 1.359 | 1.568 | 1.369 | 1.557 | 1.554 | 1.550 | - | 1.236 |
12-HRP | 1.012 | 1.520 | 1.339 | 1.142 | 1.963 | 1.604 | 1.478 | 1.564 | 1.377 | 1.579 | 1.574 | - |
Claims (3)
1.一组EV71病毒VP1重组抗原,其特征在于:由至少11种重组抗原组成,每一种重组抗原由一种抗原多肽和一种柔性短肽通过肽键间隔连接而成,连接结构为抗原多肽-柔性短肽-抗原多肽-柔性短肽-抗原多肽,
所述一种抗原多肽的氨基酸序列选自SEQ ID No.1~SEQ ID No.11,每一种抗原多肽至少形成一种重组抗原,
所述一种柔性短肽的氨基酸序列选自GGGGS、GGGGSGGGGSGGGGS或AAY。
2.如权利要求1所述EV71病毒VP1重组抗原,其特征在于:由11种重组抗原组成,其编号分别为EV71 VP1 1号~EV71 VP1 11号,其中编码EV71 VP1 1号~EV71 VP1 11号重组抗原的核苷酸序列分别为SEQ ID No.12~SEQ ID No.22。
3.权利要求1或2所述EV71病毒VP1重组抗原在制备EV71病毒抗原或抗体检测试剂盒中的应用。
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