CN114106113B - 一种表达猪细小病毒vp2蛋白的重组杆状病毒、制备方法及应用 - Google Patents
一种表达猪细小病毒vp2蛋白的重组杆状病毒、制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种表达猪细小病毒VP2蛋白的重组杆状病毒、制备方法及应用,所述重组杆状病毒的保藏编号为:CCTCCNo:V202178。本发明通过将毒株的VP2基因克隆至载体pVL1392中,将重组质粒和flashBac‑ultra杆状病毒基因共转染sf9细胞,获得表达VP2蛋白的重组杆状病毒。猪细小病毒VP2蛋白重组杆状病毒疫苗能够诱导较强的免疫应答,能提供针对猪细小病毒的临床保护并抑制病毒在机体内的复制,其免疫原性与保护效力和商品疫苗相当。该方法制备VP2蛋白简便、生产周期短、免疫效力强。
Description
技术领域
本发明涉及生物技术领域,具体为一种表达猪细小病毒VP2蛋白的重组杆状病毒、制备方法及应用。
背景技术
猪细小病毒(Porcine parvovirus,PPV)属于细小病毒科,细小病毒属,是无囊膜单链DNA病毒。PPV可导致母猪繁殖障碍,以母猪不孕、流产、产死胎、木乃伊胎及弱仔为特征。我国最早于1983年分离到PPV,此后多地出现PPV感染情况。PPV感染力强,对外界环境有很强的抵抗力,且常与其他疾病混合感染猪群。研究显示一些猪群中该病的检出率可达85%,对我国养猪业造成巨大损失。
我国对于PPV的防治以免疫预防为主,已上市的疫苗均为灭活疫苗,包括S-1株、CP-99株、WH-1株、L株等,但灭活疫苗存在免疫效果不稳定、制备过程中有生物安全隐患等问题。部分研究显示,PPV的自然弱毒株或人工致弱毒株制成弱毒疫苗可对PPV产生一定的保护效力,但弱毒疫苗存在毒力返强、与自然毒株重组等风险。因此,PPV的灭活疫苗及弱毒疫苗均存在一定的限制性,不是最理想的PPV疫苗,而通过基因工程产生PPV亚单位疫苗可以避免以上问题。
利用原核表达系统可实现蛋白的低成本大规模生产,但往往不能形成正确的空间结构,影响活性,且产物中含有内毒素,限制应用。真核表达系统中酵母和哺乳动物细胞表达量较低,不适合疫苗的大规模生产,而杆状病毒表达系统表达水平高,可对蛋白进行修饰且产物无内毒素影响。目前最常用的是杆状病毒表达系统是Bac-to-Bac表达系统,利用该系统可快速获得重组病毒,但需要通过蔗糖密度梯度超速离心技术来确保基因组稳定性。而本发明利用同为杆状病毒表达系统的flashBAC ultra表达系统,通过该系统获得的重组杆状病毒具有稳定性良好、储存时间长且保持感染能力等优势。
PPV的VP2蛋白对PPV的嗜性和毒力有极大影响,是构成病毒粒子的主要衣壳蛋白,也是PPV特异性中和抗体的靶蛋白,可诱导机体产生中和抗体。本发明利用flashBAC ultra表达系统表达PPV的VP2蛋白,并添加分泌肽,生产的蛋白生产周期短、安全性高、免疫效果好。
发明内容
鉴于上述和/或现有一种表达猪细小病毒VP2蛋白的重组杆状病毒、制备方法及应用中存在的问题,提出了本发明。
因此,本发明的目的是提供了一种昆虫细胞嗜性优化的VP2蛋白,其核苷酸序列为SEQ ID No:1。
本发明还提供一种了添加分泌肽的VP2蛋白,其核苷酸序列为SEQ ID No:2。
本发明还提供一种了表达猪细小病毒VP2蛋白的重组杆状病毒,包括上述添加分泌肽的VP2蛋白,其保藏编号为:CCTCC No:V202178。
本发明还提供一种了表达猪细小病毒VP2蛋白的重组杆状病毒的制备方法,包括如下步骤:
S1:根据PPV-NADL-2株的序列,根据昆虫细胞嗜性优化合成携带6×His标签的VP2序列一,其核苷酸序列为SEQ ID No:1;
S2:设计引物,获得S1中优化合成的VP2序列一的上游和下游引物序列;
S3:进行PCR扩增并添加分泌肽和kozak序列,获得扩增后的VP2序列二,其核苷酸序列为SEQ ID No:2;
S4:将扩增后的VP2序列二与杆状病毒载体连接、转化,获得携带有VP2序列二的重组质粒;
S5:用重组质粒和杆状病毒DNA共转染昆虫细胞,拯救获得重组杆状病毒。
进一步地,S2中,
第一对引物为上游F1、下游R1;第二对引物为上游F2、下游R1;第三对引物为上游F3、下游R1;其中,上游F1的核苷酸序列为SEQ ID No:3、下游R1的核苷酸序列为SEQ ID No:4、上游F2的核苷酸序列为SEQ ID No:5、上游F3的核苷酸序列为SEQ ID No:6。
进一步地,S4具体为:
将目的片段和pVL1392载体分别进行BglⅡ、NotⅠ双酶切,回收清洁后连接VP2序列二和载体,转化TOP10感受态细胞,筛选阳性克隆,提取重组质粒。
进一步地,S5具体为:
利用脂质体转染法,将提取的重组制粒和flashBac-ultra DNA转染sf9细胞,28℃培养,120h收获细胞培养上清,拯救获得重组杆状病毒,保存于4℃。
本发明还提供了一种如上述的表达猪细小病毒VP2蛋白的重组杆状病毒在制备猪细小病毒疫苗中的应用。
本发明还提供了一种猪细小病毒VP2蛋白的重组杆状病毒疫苗,将上述的重组杆状病毒按照MOI为0.05感染昆虫细胞,感染120h后离心收获沉淀,破碎后上清经镍柱亲和层析纯化处理,抗原与Gel02佐剂配比4:1乳化制得疫苗。
与现有技术相比,本发明具有如下有益效果:
本发明将毒株的VP2基因克隆至载体pVL1392中,将重组质粒和flashBac-ultra杆状病毒基因共转染sf9细胞,获得表达VP2蛋白的重组杆状病毒。经纯化鉴定后,配制疫苗以2mL颈部皮下接种2月龄猪,三周后二免,接种产生的抗体效价优于商品化的猪细小病毒灭活疫苗。以上结果说明,猪细小病毒VP2蛋白重组杆状病毒疫苗能够诱导较强的免疫应答,能提供针对猪细小病毒的临床保护并抑制病毒在机体内的复制,其免疫原性与保护效力和商品疫苗相当。该方法制备VP2蛋白简便、生产周期短、免疫效力强。
生物保藏说明
重组昆虫核型多角体病毒AcMNPV-fPPV-1于2021年11月01日保藏于中国典型培养物保藏中心,地址为湖北省武汉市武昌区八一路299号武汉大学校内,保藏编号为CCTCCNO:V202178。
附图说明
图1为本发明的westernblot检测VP2蛋白表达分析图,图中各泳道为:M、Maker,1、AcMNPV-fPPV-1破碎后原液,2、AcMNPV-fPPV-1破碎后上清,3、AcMNPV-fPPV-1破碎后沉淀。
图2为本发明的PPVVP2 HI抗体水平检测对比图。
图3为本发明的HI效价检测对比图。
具体实施方式
下面结合具体实施例来进一步描述本发明,但实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1
1、重组载体的构建和获得
参照PPV-NADL-2株基因(KF049424.1)的VP2序列进行昆虫细胞嗜性优化,优化后的序列羧基端添加6×His标签为SEQ ID No:1,运用生物信息学软件设计引物,在VP2基因氨基端添加分泌肽和kozak序列。
上游引物F1(SEQ ID No:3):
GTGTACATTTCTTACATCTATGCGGCCACCATGTCCGAGAATGT;
F2(SEQ ID No:4):
TAGTCAACGTTGCCCTTGTTTTTATGGTCGTGTACATTTCTTACATC;
F3(SEQ ID No:5):
CCGAGATCTATGAAATTCTTAGTCAACGTTGCCCT(下划线部分为BglⅡ酶切位点);
下游引物R1(SEQ ID No:6):
GACGCGGCCGCCTAATGATGATGATGATGATGAT(下划线部分为NotⅠ酶切位点)。
扩增后的VP2序列(SEQ ID No:2)和pVL1392载体经BglⅡ、NotⅠ双酶切,回收后连接,转化TOP 10感受态细胞,筛选阳性克隆,提取去内毒素的重组质粒AcMNPV-fPPV-1,并进行测序鉴定。
2、获得重组杆状病毒
利用脂质体转染法,将提取的重组载体和flashBac-ultra DNA转染sf9细胞,28℃培养,120h收获细胞培养上清即可获得重组杆状病毒,保存于4℃。转染方法按照baculoFECTIN II(OXFORD EXPRESSION TECHNOLOGIES)说明书进行。经westernblot鉴定收获的上清和沉淀确定表达VP2蛋白。
3、VP2蛋白的表达和纯化
将收获的重组杆状病毒接种于悬浮培养的sf9细胞中,接毒量为0.05MOI,细胞密度为2×106/mL,体积为500mL。120h后收获离心,将沉淀超声破碎后取上清进行westernblot检测VP2蛋白表达,如图1所示。纯化采用常规镍株亲和层析法,取接毒后120h的细胞沉淀,超声裂解后10000rpm离心去除细胞碎片,取上清与镍柱进行结合过柱。洗杂缓冲液(50mM咪唑,20mM Tris,200mM NaCl)过柱洗杂;洗脱缓冲液(300mM咪唑,20mM Tris,200mM NaCl)过柱洗脱;洗脱液用透析缓冲液(20mM Tris,200mM NaCl)4℃透析过夜,得到目的蛋白,经westernblot检测纯化后的蛋白。
实施例2
1、猪细小病毒VP2蛋白疫苗的制备
将实施例1中纯化得到的VP2蛋白测定浓度后过滤,抗原与Gel02佐剂配比4:1乳化制得疫苗,每毫升疫苗中VP2抗原含量为20μg。
2、猪细小病毒VP2蛋白疫苗的安全性评价
16-18g雌性Balb/C小鼠10只,分为A、B两组,每组5只。A组每只小鼠皮下注射0.3mL制得的VP2蛋白疫苗;B组每只小鼠注射0.3ml透析缓冲液(20mM Tris,200mM NaCl);连续观察14天,两组小鼠状态相同且均无异常反应,此结果表明该疫苗对小鼠是安全的。
约2月龄PPV阴性猪6头,随机分成A、B两组,每组3头。A组每头猪免疫1头份,颈部肌肉注射2ml制得的VP2蛋白疫苗,3周后进行第二次免疫;B组不免疫,作为阴性对照。连续观察至第二次免疫后28日,观察期内免疫猪健康状况良好,与非免疫猪一致,未出现因疫苗注射引起的任何局部或全身不良反应,此结果表明该疫苗对本体动物猪是安全的。
3.猪细小病毒VP2蛋白亚单位疫苗有效性评价
(1)猪细小病毒VP2蛋白亚单位疫苗在小鼠体内进行有效性评价
购买6-8周龄的雌性Balb/C小鼠20只,随机分成A、B两组,每组10只。A组每只小鼠皮下注射0.2ml,2周后加强免疫一次,B组不免疫。分别在首免2周,二免2周,二免4周采血分离血清进行血凝效价测定,PPVVP2HI抗体水平检测见图2。
(2)猪细小病毒VP2蛋白疫苗在猪体内进行有效性评价
为了进一步评价上述制备二联亚单位疫苗对本体动物猪的免疫效力,选购约2月龄PPV阴性猪16头,随机分成4组,每组4头。第1组每头猪免疫1头份(2mL)猪细小病毒VP2亚单位疫苗,第2组每头猪免疫1头份PPV灭活疫苗(由武汉科前-科细宁-猪细小病毒灭活疫苗WH-1株制得,可市售购得),均颈部肌肉注射,3周后进行第二次免疫;B组不免疫,作为阴性对照。首免后7天14天,28天和42天进行前腔静脉采血,进行HI效价检测。检测结果见图3。结果表明该疫苗具有良好的免疫原性,并且本发明的猪细小病毒VP2疫苗免疫后产生的抗体水平优于PPV灭活疫苗的免疫效果。
虽然在上文中已经参考实施方式对本发明进行了描述,然而在不脱离本发明的范围的情况下,可以对其进行各种改进并且可以用等效物替换其中的部件。尤其是,只要不存在结构冲突,本发明所披露的实施方式中的各项特征均可通过任意方式相互结合起来使用,在本说明书中未对这些组合的情况进行穷举性的描述仅仅是出于省略篇幅和节约资源的考虑。因此,本发明并不局限于文中公开的特定实施方式,而是包括落入权利要求的范围内的所有技术方案。
序列表
<110> 江苏南农高科技股份有限公司
<120> 一种表达猪细小病毒VP2蛋白的重组杆状病毒、制备方法及应用
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gacatcatgt tttatacaat tgaaaacgct gttccaatac atctcctccg cactggcgac 780
gagttctcga cgggtatcta ccatttcgac acgaaacccc tcaaacttac acacagttgg 840
cagacgaacc gcagcttggg attacctcca aaactactta ctgaaccaac cactgaaggg 900
gatcaacacc caggtaccct accggcagca aatacgcgga agggctatca ccaaacgatt 960
aacaactcat acacagaagc gaccgcgatc cgtcccgcac aggtaggcta caatacccct 1020
tatatgaatt ttgaatactc caacggtggg ccattcctaa cgcccattgt accgaccgct 1080
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ggcgtaggag tgagcacagg aacgttcaac aaccagacgg agtttcagta tctaggtgag 240
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ttgaaccccc ccacatacac tggtcagagc caacagatta ccgactcaat ccagaccggg 780
ctccatagtg acatcatgtt ttatacaatt gaaaacgctg ttccaataca tctcctccgc 840
actggcgacg agttctcgac gggtatctac catttcgaca cgaaacccct caaacttaca 900
cacagttggc agacgaaccg cagcttggga ttacctccaa aactacttac tgaaccaacc 960
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Claims (2)
1.一种猪细小病毒VP2蛋白的重组杆状病毒疫苗,其特征在于,将保藏编号为CCTCCNo:V202178的重组杆状病毒按照MOI为0.05感染昆虫细胞,感染120h后离心收获沉淀,破碎后上清经镍柱亲和层析纯化处理,抗原与Gel02佐剂配比4:1乳化制得疫苗;
所述保藏编号为CCTCC No:V202178的重组杆状病毒的制备方法包括如下步骤:
S1:根据PPV-NADL-2株的核酸,根据昆虫细胞嗜性优化合成携带6×His标签的VP2核酸一,其核苷酸序列为SEQ ID No:1;
S2:设计引物,获得S1中优化合成的VP2核酸一的上游和下游引物序列;
S3:进行PCR扩增并添加分泌肽和kozak序列,获得扩增后的VP2核酸二,其核苷酸序列为SEQ ID No:2;
S4:将扩增后的VP2核酸二与杆状病毒载体连接、转化,获得携带有VP2核酸二的重组质粒;
S5:用重组质粒和杆状病毒DNA共转染昆虫细胞,获得重组杆状病毒;
S4具体为:将目的片段和pVL1392载体分别进行BglⅡ、NotⅠ双酶切,回收清洁后连接VP2序列二和载体,转化TOP10感受态细胞,筛选阳性克隆,提取重组质粒;
S5具体为:利用脂质体转染法,将提取的重组质粒和flashBac-ultra DNA转染sf9细胞,28℃培养,120h收获细胞培养上清,获得重组杆状病毒,保存于4℃。
2.根据权利要求1所述的重组杆状病毒疫苗,其特征在于,S2中,
第一对引物为上游F1、下游R1;第二对引物为上游F2、下游R1;第三对引物为上游F3、下游R1;其中,上游F1的核苷酸序列为SEQ ID No:3、下游R1的核苷酸序列为SEQ ID No:4、上游F2的核苷酸序列为SEQ ID No:5、上游F3的核苷酸序列为SEQ ID No:6。
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