CN109456412A - 一种猪流行性腹泻病毒基因工程疫苗及其制备方法 - Google Patents
一种猪流行性腹泻病毒基因工程疫苗及其制备方法 Download PDFInfo
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- CN109456412A CN109456412A CN201810313525.9A CN201810313525A CN109456412A CN 109456412 A CN109456412 A CN 109456412A CN 201810313525 A CN201810313525 A CN 201810313525A CN 109456412 A CN109456412 A CN 109456412A
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Abstract
本发明公开了一种猪流行性腹泻病毒基因工程疫苗及其制备方法,属于兽用生物制品领域。本发明以乙肝病毒的核心蛋白为载体,在该载体的第79位氨基酸至第80位氨基酸之间用分子生物学方法插入猪流行性腹泻病毒保护性抗原,构成预防猪流行性腹泻病毒的基因工程疫苗。疫苗的制备方法简单,能大量制备抗原蛋白,耗时短,表达量高,大大降低生产成本,有利于大规模生产。本发明制备制备的包含HBcAg‑PEDV‑COE重组蛋白的猪流行性腹泻亚单位疫苗,免疫效果良好,免疫剂量小,能有效预防猪流行性腹泻病毒的感染。
Description
技术领域
本发明涉及一种猪流行性腹泻病毒基因工程疫苗及其制备方法,属于兽用生物制品领域。
背景技术
猪流行性腹泻(porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)引起的一种肠道传染性疾病,其主要临床特征为水样腹泻、呕吐和脱水。该病于20世纪70年代首次在欧洲发现,随后在全球广泛流行,给养猪业带来了巨大的经济损失。该病主要引起新生仔猪发生致死性腹泻,病死率甚至达到了100%,主要病理变化是仔猪小肠绒毛脱落、变短,最终造成仔猪因脱水及营养缺乏而死亡。
PEDV的S糖蛋白携带主要的B淋巴细胞抗原表位,是诱导机体产生中和抗体和提供免疫保护的主要结构蛋白,试验证实了PEDV S蛋白上包括多个具有抗原性的表位序列。使用具有颗粒性结构的人乙肝核心抗原作为携带抗原基因的蛋白载体,可以充分的将抗原展示于颗粒表面,高效地刺激机体产生免疫反应。但是值得注意的是,人乙肝核心抗原作为蛋白载体插入抗原序列的容量有限,较大长度的抗原序列插入到该蛋白载体会影响其二级结构,从而影响颗粒的形成(Borisova,G.1996,Intervirology,Vol.39,p.16-22)。
疫苗接种是预防、控制甚至消灭猪流行性腹泻病毒的主要措施之一。亚单位疫苗不含有核酸物质,安全性较好,接种后不会产生持续感染或潜伏感染,产生的免疫应答可以与野毒感染相区分,有利于疫病的控制和消灭。
因此研制一种生产成本低、生产效率高以及疫苗免疫效果好的猪流行性腹泻病毒亚单位疫苗的生产方法具有重要的现实意义。
发明内容
本发明首先提供一种用于预防猪流行性腹泻病的基因工程疫苗,所用的抗原为灭活的重组抗原蛋白HBcAg-PEDV-COE,所述重组抗原蛋白HBcAg-PEDV-COE是以乙肝病毒核心抗原为载体,在该载体的第79位氨基酸至80位氨基酸之间通过linker插入猪流行性腹泻病毒的保护性抗原COE。
所述重组抗原蛋白的氨基酸序列如SEQ ID NO.7所示,编码所述重组抗原蛋白的核苷酸序列如SEQ ID NO.8所示。
所述重组抗原蛋白经过BEI灭活。
所述乙肝病毒核心抗原的氨基酸序列如SEQ ID NO.1所示,编码该乙肝病毒核心抗原的核酸序列如SEQ ID NO.2所示。
所述猪流行性腹泻病毒的保护性抗原COE的氨基酸序列如SEQ ID NO.3所示,编码所述COE的核酸序列如SEQ ID NO.4所示。
所述基因工程疫苗中重组抗原蛋白的含量为75~100μg/mL、例如75μg/mL、100μg/mL。
所述基因工程疫苗中所述佐剂为纳米佐剂。
本发明还提供了制备所述重组抗原蛋白的方法,采用昆虫细胞-杆状病毒表达系统表达所述重组抗原蛋白,主要包括以下步骤:
(1)将编码猪流行性腹泻病毒S蛋白COE的基因插入到编码人乙肝核心抗原的基因中,插入的位置是编码第79位氨基酸和编码第80位氨基酸的核苷酸序列之间,构建融合基因片段HBcAg-PEDV-COE;
(2)将融合基因片段与pFastBac I载体分别进行BamH I和Hind I双酶切,并进行连接,连接产物转化E.coli DH5α;获得阳性质粒pFastBac I-HBcAg-PEDV-COE;
(3)以所述步骤(2)中获得的pFastBac I-HBcAg-PEDV-COE质粒转化E.coliDH10Bac感受态细胞,通过转座,获得重组杆粒Bacmid-HBcAg-PEDV-COE;
(4)将所述步骤(3)中获得的重组杆粒Bacmid-HBcAg-PEDV-COE转染昆虫细胞sf9,获得重组杆状病毒rBac-HBcAg-PEDV-COE;
(5)培养所述步骤(4)中获得的重组杆状病毒rBac-HBcAg-PEDV-COE,收获上清,得到HBcAg-PEDV-COE重组蛋白。
本发明提供的重组HBcAg-PEDV-COE蛋白,可在体外高效组装为类病毒样颗粒,并将COE多肽展示于该颗粒表面。
本发明还提供一种抗体,能够与重组HBcAg-PEDV-COE蛋白特异性结合,该抗体还可以用于制备基于酶联免疫法的检测猪流行性腹泻病毒的试剂盒。
本发明将表达的重组蛋白的重组杆状病毒rBac-HBcAg-PEDV-COE,接入昆虫细胞高效的表达HBcAg-PEDV-COE蛋白,经过离心去除细胞碎片,加入BEI灭活后,本发明制备的疫苗能提高免疫后的抗体水平,提高免疫后抗体的整齐度,保证疫苗的免疫效果,此疫苗具有高效、安全性好的优点。本发明的目的是提供一种以人乙肝核心抗原为载体,在其79~80位氨基酸之间插入猪流行性腹泻病毒S蛋白COE基因,经过杆状病毒-昆虫细胞表达系统表达的重组蛋白,该重组蛋白在体外能够自组装为病毒样颗粒(Virus Like Particles,VLPs),并且PEDV S蛋白的主要免疫原COE多肽展示于VLP表面。向重组抗原蛋白HBcAg-PEDV-COE中加入纳米佐剂混合乳化制成疫苗。该疫苗具有高效、安全性好、抗体整齐度高、保护率高的优点。
附图说明
图1是一个融合的携带了外源基因的HBcAg分子示意图;
图2是转移载体构建PCR鉴定的电泳图,M:DL5000DNA Marker;1:PCR产物;
图3是重组杆粒PCR鉴定的电泳图,M:DL5000DNA Marker;1~7:7个不同菌落PCR;
图4是SDS-PAGE检测重组杆状病毒表达产物,M:预染蛋白质Marker;1:F3代重组杆状病毒上清;
图5是Western Blot鉴定重组杆状病毒表达产物,M:预染蛋白质Marker;1:F3代重组杆状病毒;2:感染空杆状病毒的Sf9细胞;
图6是纯化后重组蛋白透射电镜照片。
具体实施方式
实施例1:融合基因片段HBcAg-PEDV-COE的构建
1、融合基因片段HBcAg-PEDV-COE的构建:如图1所示,利用分子生物学技术和方法,将PEDV COE基因插入到编码HBcAg(人乙肝核心抗原)分子的基因中,插入的位置是编码第79位氨基酸和编码第80位氨基酸的核苷酸序列之间,构建融合基因片段HBcAg-PEDV-COE(SEQ ID NO.8)。
2、目的基因与中转质粒连接:将昆虫表达载体pFastBac I和HBcAg-PEDV-COE基因扩增片段用BamH I和Hind I酶切后分别回收、纯化,用T4DNA连接酶4℃连接过夜。
3、连接产物转化感受态细胞:将连接产物在无菌条件下转化T1感受态细胞中,具体地,混匀,冰浴30min,42℃热激90s,立即冰浴2min,无菌条件下加入800μL LB培养基于37℃震荡培养60min。将培养物14000rpm离心1min,吸取800μL上清,将剩余培养物涂布于LB(含氨苄青霉素抗性)固体培养基中,37℃过夜培养。挑取单菌落作菌落PCR鉴定,阳性质粒送检测序。测序正确的重组质粒命名为pFastBac I-HBcAg-PEDV-COE。
4、重组杆状病毒构建:将鉴定正确的中转质粒pFastBac I-HBcAg-PEDV-COE转入大肠杆菌DH10Bac感受态细胞中,挑选阳性克隆用M13引物作PCR鉴定。
M13-F:TGTAAAACGACGGCCAGT
M13-R:CAGGAAACAGCTATGAC
PCR反应体系为(总体积25μL):DNA模板0.5μL、M13-F和M13-R个0.5μL、DNA聚合酶12.5μL及无菌水11μL。PCR反应条件为:95℃,5min;95℃ 30s,65℃ 30s,72℃90s,30个循环;72℃ 10min。1%琼脂糖凝胶电泳显示,成功扩增出约3000bp的特异性条带,与预期大小相符。阳性重组杆粒命名为rBacmid-HBcAg-PEDV-COE。
5、重组杆粒转染sf9细胞:将提纯的重组杆粒用脂质体转染的方法转染sf9细胞,具体操作方法参照赛默飞世尔科技(中国)有限公司的cellfectin转染试剂说明书进行,获得f1代重组杆状病毒rBac-HBcAg-PEDV-COE。
实施例2:重组HBcAg-PEDV-COE蛋白的制备
1、重组杆状病毒扩增:将重组杆状病毒rBac-HBcAg-PEDV-COE接种昆虫细胞sf9,27℃培养4天,收集培养物,离心取上清即获得f2代重组杆状病毒;
2、表达蛋白鉴定:
(1)将上述f2代重组杆状病毒以MOI=5~10的接种量接入昆虫细胞sf9,27℃培养4天,收集培养物,离心取上清即获得重组VP3蛋白;
(2)SDS-PAGE鉴定:将上述上清液进行SDS-PAGE电泳;电泳结束后,经染色和脱色后发现,大约在32kDa位置,其分子量与理论大小相符,说明表达成功。
(2)Western Blot鉴定:取SDS-PAGE电泳后凝胶,直接用BIO-LAB转印装置将其转印到NC膜上,转印结束后,按常规方法进行Western blot鉴定。用猪流行性腹泻阳性血清参考品(1:200)作为一抗(PEDV攻毒后阳性血清);用辣根过氧化物酶标记的羊抗猪IgG(1:2000)作为酶标二抗;最后用TMB显色(碧云天生物技术研究所)。结果表明,在32kDa处出现1条明显的特异性条带,而阴性对照无此特异性反应,说明该重组蛋白可被猪流行性腹泻病毒阳性血清中的抗体识别,具有良好的特异性和反应原性。
3、重组HBcAg-PEDV-COE蛋白的大量表达:将鉴定正确的重组病毒以MOI=1~10的接毒量接种High Five细胞大量培养,离心收集培养液上清,即获得含有大量的重组HBcAg-PEDV-COE蛋白。
4、重组HBcAg-PEDV-COE蛋白纯化:利用GE公司的镍柱(HisTrap HP,5mL)对重组蛋白进行纯化,并对纯化后的重组蛋白进行透析处理,去除纯化样品中的咪唑和氯化钠。
5、重组HBcAg-PEDV-COE蛋白定量:利用碧云天生物技术研究所BCA蛋白浓度测定试剂盒(增强型)进行蛋白定量,结果显示纯化后的目的蛋白浓度为2.03mg/mL。
实施例3猪流行性腹泻病毒基因工程亚单位疫苗的制备
取实施例2中获得的纯化的HBcAg-PEDV-COE重组蛋白,加入佐剂进行乳化,混匀,4℃保存。疫苗具体配比见表1。
表1猪流行性腹泻病毒基因工程亚单位疫苗成分配比
实施例3猪流行性腹泻病毒基因工程疫苗免疫原性试验
1、妊娠母猪免疫试验
选取产前5~6周猪流行性腹泻中和抗体、抗原均为阴性,且预产期均相同的怀孕母猪20头,随机分为4组,每组5头。1~3组分别免疫实施例2中制备的疫苗1、疫苗2、疫苗3,免疫方式为颈部肌肉注射,注射剂量为1mL/头,第4组注射相同剂量的无菌PBS。产前3周进行二免,免疫方式与免疫剂量与首免相同。免疫后,观察各组母猪精神状态、采食、饮水等临床症状。统计母猪产仔情况,并且分娩后第0天、7天、14天和21天分别采集母猪乳汁,比较乳汁中PEDV IgA抗体水平。
免疫组母猪经两次免疫后精神状态、采食及饮水等均未出现异常。免疫组与对照组产仔数量相当,未出现弱胎、死胎和木乃伊胎,结果见表2。
表2妊娠母猪免疫后产仔情况统计表
采用韩国安捷公司的PEDV IgA抗体检测试剂盒测定产仔后母猪第0天、7天、14天和21天的乳汁中PEDV IgA抗体,结果如表3所示。从表3中可以看出,免疫组母猪产后乳汁IgA抗体水平均为阳性,且3组免疫组抗体均在14天达到最高,21天有所下降。另外,从免疫组乳汁IgA抗体水平来看,疫苗中抗原含量直接影响抗体水平的高低:75μg和100μg要明显高于50μg,但也不是抗原含量越高越好,从数据上来看,75μg和100μg的免疫效果相当,抗体水平十分接近。因而,确定疫苗中重组蛋白的抗原含量在75μg~100μg之间。
表3母猪产后乳汁中IgA抗体水平
注:阴性对照平均OD值:0.078;判定值=0.35+0.078=0.428
2、仔猪攻毒试验
从实施例3的1中免疫组和对照组中各母猪所产仔猪各随机选取8头(已母乳饲养21天)。采用猪流行性腹泻病毒变异株(扬州优邦生物药品有限公司于2014分离于河南某猪场送检病料,命名为HN-14)进行口服攻毒。攻毒后每天观察仔猪临床表现(精神状态、采食、呕吐、腹泻)。
结果显示:免疫组仔猪仅有免疫组1在攻毒后第3天出现不同程度的腹泻,且持续3天;期间免疫组1仔猪出现食欲下降、精神沉郁。免疫组2和免疫组3仔猪攻毒后未出现临床症状,精神状态、采食均为显示异常。对照组仔猪在攻毒后48小时全部出现精神沉郁和食欲下降等典型临床症状,并且在攻毒后48小时7/8仔猪有水样腹泻,粪便呈黄色,有腥臭味,部分仔猪伴有呕吐现象。
从表4中数据可以得出,该发明所述的预防猪流行性腹泻病毒基因工程疫苗具有良好的免疫效果,可以保护仔猪抵御变异株的攻击。
表4仔猪攻毒后临床表现统计
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 扬州优邦生物药品有限公司
<120> 一种猪流行性腹泻病毒基因工程疫苗及其制备方法
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 149
<212> PRT
<213> 乙型肝炎病毒
<400> 1
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tgctgggggg aattgatgac tctagctacc tgggtgggta ataatttgga agatccagca 240
tccagggatc tagtagtcaa ttatgttaat actaacatgg gtttaaagat caggcaacta 300
ttgtggtttc atatatcttg ccttactttt ggaagagaga ctgtacttga atatttggtc 360
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atggacattg acccttataa agaatttgga gctactgtgg agttactctc gtttttgcct 60
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ggcggctccg gcggcggcgg cacagttact ttgccatcat ttaatgatca ttcttttgtt 300
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actactatca atgggtttag ttctttctgt gttgacacta gacaatttac catttcactg 420
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ccacttgaag gtgttacgga cgttggcggc ggcggctccg gcggcggcgg ctccagggat 720
ctagtagtca attatgttaa tactaacatg ggtttaaaga tcaggcaact attgtggttt 780
catatatctt gccttacttt tggaagagag actgtacttg aatatttggt ctctttcgga 840
gtgtggattc gcactcctcc agcctataga ccaccaaatg cccctatctt atcaacactt 900
ccggaaacta ctgttgttta a 921
Claims (10)
1.一种重组抗原蛋白,其特征在于,是以乙肝病毒核心抗原为载体,在该载体的第79位氨基酸至80位氨基酸之间通过linker插入猪流行性腹泻病毒的保护性抗原COE;所述乙肝病毒核心抗原的氨基酸序列如SEQ ID NO.1所示,所述猪流行性腹泻病毒的保护性抗原COE的氨基酸序列如SEQ ID NO.3所示。
2.编码权利要求1所述重组抗原蛋白的基因,其特征在于,核苷酸序列如SEQ ID NO.8所示。
3.一种用于预防猪流行性腹泻病的基因工程疫苗,其特征在于,含有权利要求1所述的重组抗原蛋白。
4.根据权利要求3所述的基因工程疫苗,其特征在于,所述重组抗原蛋白经过BEI灭活。
5.根据权利要求3或4所述的基因工程疫苗,其特征在于,所述基因工程疫苗中重组抗原蛋白的含量为75~100μg/mL。
6.根据权利要求所述的基因工程疫苗,其特征在于,所述基因工程疫苗中重组抗原蛋白的含量为75μg/mL或100μg/mL。
7.根据权利要求3~6任一所述的基因工程疫苗,其特征在于,还含有包括纳米佐剂在内的佐剂。
8.制备权利要求1所述重组抗原蛋白的方法,其特征在于,采用昆虫细胞-杆状病毒表达系统表达所述重组抗原蛋白,主要包括以下步骤:
(1)将编码猪流行性腹泻病毒S蛋白COE的基因插入到编码人乙肝核心抗原的基因中,插入的位置是编码第79位氨基酸和编码第80位氨基酸的核苷酸序列之间,构建融合基因片段HBcAg-PEDV-COE;
(2)将融合基因片段与pFastBac I载体分别进行BamH I和Hind I双酶切,并进行连接,连接产物转化E.coli DH5α;获得阳性质粒pFastBac I-HBcAg-PEDV-COE;
(3)以所述步骤(2)中获得的pFastBac I-HBcAg-PEDV-COE质粒转化E.coli DH10 Bac感受态细胞,通过转座,获得重组杆粒Bacmid-HBcAg-PEDV-COE;
(4)将所述步骤(3)中获得的重组杆粒Bacmid-HBcAg-PEDV-COE转染昆虫细胞sf9,获得重组杆状病毒rBac-HBcAg-PEDV-COE;
(5)培养所述步骤(4)中获得的重组杆状病毒rBac-HBcAg-PEDV-COE,收获上清,得到HBcAg-PEDV-COE重组蛋白。
9.一种抗体,其特征在于,能够与权利要求1所述重组抗原蛋白特异性结合。
10.一种检测猪流行性腹泻病毒的试剂盒,其特征在于,含有权利要求9所述的抗体。
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