CN102382845B - 猪细小病毒抗原的制备方法及其产品 - Google Patents
猪细小病毒抗原的制备方法及其产品 Download PDFInfo
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- CN102382845B CN102382845B CN201110310761.3A CN201110310761A CN102382845B CN 102382845 B CN102382845 B CN 102382845B CN 201110310761 A CN201110310761 A CN 201110310761A CN 102382845 B CN102382845 B CN 102382845B
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Abstract
本发明公开了一种猪细小病毒空衣壳抗原的制备方法及其产品。本发明方法包括:将猪细小病毒衣壳蛋白VP2基因或优化后的VP2基因克隆到杆状病毒运载载体中,构建得到转移表达载体;将所构建的转移表达载体与杆状病毒DNA进行共转染,获得重组杆状病毒;将重组杆状病毒感染昆虫宿主或细胞;培养被感染的昆虫宿主表达相应的猪细小病毒空衣壳蛋白;收获并纯化所表达的抗原,即得。本发明方法采用杆状病毒表达系统在家蚕生物反应器中安全、高效的生产猪细小病毒抗原空衣壳颗粒,所制备的抗原纯化后,安全性极高,可直接制备成疫苗免疫动物。本发明抗原制备方法具有表达效率高、所表达的抗原免疫活性高、生产成本低、可实现规模化生产等优点。
Description
技术领域
本发明涉及一种猪细小病毒(Porcine parvovirus,PPV)抗原的制备方法,尤其涉及一种利用重组杆状病毒在昆虫体内表达猪细小病毒空衣壳抗原的方法,本发明还涉及由该方法制备得到的抗原产品以及用该抗原制备预防或治疗猪细小病毒的口服或注射用疫苗的用途,属于猪细小病毒抗原的制备领域。
背景技术
猪细小病毒(Porcine parvovirus,PPV)由Mary和Mahnel于1966年进行猪瘟病毒组织培养时发现,由Cartwright首次分离得到,随后在欧、美、亚、非及大洋洲很多国家均有报道,呈世界性分布。PPV是引起母猪繁殖障碍的重要病原之一,也可以引起胎儿木乃伊、新生仔猪死亡以及仔猪的皮炎和腹泻等,是危害全球养猪业重要病原之一。近年来,PPV的感染呈扩大上升趋势,给全球养猪业带来了巨大的经济损失(刘秀芬,中国兽医杂志,2010,46(5):9-11;谢巧等,畜牧与兽医,2010,42(2):92-95)。
猪细小病毒属于细小病毒科细小病毒属成员,其病毒粒子呈圆形或六角形,直径约20nm,二十面体立体对称,衣壳由32个壳粒组成,无囊膜,是一种自主复制型病毒(甄洪花等,动物医学进展,2009,20(8):85-88)。猪细小病毒能够凝集人、猴、豚、鼠、猪、鸡、大鼠、小鼠的红细胞,但血凝试验时常用豚鼠的红细胞(孙绍元等,中国预防兽医学报,2000,22:214-215)。PPV基因组为单股负链DNA,大小约为5000bp,基因组有两个主要的开放阅读框,共编码VP1、VP2、VP3三种结构蛋白和NS1、NS2、NS3三种非结构蛋白。VP2的C端暴露在该蛋白的表面,因此C端的完整性是保持该衣壳蛋白二级结构所必须的,而其N端位于二级结构的内部,可以利用VP2自我包装成空壳粒子的特性。结构蛋白VP1、VP2和VP3都有免疫原性,其中VP2的免疫原性最好,VP1最差(甄洪花等,动物医学进展,2009,20(8):85-88)。
目前用于猪细小病毒病的疫苗主要有弱毒活疫苗和灭活疫苗两种,但是这两种疫苗均有其缺陷或不足,譬如,弱毒疫苗存在发生重组及毒力反强的潜在威胁,灭活疫苗免疫存在效果不稳定等缺陷(周斌等,畜牧与兽医,2007,39(2)54-56)。任雪枫等将VP2蛋白主要抗原表位基因VP2 I基因插入真核表达载体pIREShyg转CHO细胞经潮霉素筛选,所有得到的阳性克隆经间接免疫荧光鉴定均表达目的基因,建立了稳定表达VP2蛋白的细胞株,可在一定程度上取代全病毒作为抗原应用,给PPV的研究带来较多方便,为探索新的血清学诊断方法奠定了基础(任雪枫等,中国病毒学,2004,19(6):636-638)。猪细小病毒VP2蛋白主要抗原域在毕氏酵母中表达并且具有免疫活性,但是表达量很低(李斐等,农业生物技术学报,2006,14(6):988-989)。将猪细小病毒VP2基因插入干酪乳杆菌细胞表面表达载体pPG611.1中构建了重组表达载体,重组蛋白Lc393-rPPV-VP2在干酪乳酸菌表面获得表达,经口服免疫Balb/c小鼠后能够诱导小鼠产生针对PPV的局部黏膜免疫应答和全身免疫应答,为进行口服疫苗有效预防PPV的研究奠定了基础(Xu Y G etal,Appl Environ Microbiol,2007,73(21):7041-7047)。之后,又构建了另一种表达载体分泌型重组表达载体pPG612.1,将这两种不同的重组菌以口服方式免疫小鼠比较发现,两种重组菌免疫的小鼠均能产生针对VP2的特异性免疫应答,口服免疫46d后IgA和IgG达到最高水平(Xu Y Getal,Immunology,2008,124(1):68-75)。
随着生物技术、基因工程等高新技术的发展,人们意识到通过基因工程的手段,利用生物反应器来高效表达猪细小病毒基因,可望达到大幅度提高猪细小病毒抗原产量、降低生产成本的目的。动物疾病的免疫疫苗和治疗药物的研究和开发随着家蚕生物反应器的成熟也越来越受到重视。如口蹄疫病毒的空衣壳表达、狂犬病的N、P、N-P的联合表达均已获得了中国农业部遗传工程安全生产委员会颁发的商品化生产许可证。1997年,田鄂等在家蚕细胞和幼虫中成功表达了日本血吸虫中国大陆株28KD谷胱甘肽S-转移酶基因,在家蚕培养细胞(106个/mL)中的表达产量为0.77mg,在家蚕幼虫中则超过5mg/条蚕(田鄂等,生物化学与生物物理学报,1997,29(1):33-38)。日本国家动物卫生研究所的研究人员利用家蚕表达体系对猪的细胞因子,包括IL系列、GM-CSF、TNF及其受体、Caspase等进行了系统深入的基础和开发研究,用BmNPV体系生产的宠物(猫和狗)的预防重组IL制剂(Intercat)最近已由日本“TORAY”株式会社生产并投入市场(木村滋,昆虫生物工厂[M],日本:工业调查会,2000,124-127)。
现有的采用基因工程的手段制备猪细小病毒抗原的方法不同程度的存在着表达效率较低、生产成本高、所表达的抗原免疫活性低等缺陷,有待改进。
发明内容
本发明所要解决的技术问题是克服现有技术的不足,提供一种新的猪细小病毒空衣壳抗原的制备方法,该制备方法利用重组杆状病毒在昆虫体内表达猪细小病毒空衣壳抗原,具有表达效率高、所表达的蛋白免疫活性高、生产成本低、可实现规模化生产等优点。
本发明所要解决的技术问题是通过以下技术方案来实现的:
一种猪细小病毒空衣壳抗原的制备方法,包括:将猪细小病毒空衣壳蛋白VP2基因或优化后的VP2基因克隆到杆状病毒运载载体中,构建得到转移表达载体;将所构建的转移表达载体与杆状病毒DNA进行共转染,获得重组杆状病毒;用重组杆状病毒感染昆虫宿主或细胞;培养被感染的昆虫宿主或昆虫细胞表达重组猪细小病毒空衣壳蛋白;收获并纯化所表达的重组猪细小病毒空衣壳蛋白,即得。
其中,所述的猪细小病毒空衣壳蛋白基因VP2的碱基为SEQ ID NO:1所示,所述优化后的猪细小病毒空衣壳蛋白VP2基因的碱基为SEQ ID NO:2所示。
所述的杆状病毒运载载体优选自AcRP23-lacZ,AcRP6-SC,AcUWl-lacZ,BacPAK6,Bac to Pac,Bacmid,BlucBacII(pETL),p2Bac,p2Blue,p89B310,pAc360、pAc373,pAcAB3、pAcAB 4,PAcAS3,pAcC129,pAcC4,DZI,pAcGP67,pAcIEl,pAcJPl,pAcMLF2,pAcMLF 7,pAcMLF 8,pAcMP1,pAcMP2,pAcRP23,pAcRP 25,pAcRW4,pAcsMAG,pAcUW1,pAcUW21,pAcUW2A,pAcUW2B,pAcUW3,pAcUW31,pAcUW41,pAcUW42,pAcUW43,pAcUW51,pAcVC2,pAcVC 3,pAcYMl,pAcJcC5,pBac1、pBac2,pBlueBacIII,pBlueBacHis,pEV55,mXIV,pIEINeo,pJVETL,pJVNhel,pJVP10,pJVrsMAG,pMBac,pP10,pPAKl,pPBac,pSHONEX 1.1,pSYN XIV VI+,pSYNVI+wp,pSYNXIV VI-,pVL1391,pVL 1392,pVL 1393,pVL941,pVL 945,pVL985,pVTBac,pBM030,pUAC-5或其它类似的杆状病毒同源重组或转座载体中的任一种,更优选为pVL1393。
所述的杆状病毒优选自BmNPV、AcMNPV、ApNPV、HaNPV、HzNPV、LdMNPV、MbMNPV、OpMNPV、S1MNPV、SeMNPV或SpltNPV,更优选为家蚕杆状病毒亲本株Bm-NPV-ZJ8。
所述的重组杆状病毒选自以下任意一种重组病毒:(1)重组家蚕核型多角体病毒rBmNPV(PPV-VP2),(2)重组家蚕核型多角体病毒rBmNPV(PPV-VP2-M)。
所述的昆虫宿主选自家蚕(Bombyx mori)、野蚕(Bombyx mandarina)、蓖麻蚕(Philosamia cynthia ricim)、樟蚕(Dictyoplocajapanica)、樗蚕(Philosamia cynthiapryeri)、柞蚕(Antheraea pernyi)、日本柞蚕(Antheraea yamamai)、野天蚕(Antheraeapolyphymus)、苜蓿尺蠖(Atographa califorica)、茶尺蠖(Ectropis obliqua)、甘兰夜蛾(Mamestra brassicae)、斜纹夜蛾(Spodoptera littoralis)、秋粘虫(Spodopterafrugiperda)、粉纹夜蛾(Trichoplusia ni)、行军虫(Thaumetopoea wilkinsoni)、棉铃虫(Heliothis armigera)、美国棉铃虫(Heliothis zea)、烟青虫(Heliothis assulta)、烟草夜蛾(Heliothis virescens)、东方粘虫(Pseudaletia separata)、舞毒蛾(Lymantriadispar)等;更优选为家蚕(Bombyx mori)。
所述的感染是指重组杆状病毒通过口食或透过表皮来感染1-5龄的昆虫幼虫或蛹体(更优选为:将重组家蚕杆状病毒感染家蚕细胞或将重组家蚕杆状病毒穿刺接种1-5龄的家蚕幼虫或蛹,在感染3-6天后收集含猪细小病毒抗原的家蚕幼虫或蛹的体液或组织匀浆);其中,所述的蛹体最优为1-2天的早期嫩蛹。
本发明在不改变猪细小病毒空衣壳蛋白VP2的氨基酸序列前提下,根据家蚕密码子偏好性对猪细小病毒空衣壳蛋白基因原始序列VP2(SEQ ID NO:1)进行优化,优化后的序列(VP2-M)如SEQ ID NO:2所示。VP2原始序列中含有串联的稀有密码子,这减少了翻译序列或者甚至解除翻译装置,优化后的序列中CAI值由0.86提高为0.89,调整了GC含量及不宜峰以延长mRNA的半衰期,GC含量由38.32%调整为48.70%,原有的影响mRNA稳定性及其与核糖体结合的茎环结构均被破坏。本发明将优化后的猪细小病毒VP2-M基因在家蚕蛹体中进行表达,根据表达结果可见,PPV-VP2-M基因在蚕血或蚕蛹中的表达量高,比猪细小病毒空衣壳蛋白基因原始序列(PPV-VP2)在家蚕中表达量高出了2-3倍,达到每克蛹体2-3毫克,在12800倍稀释甚至更高稀释度下仍能检测到所表达的PPV-VP2-M抗原与抗体的特异性反应。血凝实验检测表明,PPV-VP2-M在家蚕中所表达的含病毒蚕血稀释到6.4×104倍时为仍为阳性。
本发明采用基因重组技术将猪细小病毒空衣壳蛋白的原始序列(SEQ ID NO:1)或将原始序列优化后的序列(SEQ ID NO:2)克隆到杆状病毒运载载体上,在多角体启动子、p10启动子或别的病毒和真核生物的强启动子控制之下,通过体内或体外(invivo/in vitro)重组,使PPV空衣壳蛋白的原始序列或优化后的序列整合到杆状病毒的基因组上,得到重组病毒;重组病毒可通过经口食下或采用各种手段透过表皮感染1-5龄(最优时间为四或五龄)的昆虫幼虫或蛹体(最优时间为1-2天的早期嫩蛹),表达生产猪细小病毒空衣壳抗原。
本发明的最优选的一个整体技术方案如下:将空衣壳蛋白的原始序列VP2(SEQID NO:1所示)或优化后的序列VP2-M(SEQ ID NO:2所示)分别克隆到杆状病毒运载载体pVL1393上,再通过体内重组将猪细小病毒衣壳蛋白基因的原始序列VP2或密码子优化后的序列VP2-M分别转移到家蚕杆状病毒亲本株Bm-NPV-ZJ8的基因组上,替代基因组上的Polyhedrin基因,通过空斑筛选技术和PCR检测技术,分别获得携带猪细小病毒衣壳蛋白基因VP2的重组家蚕杆状病毒rBmNPV(PPV-VP2)以及rBmNPV(PPV-VP2-M);再将所获的重组家蚕杆状病毒感染家蚕细胞系或穿刺接种1-5龄的家蚕幼虫或蛹,大量繁殖rBmNPV(PPV-VP2)、rBmNPV(PPV-VP2-M);当rBmNPV(PPV-VP2)、rBmNPV(PPV-VP2-M)在蚕体内复制时,VP2及VP2-M基因在多角体蛋白基因(polh)启动子控制下表达,并自我组装成为猪细小病毒空衣壳抗原;在感染3-6天(最佳为5天,25℃饲养或保护温度)后收集含猪细小病毒空衣壳抗原的家蚕幼虫或蛹的体液(或整体匀浆),经过辐射照射杀灭杆状病毒、将蛋白纯化后便得到安全、高效的猪细小病毒空衣壳抗原,此抗原可直接用于制备预防猪细小病毒病的注射用或口服疫苗。
本发明还提供了一种用于预防或治疗猪细小病毒病的疫苗,该疫苗由有效量的本发明方法所制备的猪细小病毒空衣壳抗原与药学上可接受的载体或辅料组成;其中,所述的载体或辅料可以是各种常用的佐剂、稀释剂或表面活性剂等;可以按照疫苗制剂中常用的方法将所述的疫苗制备成口服或注射用疫苗,这些制备方法均为本领域技术人员所习知。
本发明方法采用杆状病毒表达系统在家蚕生物反应器中安全、高效的生产猪细小病毒抗原空衣壳颗粒,其生产成本显著低于传统的制备猪细小病毒抗原方法(例如通过细胞繁殖病毒制备猪细小病毒抗原),无需投资建厂,无三废,电力和水资源等能源消耗极少。由于家蚕已经我国卫生部批准为食药兼用昆虫,所以将本发明方法所制备的抗原纯化后,安全性极高,可直接制作疫苗免疫动物。本发明方法可以大幅度降低猪细小病毒抗原空衣壳颗粒的生产成本,具有表达效率高、所表达的蛋白免疫活性高、生产成本低、可实现规模化生产等优点。
附图说明
图1PPV血凝试验结果(稀释倍数为:103);A:VP2;B:VP2-M;C:阴性蚕血。
图2本发明方法所表达的猪细小病毒空衣壳粒子的电镜观察结果。
图3免疫电镜观察所表达的猪细小病毒空衣壳粒子结果(样品40X稀释)。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
试验材料
1.大肠杆菌株E.coli DH5α购自Promega公司;克隆载体pEASY-T3购自全式金公司;克隆载体pMD18-T购自TaKaRa公司;原核表达载体pET-28a+、受体菌E.coliTOP10和BL21(DE3)均为中国农业科学院生物技术研究所分子微生物实验室保存;运载载体pVL1393购自于Invitrogen公司;猪细小病毒衣壳蛋白基因的原始序列VP2是自发病动物病变组织中提取并克隆到载体上得到;密码子优化后的序列VP2-M直接生物合成;家蚕细胞BmN、家蚕核型多角体病毒亲本株Bm-NPV-ZJ8由中国农业科学院生物技术研究所分子微生物实验室保存(也可向中国科学院武汉病毒研究所或中国农业科学院蚕业研究所购买获得);高表达家蚕品种JY1由中国农业科学院生物技术研究所保存(也可向中国科学院武汉病毒研究所或中国农业科学院蚕业研究所购买获得)。
2.酶与试剂:所用限制性内切酶和配套的缓冲液均购自Promega公司。T4DNALigase及缓冲液为Promega公司产品。LA Taq聚合酶及缓冲液购自TaKaRa公司。RnaseA、dNTPs购自Sigma公司。各种规格的DNA和蛋白质分子量标准为TranGenBiotech公司产品。
3.生化试剂:bisacrylamide,acrylamide、IPTG、X-Gal购自Promega公司;Tris、Ampicillin、Kanamycin、IPTG、SDS、尿素、咪唑、TritonX-100、TEMED(N,N,N′,N′-Tetramethylethylene diamine)、过硫酸铵(Ammonium Persulfate)、卡那霉素(Kanamycin)购自Sigma公司,细胞培养基TC-100购自GIBCO公司。琼脂糖为Sunbiotech公司产品;酵母抽提物(Yeast Extract)、胰蛋白胨均购自英国OXOID公司;0.2um、0.45um滤器购自GelmanSciences公司;溴化乙锭(EB)、考马斯亮兰R-250购自Fluka公司。琼脂粉为日本进口分装。Ni-NTA树脂、Proteinase K和胎牛血清购自Invitrogen公司。
4.培养基:大肠杆菌培养基为LB(1%蛋白胨、0.5%酵母提取物、1%NaCl,pH7.0);家蚕细胞培养基为TC-100。
实施例1猪细小病毒抗原的制备、纯化及动物免疫实验及病毒攻击保护实验
1.有关溶液和培养基的配制
溶液I:50mmol/L葡萄糖,25mmol/L Tris-HCl(pH8.0),10mmol/L EDTA。
溶液II:0.2mol/L NaOH,1%SDS(现配现用)。
溶液III:100mL体系,5mol/L醋酸钾80mL,冰乙酸12mL,ddH208mL。TAE(50×):242gTris碱,57.1mL冰乙酸,100mL 0.5mol/L EDTA(pH8.0),无菌水容至1000mL。
TER溶液:胰RNAse(RNAse A)溶解于10mM Tris-HCl、15mMNaCl中,配成10mg/mL的储存液-20℃冻存,用1×TE buffer稀释成20μg/mL的工作液4℃保存。
PPt Buffer:异丙醇22mL;5mol/mL KAc 1mL;ddH2O2mL。
1×TE buffer:10mmol/L Tris·Cl(pH8.0),1mmol/L EDTA(pH8.0),121℃高温高压蒸汽灭菌20min后储存于4℃。
溴化乙锭(EB)溶液母液:将EB配成浓度为10mg/mL,用铝箔或黑纸包裹容器储存于室温即可。
6mol/L NaI:将0.75g Na2SO3溶于40mL ddH2O中,加入45gNaI并搅拌至完全溶解,4℃储存。
玻璃奶(Glassmilk):将10g(100mg/mL,Sigma S-5631)的Silica溶于100mL PBS中,沉淀2h,弃上清,重复该步骤2~3次;2000g离心2min,将沉淀物溶于3mol/L的NaI中,终浓度为100mg/mL,4℃下避光保存。
New Wash洗液:Tris-HCl(pH 7.4)20mmol/L;EDTA 1mmol/L;NaCl 100mmol/L;与等体积的无水乙醇配制而成。
蛋白表达诱导物IPTG为1mol/L,超纯水配制后用0.2mm滤器过滤除菌。
蛋白上样缓冲液(2×):100mmol/L Tris-HCl(pH6.8)、200mmol/L二硫苏糖醇(DTT)、4%SDS、0.2%溴酚蓝、10%甘油。
30%丙烯酰胺溶液29g:丙烯酰胺,1gN,N′-亚甲叉丙烯酰胺,溶于100mL水,过滤。
考马斯亮蓝染液:0.24g考马斯亮蓝R250溶于90mL甲醇∶水(1∶1,v/v)和10mL冰乙酸中。
脱色液:90mL甲醇∶水(1∶1,v/v)和10mL冰乙酸。
裂解缓冲液(pH8.0):50mmol/L Tris-Base、0.1M NaCl。
包涵体洗涤液I(pH8.0):20mmol/L Tris-Base、0.2M NaCl、1%TritonX-100。
包涵体洗涤液II(pH8.0):20mmol/L Tris-Base、0.2M NaCl、2M尿素
包涵体蛋白溶解液(pH8.0):20mmol/L Tris-Base、0.2M NaCl、8M尿素
脲NTA-0Buffer:20mM Tris-HCl pH7.9,0.5MNaCl,10%Glycerol,8M Urea;
脲NTA-500Buffer:20mM Tris-HCl pH7.9,0.5MNaCl,10%Glycerol,8M Urea0.5M Imidazole。
Bradford试剂为Bio-Rad公司Protein Assay Dye-Reagent Concentrate。
ELISA所需试剂:包被液(pH9.6)1.59g Na2CO3、2.93g NaHCO3、ddH2O定容至1L,保存于4℃;临用前配制封闭液,BSA溶于PBS中至终浓度为1%;洗涤液为含0.05%Tween-20的PBS;底物缓冲液为1.84g Na2PO4·12H2O、0.51g柠檬酸、ddH2O定容至100mL;OPD显色液是4mg OPD溶于10mL底物缓冲液,加15μL 30%H2O2,临用前配制;终止液为2mol/L H2SO4。
Western blotting试剂:半干转电转膜缓冲液是14.41g甘氨酸、12.11g Tris-Base、50ml甲醇、ddH2O定容至1L、4℃保存;1×PBS加Tween-20至终浓度为0.1%配成洗涤液;BSA溶于PBS中至终浓度为3%为封闭液;纯化的多克隆抗体用封闭液按1∶1000稀释;HRP标记的羊抗兔IgG抗体用封闭液按1∶1000稀释;临用前配制DAB显色液,4mg DAB溶于10mL 100mmol/L pH7.5的Tris-Cl中,加15μL 30%H2O2。
LB培养基:胰蛋白胨10g/L,酵母粉5g/L,氯化钠10g/L,调整pH值到7.0(固体培养基含1.5%琼脂)。液体培养基中加入1.5%琼脂粉,121℃高温高压蒸汽灭菌15min,在温度低于45℃且还未凝固时,加入相应抗生素溶液,混匀后倒入平板,为LB固体培养基4℃保存备用。
2猪细小病毒空衣壳蛋白基因的原始序列VP2的获得和载体构建
2.1病毒基因组的提取
2.1.1取发病样品(猪流产胎儿)1克左右磨碎,反复冻融3次后,取5000rpm离心10分钟后的上清液,加入等体积的裂解缓冲液(20mmol Tris,20mmolEDTA,1%SDS),然后加入蛋白酶K至终浓度为200μg/ml,56℃水浴30min。
2.1.2用等体积的Tris-cl饱和酚、酚/氯仿(1∶1)、氯仿各抽提一次,12000rpm,离心5min。
2.1.3取上清加入1/10体积的3mol/L醋酸钠(pH5.2),2倍体积的无水乙醇,-20℃放置30min,12000rpm,10min,弃上清,沉淀用70%的乙醇洗涤1次,自然干燥后,加入50μl TER(含RNase)溶解。
2.2设计引物,通过PCR的方法扩增出猪细小病毒VP2基因。
所设计的VP2基因扩增引物为:
VP2上游:5’-GATAGGATCCATGAGTGAAAATGTGGAACAAC3’,
BamH I酶切位点
VP2下游:5’-CGCTCTAGACTAGTATAATTTTCTTGGTATAAG-3’。
Xba I酶切位点
PCR反应体系如下:
表1PCR反应条件
PCR反应过程:94℃变性10分钟;94℃1分钟,58℃1分钟,72℃8分钟,共30个循环。最后延伸反应10分钟。
2.3PCR产物的纯化
将PCR扩增的VP2基因产物进行1%琼脂糖凝胶电泳,发现扩增出约1.7kb的片段。在紫外灯下用灭菌手术刀切取含相应DNA片段的凝胶,然后用Geneclean试剂盒进行纯化。方法如下:切取凝胶片段并称重,将其放入灭菌的1.5ml小离心管中,加入3倍(v/w)体积的6M NaI,37℃将凝胶溶解后,加入10μl玻璃奶(Glass milk),混匀后室温下放置5分钟,使DNA充分吸附在玻璃奶上,12000rpm离心5秒钟,再用New Wash溶液洗三次,每次均将沉淀弹起,并离心。最后将沉淀晾干后加入30μl0.1×TE Buffer溶解DNA,离心后去沉淀,取上清作进一步分析。
2.4片段与克隆载体的连接
回收的PCR产物2.5μl、pMD18-T 0.5μl和soul I 3μl混匀后,16℃反应2h。
2.5大肠杆菌的遗传转化
用75mM CaCl2制备大肠杆菌TG1感受态细胞。取制备的连接混合物5μl,加到200μl感受态细胞中,轻轻混匀,冰浴30分钟,42℃热激2分钟,迅速置于冰上2~3分钟,加入已温育至37℃的LB培养基500μl,37℃培养1小时,取100~200μl涂布于含100μg/ml氨苄青霉素(Amp)的LB固体培养基平板上,37℃倒置培养过夜。
2.6质粒DNA的制备
(1)从转化的LB平板上挑取单个菌落,接种于3ml含100μg/ml Amp的LB培养基中,37℃培养过夜。
(2)取1.5ml菌液于小离心管中,3500rpm离心4min,去上清。
(3)加入Solution I 150μl,混匀后置于冰上15min。
(4)加入Solution II 300μl,氯仿150μl,轻轻混匀后静置5min。
(5)加入Solution III 450μl,混匀后置于冰上15min。
(6)12000g离心10min,上清移入新管。
(7)加入异丙醇450μl,混匀后置于4℃15min。
(8)12000g离心6min,去上清。
(9)加入TER 250μl,混匀后置于37℃20min。
(10)加入PPt Buffer 300~350μl,混匀后静置15min。
(11)12000g离心6min,去上清。
(12)加入75%乙醇400μl。
(13)12000g离心3min,倒掉乙醇,抽干后加入0.1×TE Buffer 40μl溶解,于-20℃保存。
2.7重组子的鉴定
用BamHI/Xba I双酶切2.6中制备的质粒DNA,电泳后出现约2.6kb、1.7kb的DNA条带的质粒为阳性的T-VP2。将鉴定为阳性克隆(全长基因分别命名为T-VP2)的菌种重新加入含有Amp的LB培养液,220r/min摇培过夜后,吸取1mL新鲜菌液至无菌的Eppendorf管中,加入少量甘油,以封口膜封好后进行测序,结果如SEQ ID NO:1所示,氨基酸序列如SEQ ID NO:3所示。测序结果用DNAStar、DNAMAN软件对序列进行分析。
2.8酶切与连接反应
酶切反应:T-VP2用BamHI和Xba I双酶切分析,反应总体积为50μl,质粒10μl,10×酶相应缓冲液5μl,两种酶各为1μl,无菌水补足体积。37℃反应2小时以上。将转移质粒pVL1393作同样酶切反应。反应结束后于65℃灭活10分钟。
连接反应:连接总体积10μl,酶切回收产物7μl,载体1μl,5×T4DNA连接缓冲液1μl,T4DNA连接酶1μl,无菌水补足体积,16℃连接过夜。
2.9酶切产物的回收
将T-VP2用BamHI和Xba I双酶切产物进行1%琼脂糖凝胶电泳,回收1.7kb大小的片段。在紫外灯下用灭菌手术刀切取含相应DNA片段的凝胶,然后用Geneclean试剂盒进行纯化。方法如下:切取凝胶片段并称重,将其放入灭菌的1.5ml小离心管中,加入3倍(v/w)体积的6M NaI,37℃将凝胶溶解后,加入10μl玻璃奶(Glassmilk),混匀后室温下放置5分钟,使DNA充分吸附在玻璃奶上,12000rpm离心5秒钟,再用New Wash溶液洗三次,每次均将沉淀弹起,并离心。最后将沉淀晾干后加入30μl 0.1×TE Buffer溶解DNA,离心后去沉淀,取上清作进一步分析。
2.10质粒的转化与鉴定。
参照2.5相关方法将连接产物转化感受态细胞,在含氨苄青霉素抗性的琼脂平板上筛选,挑选克隆后,提取质粒。重组质粒经BamHI和Xba I双酶切、菌液PCR鉴定和基因测序,鉴定正确的重组质粒命名为pVL1393-PPV-VP2。
3家蚕核多角体病毒亲本株Bm-NPV-ZJ8的繁殖及病毒DNA的制备
按GIBCO公司产品说明配制1×TC-100培养基,用2N NaOH将pH调至6.22,过滤除菌后的培养基补加10%胎牛血清,27℃下培养家蚕细胞BmN。用家蚕核多角体病毒亲本株Bm-NPV-ZJ8感染对数生长期的细胞约50ml,感染复数为1,3~4天后收集病毒感染液,离心(5000rpm×10min),除去沉淀,上清用25000rpm离心1小时,除上清,用1ml病毒DNA抽提液(1000ml中含Tris 12.1g,EDTA 33.6g,KCl 14.1g,pH7.5)悬浮病毒粒子沉淀,转移至1.5ml离心管中,加入蛋白酶K至终浓度为50μg/ml,50℃保温2小时,再加入35%的Sarkorsel至终浓度为1%,继续于50℃保温2小时,分别用等体积的苯酚、苯酚∶氯仿(1∶1)氯仿依次抽提,将上层水相转移到一个新管中,加入1/10体积的3MNaCl,再加入2倍体积的无水乙醇,-20℃放置2小时以上沉淀病毒DNA,5000rpm离心10分钟,沉淀用75%乙醇洗一次,冷冻干燥。溶解在100μl TE Buffer中,放4℃保存备用。
4重组家蚕杆状病毒rBmNPV(PPV-VP2)的构建和获得
4.1重组杆状病毒rBmNPV(PPV-VP2)的构建
接种大约1×106细胞于15cm2培养瓶中,细胞贴壁后,除去含胎牛血清(FBS)培养基,用不含FBS的培养基洗三次,加1.5ml无FBS培养基。向一灭菌管中依次加入1μg家蚕杆状病毒亲本株Bm-NPV-ZJ8 DNA,2μg重组转移质粒pVL1393-PPV-VP2的DNA和5μl脂质体,用无菌双蒸水补足体积到60μl,轻轻混匀,静置15分钟后,逐滴加入到培养瓶中进行共转染。27℃培养4小时后补加1.5ml无血清培养基和300μl FBS。27℃恒温培养4~5天,收集上清液用于重组病毒的筛选。
4.2重组家蚕杆状病毒rBmNPV(PPV-VP2)的筛选和纯化
接种适量细胞(约70~80%)于35mm小平皿中,细胞贴壁后,吸去培养基,将共转染上清进行不同浓度稀释,取1ml共转染液加到贴壁细胞中,分布均匀。27℃感染1小时后,吸去感染液,将2%低融点琼脂糖凝胶于60℃水浴中融化,冷至40℃与40℃预热的2×TC-100培养基(含20%FBS)混合均匀,每平皿加4ml胶,待凝固后用Parafilm封口,27℃倒置培养3~5天,显微镜观察。将不含有多角体的空斑挑选出来,重复以上步骤,经过2~3轮的纯化获得纯的重组家蚕杆状病毒rBmNPV(PPV-VP2)。
4.3重组病毒rBmNPV(PPV-VP2)在家蚕细胞中的扩增
将重组家蚕杆状病毒rBmNPV(PPV-VP2)感染正常生长的BmN细胞,培养3天后收集上清液,上清液中即含有大量的重组病毒rBmNPV(PPV-VP2)。
4.4重组病毒的鉴定
利用PCR方法分析外源基因整合。游离病毒基因组DNA的提取方法如下:取病毒上清150μl,加入150μl(0.5mol/L)的NaOH后混匀,再加入20μl(8mol/L)的醋酸铵,混匀后用等体积的酚和氯仿分别抽提一次,酒精沉淀后用20μl的TE溶解DNA。寡核苷酸引物为:
FP:5-ATGAGTGAAAATGTGGAACAAC-3’,
RP:5-CTAGTATAATTTTCTTGGTATAAG-3’
取上述病毒基因组DNA 1μl进行PCR扩增,反应条件为:94℃变性5min、94℃1min、58℃1min、72℃1.5min,30个循环,最后72℃延伸5min。取15μl反应产物电泳分析,结果证明获得了重组病毒。
5猪细小病毒VP2基因在家蚕中的表达
本实验所用的家蚕高表达品种为JY1(由本实验室保存;也可向中国科学院武汉病毒研究所或中国农业科学院蚕业研究所购买获得)。JY1品种家蚕饲养按吕鸿声主编的《中国养蚕学》(上海科学技术出版社,1991)的常规方法进行。饷食后48h选择平均体重相同的家蚕,每头蚕接种约1.0×105rBmNP(PPV-VP2),4-5天后收集发病蚕血淋巴,-20℃冻存以进行ELISA法检测,结果如表2。
表2ELISA检测家蚕生物反应器表达的PPV-VVP2基因
由表2可知PPV-VP2基因在蚕血中的表达产物稀释到6400倍甚至更高的稀释倍数仍能被检测到。
6猪细小病毒VP2病毒样颗粒的收集与纯化。
将含表达有PPV-VP2的蚕蛹用预冷的PBS(料液比为1∶9)在匀浆器中研磨后,超声破碎,10000g离心15min,取上清。用饱和的硫酸铵在20%浓度下沉淀病毒粒子后,溶解并用1%的TritonX-100灭活样品中的杆状病毒,再经超滤膜渗滤,可以将体积缩小到1/10,产量可以达到81%-95%。
7表达产物的检测
注射病毒后的家蚕,待发病时收集蚕血(或蚕蛹)。ELISA方法检测PPV-VP2病毒空衣壳颗粒抗原蛋白表达量的高低,包被液作为空白对照,以正常蚕血(或蚕蛹)作为阴性对照,原核表达的PPV-VP2-2蛋白免疫新西兰大白兔之后的兔血清为第一抗体,HRP标记的羊抗兔抗体为第二抗体。任取所收蚕血(或蚕蛹)中的样品,用包被液做梯度稀释,从50×两倍倍比稀释到25600倍。每个梯度处做双孔检测,各取100μl包被到酶标板上。结果如表2,表明PPV-VP2基因在家蚕中表达量高,据计算平均每头家蚕或蚕蛹中的表达量至少1mg,在6400倍稀释甚至更高稀释度下仍能检测到抗原与抗体的特异性反应。Western blotting结果表明在重组病毒感染后家蚕的血淋巴样品的上清液中可检测到大约64kD大小的特异性条带。
8动物免疫实验及HI检测
将收集的纯化抗原PPV病毒衣壳粒子经肌肉注射免疫10只豚鼠为试验组,另设5只豚鼠只注射佐剂为对照组。400-600g的雌性豚鼠,对PPV的抗体为阴性,喂养水和干饲料,每5只豚鼠饲养在一个1m2的饲养箱中,箱中铺上锯末。每支疫苗为0.5微克的病毒粒子与0.5ml的弗氏佐剂混合试制,肌肉注射后0天和14天眼窝取血,28天心脏取血,-20℃冻存以进行HI法检测。
在96孔V型板上进行,用50μl移液管加样和稀释。先取生理盐水50μl,加入第1孔,再取浓度为4个血凝单位的保存的粗纯化病毒抗原依次加入3~12孔,每孔50μl,第2孔加浓度为8个血凝单位的抗原50μl。用稀释器吸稀释2倍的被检血清50μl于第1孔(血清对照)中,挤压混匀后吸50μl于第二孔,依次倍比稀释至第12孔,最后弃去50μl,血清最大的稀释倍数为1∶8192。至室温(18~20℃)下作用45min。加入50μl1%豚鼠红细胞悬液于各孔中,振荡混匀后,室温下静置90min,判定结果,被检测血清稀释到2048倍时仍为阳性。
实施例2猪细小病毒空衣壳蛋白基因经密码子优化后的序列VP2-M在家蚕生物反应器中的表达及表达产物的检测
1.有关溶液和培养基的配制:同实施例1。
2.猪细小病毒VP2基因的优化和载体构建
2.1PPV-VP2基因的密码子优化
根据家蚕密码子偏好性对实施例1测得的猪细小病毒衣壳蛋白基因原始序列VP2进行优化,不改变氨基酸序列,优化后序列VP2-M如SEQ ID NO:2所示,原始序列中含有串联的稀有密码子,这减少了翻译序列或者甚至解除翻译装置,优化后序列CAI值由0.86提高为0.89,调整了GC含量及不宜峰以延长mRNA的半衰期,GC含量由38.32%调整为48.70%,那些影响mRNA稳定性及其与核糖体结合的茎环结构被破坏。直接合成优化后的序列VP2-M克隆到载体pUC57上,两端分别带有Bam HI/Xba I酶切位点。测序鉴定其碱基序列为SEQ ID NO:2所示,氨基酸序列为SEQ ID NO:3所示。
2.2酶切产物的纯化
将pUC57-VP2-M用BamH I/Xba I酶切后,进行1%琼脂糖凝胶电泳,出现2.6kb和1.7kb两条带,回收1.7kb的条带。在紫外灯下用灭菌手术刀切取含相应DNA片段的凝胶,然后用Geneclean试剂盒进行纯化。方法如下:切取凝胶片段并称重,将其放入灭菌的1.5ml小离心管中,加入3倍(v/w)体积的6M NaI,37℃将凝胶溶解后,加入10μl玻璃奶(Glass milk),混匀后室温下放置5分钟,使DNA充分吸附在玻璃奶上,12000rpm离心5秒钟,再用New Wash溶液洗三次,每次均将沉淀弹起,并离心。最后将沉淀晾干后加入30μl 0.1×TE Buffer溶解DNA,离心后去沉淀,取上清作进一步分析。
2.3酶切与连接反应
酶切反应:pUC57-VP2-M用BamHI和Xba I双酶切分析,反应总体积为50μl,质粒10μl,10×酶相应缓冲液5μl,两种酶各为1μl,无菌水补足体积。37℃反应2小时以上。将转移质粒pVL1393作同样酶切反应。反应结束后于65℃灭活10分钟。连接反应:连接总体积10μl,酶切回收产物7μl,载体1μl,5×T4DNA连接缓冲液1μl,T4DNA连接酶1μl,无菌水补足体积,16℃连接过夜。
2.4大肠杆菌的遗传转化
用75mM CaCl2制备大肠杆菌TG1感受态细胞。取制备的连接混合物5μl,加到200μl感受态细胞中,轻轻混匀,冰浴30分钟,42℃热激2分钟,迅速置于冰上2~3分钟,加入已温育至37℃的LB培养基500μl,37℃培养1小时,取100~200μl涂布于含100μg/ml氨苄青霉素(Amp)的LB固体培养基平板上,37℃倒置培养过夜。
2.5质粒DNA的制备:同实施例1。
2.6重组子的鉴定
用BamHI/Xba I双酶切2.5中制备的质粒DNA,电泳后出现约9.6kb、1.7kb的DNA条带的质粒为重组的杆状病毒转移质粒pVL1393(PPV-VP2-M)。
3、家蚕核多角体病毒亲本株Bm-NPV-ZJ8的繁殖及病毒DNA的制备同实施例1。
4、重组家蚕杆状病毒rBmNPV(PPV-VP2-M)的构建和获得
4.1重组杆状病毒rBmNPV(PPV-VP2-M)的构建
接种大约1×106细胞于15cm2培养瓶中,细胞贴壁后,除去含胎牛血清(FBS)培养基,用不含FBS的培养基洗三次,加1.5ml无FBS培养基。向一灭菌管中依次加入1μg家蚕杆状病毒亲本株Bm-NPV-ZJ8 DNA,2μg重组转移质粒pVL1393(PPV-VP2-M)DNA和5μl脂质体,用无菌双蒸水补足体积到60μl,轻轻混匀,静置15分钟后,逐滴加入到培养瓶中进行共转染。27℃培养4小时后补加1.5ml无血清培养基和300μl FBS。27℃恒温培养4~5天,收集上清液用于重组病毒的筛选。
4.2重组家蚕杆状病毒rBmNPV(PPV-VP2-M)的筛选和纯化
接种适量细胞(约70~80%)于35mm小平皿中,细胞贴壁后,吸去培养基,将共转染上清进行不同浓度稀释,取1ml共转染液加到贴壁细胞中,分布均匀。27℃感染1小时后,吸去感染液,将2%低融点琼脂糖凝胶于60℃水浴中融化,冷至40℃与40℃预热的2×TC-100培养基(含20%FBS)混合均匀,每平皿加4ml胶,待凝固后用Parafilm封口,27℃倒置培养3~5天,显微镜观察。将不含有多角体的空斑挑选出来,重复以上步骤,经过2~3轮的纯化获得纯的重组家蚕杆状病毒rBmNPV(PPV-VP2-M)
4.3重组病毒rBmNPV(PPV-VP2-M)在家蚕细胞中的扩增
将重组家蚕杆状病毒rBmNPV(PPV-VP2-M)感染正常生长的BmN细胞,培养3天后收集上清液,上清液中即含有大量的重组病毒rBmNPV(PPV-VP2-M)。
4.4重组病毒的鉴定
利用PCR方法分析外源基因整合。游离病毒基因组DNA的提取方法如下:取病毒上清150μl,加入150μl(0.5mol/L)的NaOH后混匀,再加入20μl(8mol/L)的醋酸铵,混匀后用等体积的酚和氯仿分别抽提一次,酒精沉淀后用20μl的TE溶解DNA。寡核苷酸引物为:
F2:GCAAGCCTTATGGTAGCGCT-3′
R2:CTAAAGTTCCTGAGATGACGTGGT-3′
取上述病毒基因组DNA 1μl进行PCR扩增,反应条件为:94℃变性5min、94℃1min、58℃1min、72℃1min,30个循环,最后72℃延伸5min。取15μl反应产物电泳分析,结果证明获得了重组病毒。
5猪细小病毒VP2-M基因在家蚕中的表达
本实验所用的家蚕高表达品种为JY1(由本发明人实验室保存;也可向中国科学院武汉病毒研究所或中国农业科学院蚕业研究所购买获得)。JY1品种家蚕饲养按吕鸿声主编的《中国养蚕学》(上海科学技术出版社,1991)的常规方法进,饷食后48h选择平均体重相同的家蚕,每头蚕接种约1.0×105rBmNP(PPV-VP2-M),4-5天后收集发病蚕血淋巴,-20℃冻存以进行ELISA法检测,结果如表3。
表3ELISA检测家蚕生物反应器中表达的PPV-VP2-M基因
由表3可知PPV-VP2-M基因在蚕血中的表达产物稀释到12800倍甚至更高的稀释倍数后仍能被检测到。
6猪细小病毒检测抗体的制备
6.1设计特异引物PCR扩增三段部分VP2基因进行原核表达
引物设计如下:
F1:5′-CGGGATCCGCTGGAGGCGTCGGCGTATC-3′(BamH I)
R1:5′-CAAGCTTCTACGAATGCAGGCCCGTCTGGAT-3′(HindIII)
F2:5′-CGGGATCCGCAAGCCTTATGGTAGCGCT-3′(BamH I)
R2:5′-CAAGCTTCTAAAGTTCCTGAGATGACGTGGT-3′(HindIII)
F3:5′-CGGGATCCTACTATGACGATGAACCCAACG-3′(BamH I)
R3:5′-CAAGCTTTTAATACAGTTTACGGGGAATCAG-3′(HindIII)
6.2以质粒T-VP2为模板,用引物F1、R1;F2、R2;F3、R3扩增目的片段VP2-1,VP2-2,VP2-3;玻璃奶法纯化回收PCR产物,PCR纯化产物与pMD18-T克隆载体连接,连接产物转化已制备的大肠杆菌热击感受态细胞,挑斑培养进行重组质粒的鉴定,碱裂解法提取重组质粒,用BamHI/HindIII进行双酶切鉴定,并将酶切鉴定正确的质粒进行测序验证。
6.3测序正确的重组质粒经BamHI/HindIII双酶切消化后,玻璃奶法纯化回收酶切产物,将其连接到已被BamHI/HindIII双酶切消化的表达载体pET-28a上构建pET-28a-VP2-1,pET-28a-VP2-2,pET-28a-VP2-3,连接产物转化Top10感受态细胞,提取质粒进行酶切鉴定。
酶切正确的重组质粒转化BL21感受态细胞,IPTG诱导4h后收集菌液,用SDS-PAGE电泳分析表达情况,结果显示与阴性对照相比,转化重组质粒的菌株出现浓集的融合蛋白表达条带,挑取成功重组的表达菌株单菌落振荡培养后进行诱导,经超声破碎后,SDS-PAGE电泳分析显示表达的融合蛋白(His-PEDV-F3R3)存在于沉淀中,表明表达蛋白以包涵体形式存在并且比较发现pET-28a-VP2-2表达量最大。
6.4融合蛋白的纯化及其抗体的制备
挑取成功重组的pET-28a-VP2-2表达菌株单菌落振荡培养后进行大量诱导表达,采用处理包涵体蛋白的相关溶液与方法将包涵体溶解后,用Ni+-NTA树脂层析柱纯化该表达蛋白,在脲NTA-25、脲NTA-50、脲NTA-100、脲NTA-250、脲NTA-500,5个梯度收集洗脱液,收集穿透液、洗脱液,每管收集一个NTA体积,SDS-PAGE分析确定蛋白质的结合情况、目标蛋白在洗脱液中的分布情况。结果显示单一条带,表达的目标蛋白His-VP2-2大小与阳性对照一致,用脲NTA-100进行洗脱时,洗脱峰最大,SDS-PAGE胶纯化蛋白后用灭菌小刀割下纯化条带蛋白,并切成1mm3的小胶块进行质谱分析,方法为二级质谱的数据库搜索鉴定法,显示红色标出色序列表示吻合的蛋白序列,样品蛋白序列质量检测覆盖率40.29%,分析显示,该样品Score Delta Cn超过30,高达505.3,有6段肽段能够匹配得上且其中2段肽段氨基酸序列大于15,能够检测出来的AA序列与预期序列匹配率高达39.45%,所以能够确定纯化蛋白就是目标基因VP2-2的表达产物。纯化表达的融合蛋白并进行蛋白浓缩,采用Bradford法测浓缩后蛋白溶液的浓度。将纯化好的浓度大于1mg/ml,总量不低于3mg的融合蛋白进行SDS-PAGE后,割下目的条带,把胶粒磨碎后免疫兔子制备多克隆抗体。
6.5多克隆抗体效价检测
纯化的VP2-2蛋白免疫新西兰大白兔4次后获得多克隆抗体,用纯化的融合蛋白作包被抗原,用PBS稀释不同倍数100、200、400、800、1600、3200、6400、12800、25600、51200的多克隆抗血清作一抗,夹心ELISA检测法测抗体效价图,免疫前血清作阴性对照,在A595nm处吸光值,以各个稀释倍数为X轴,各稀释倍数下的吸光值平均值为Y轴。通常大于规定的阴性对照OD值的2.1倍,即为阳性(以空白对照孔调零后计算),效价检测结果显示:所制备的抗体在抗原蛋白浓度为10μg/ml时抗体的效价可达1∶3200。
7猪细小病毒VP2-M基因表达产物的检测
注射病毒后的家蚕,待发病时收集蚕血(或蚕蛹)。ELISA方法检测PPV-VP2-M抗原蛋白表达量的高低,包被液作为空白对照,以正常蚕血(或蚕蛹)作为阴性对照,原核表达的VP2-2蛋白免疫新西兰大白兔之后的兔血清为第一抗体,HRP标记的羊抗兔抗体为第二抗体。任取所收蚕血(或蚕蛹)中的样品,用包被液做梯度稀释,从1000×两倍倍比稀释到6400倍。每个梯度处做双孔检测,各取100μL包被到酶标板上。结果如表3,表明PPV-VP2-M基因在蚕血或蚕蛹中的表达量高,比PPV-VP2基因在家蚕中表达量高出2-3倍,达到每克蛹体2-3毫克,在12800倍稀释甚至更高稀释度下仍能检测到抗原与抗体的特异性反应
8猪细小病毒的血凝实验检测
取新鲜豚鼠的红细胞于阿氏液中保存,用20倍的生理盐水洗涤红细胞三次,最后将红细胞用生理盐水配成1%悬液。再U形板的每空加入灭菌的生理盐水50ul;用微量移液器取适当浓度的粗纯化表达产物的稀释液50ul加入第1孔内,混匀后,吸取50ul后加入到第二孔中,如此连续稀释至第10孔,第10孔吸取50ul液体弃掉;第11孔和第12孔为生理盐水对照,阴性蚕血和样品一样稀释。每孔加入50ul的洗涤后的豚鼠红细胞,混匀后,作用45分钟左右,待对照的红细胞完全沉积后观察结果,结果如图1。由图1可知,含病毒的蚕血稀释到6.4×104倍时为仍为阳性。
9猪细小病毒抗原的纯化
将含表达有PPV-VP2的蚕蛹用预冷的PBS(料液比为1∶9)在匀浆器中研磨后,超声破碎,10000g离心15min,取上清。用饱和的硫酸铵沉淀病毒粒子后,溶解并用1%的TritonX-100灭活样品中的杆状病毒,再经超滤膜渗滤,可以将体积缩小到1/10,产量可以达到81%-95%。
10电镜及胶体金免疫电镜观察
病毒样颗粒收集与纯化后的样品用水稀释10倍,放上铜网,滤纸片吸干液体后用ddH2O滴洗后再用滤纸片吸干,将铜网置于醋酸铀液滴上,滤纸片吸干液体后放入新的醋酸铀液滴上,重复3次后电镜观察(图2),可观察到猪细小病毒空衣壳粒子,大小与预期相符。样品用水稀释40倍后放铜网,滤纸片吸干液体后用ddH2O滴洗后再用滤纸片吸干,将铜网置于稀释的一抗液滴上约3min,滴洗吸干后置于稀释的胶体金二抗液滴上,滴洗吸干后铜网置于醋酸铀液滴上,洗染3次后电镜观察,如图3,可观察到吸附免疫胶体金的猪细小病毒空衣壳粒子数量不少。
<110> 中国农业科学院生物技术研究所
<120> 猪细小病毒抗原的制备方法及其产品
<130> dqxl0056
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1740
<212> DNA
<213> Porcine parvovirus
<400> 1
atgagtgaaa atgtggaaca acacaaccct attaatgcag gcactgaatt gtctgcaaca 60
ggaaatgaat ctgggggtgg gggcggcggt ggcgggggta ggggtgctgg gggggttggt 120
gtgtctacag gtagtttcaa taatcaaaca gaatttcaat acttggggga gggcttggtt 180
agaatcactg cacacgcatc aagactcata catctaaata tgccagaaca cgaaacatac 240
aaaagaatac atgtactaaa ttcagaatca ggggtggcgg gacaaatggt acaagacgat 300
gcacacacac aaatggtaac accttggtca ctaatagatg ctaacgcatg gggagtgtgg 360
ttcaatccag cggactggca gttaatatcc aacaacatga cagaaataaa cttagttagt 420
tttgaacaag caatattcaa tgtagtactt aaaacaatta cagaatcagc aacctcacca 480
ccaaccaaaa tatataataa tgatctaact gcaagcttaa tggtcgcact agacaccaat 540
aacacacttc catacacacc agcagcacct agaagtgaaa cacttggttt ttatccatgg 600
ttacctacaa aaccaactca atacagatat tacctatcat gcatcagaaa cctaaatcca 660
ccaacataca ctggacaatc acaacaaata acagactcaa tacaaacagg actacacagt 720
gacattatgt tctacacaat agaaaatgca gtaccaattc atcttctaag aacaggagat 780
gaattctcca caggaatata tcactttgac acaaaaccac taaaattaac tcactcatgg 840
caaacaaaca gatctctagg actgcctcca aaactactaa ctgaacctac cacagaagga 900
gaccaacacc caggaacact accagcagct aacacaagaa aaggttatca ccaaacaatt 960
aataatagct acacagaagc aacagcaatt aggccagctc aggtaggata taatacacca 1020
tacatgaatt ttgaatactc caatggtgga ccatttctaa ctcctatagt accaacagca 1080
gacacacaat attatgatga tgaaccaaat ggtgctataa gatttacaat gggttaccaa 1140
catggacact taaccacatc ttcacaagag ctagaaagat acacattcaa tccacaaagt 1200
aaatgtggaa gagctccaaa gcaacaattt aatcaacagg caccactaaa cctagaaaat 1260
acaaataatg gaacactttt accttcagat ccaataggag ggaaatctaa catgcatttc 1320
atgaatacac tcaatacata tggaccatta acagcactaa acaatactgc acctgtattt 1380
ccaaatggtc aaatatggga taaagaactt gatacagatc taaaacctag actacatgtt 1440
acagctccat ttgtttgtaa aaacaatcca ccaggacaac tatttgtaaa aatagcacca 1500
aacctaacag atgatttcaa tgctgactct cctcaacaac ctagaataat aacttattca 1560
aacttttggt ggaaaggaac actaacattc acagcaaaaa tgagatccag taatatgtgg 1620
aaccctattc aacaacacac aacaacagca gaaaacattg gtaaatatat tcctacaaat 1680
attggtggca taaaaatgtt tccagaatat tcacaactta taccaagaaa attatactag 1740
<210> 2
<211> 1740
<212> DNA
<213> Artifical sequence
<400> 2
atgagcgaga acgtggagca acacaatccc ataaatgcgg gcactgaact gtctgcgact 60
ggaaacgaat cgggtggcgg cggaggtggt ggaggcggta gaggtgctgg aggcgtcggc 120
gtatcaacag gttctttcaa caaccaaact gaattccagt acttgggaga gggcctcgtg 180
agaattaccg ctcacgccag ccgcctcata catttaaata tgcctgaaca cgagacgtac 240
aaacgcatac atgttcttaa ctcggaaagt ggtgtggcag gacaaatggt tcaggacgat 300
gcgcacacac agatggtgac tccgtggtcc ttaatcgacg caaatgcgtg gggcgtttgg 360
ttcaaccccg ccgattggca actcatctcg aacaacatga ccgaaatcaa tttagtcagt 420
ttcgagcagg caatctttaa cgtggttctg aaaacgatta cagaaagcgc gacctcccct 480
ccaacgaaga tttacaacaa tgacttaaca gcaagcctta tggtagcgct ggataccaac 540
aatacgttgc cttatacccc agctgctcct agatccgaga cgcttggttt ttacccctgg 600
ctgcctacaa agccaactca gtatcgttac tatttgtcgt gcataaggaa cctcaatccg 660
cccacttaca ccggacaatc gcaacagata accgacagta tccagacggg cctgcattcg 720
gatatcatgt tctacactat agaaaacgca gtgcctattc acctgttgcg tacaggcgac 780
gagttcagta ctggtatcta ccatttcgat acaaagccat tgaagctcac acactcatgg 840
caaactaata ggtctttagg acttcctcca aaactcttaa ctgaaccaac aactgagggt 900
gaccaacatc caggaaccct cccagcagca aacacgcgta agggctacca ccagaccata 960
aacaattcat ataccgaagc tacggccatc aggccggccc aagtcggtta caacaccccc 1020
tatatgaatt tcgagtactc taacggtgga ccgtttctga cgccgatcgt acccacagct 1080
gatactcagt actatgacga tgaacccaac ggcgccattc gtttcactat gggataccaa 1140
cacggccatt taaccacgtc atctcaggaa cttgagagat acaccttcaa ccctcaatca 1200
aagtgcggac gcgctccaaa gcaacagttc aaccaacagg ccccgctgaa cttggaaaat 1260
acaaacaatg gcactcttct gcctagcgac ccaattggcg gtaaatccaa catgcatttc 1320
atgaacacct tgaatacgta cggtcccctt accgctctga acaatacggc cccggttttc 1380
cccaacggac agatatggga taaagagttg gacacagatc tcaagcctag attacacgtc 1440
actgctccat tcgtatgtaa aaacaatccg cccggacaac tgtttgtgaa gatcgcaccg 1500
aatttgacag acgatttcaa cgcggactca cctcaacagc caagaatcat tacttactct 1560
aacttctggt ggaagggtac gctcacattt actgctaaga tgcgcagctc caacatgtgg 1620
aatcctattc aacagcacac aactaccgcc gaaaatattg gaaagtacat accaacaaac 1680
ataggtggca taaagatgtt ccccgagtat tcgcaactga ttccccgtaa actgtattaa 1740
<210> 3
<211> 579
<212> PRT
<213> Porcine parvovirus
<400> 3
Met Ser Glu Asn Val Glu Gln His Asn Pro Ile Asn Ala Gly Thr Glu
1 5 10 15
Leu Ser Ala Thr Gly Asn Glu Ser Gly Gly Gly Gly Gly Gly Gly Gly
20 25 30
Gly Arg Gly Ala Gly Gly Val Gly Val Ser Thr Gly Ser Phe Asn Asn
35 40 45
Gln Thr Glu Phe Gln Tyr Leu Gly Glu Gly Leu Val Arg Ile Thr Ala
50 55 60
His Ala Ser Arg Leu Ile His Leu Asn Met Pro Glu His Glu Thr Tyr
65 70 75 80
Lys Arg Ile His Val Leu Asn Ser Glu Ser Gly Val Ala Gly Gln Met
85 90 95
Val Gln Asp Asp Ala His Thr Gln Met Val Thr Pro Trp Ser Leu Ile
100 105 110
Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Ala Asp Trp Gln Leu
115 120 125
Ile Ser Asn Asn Met Thr Glu Ile Asn Leu Val Ser Phe Glu Gln Ala
130 135 140
Ile Phe Asn Val Val Leu Lys Thr Ile Thr Glu Ser Ala Thr Ser Pro
145 150 155 160
Pro Thr Lys Ile Tyr Asn Asn Asp Leu Thr Ala Ser Leu Met Val Ala
165 170 175
Leu Asp Thr Asn Asn Thr Leu Pro Tyr Thr Pro Ala Ala Pro Arg Ser
180 185 190
Glu Thr Leu Gly Phe Tyr Pro Trp Leu Pro Thr Lys Pro Thr Gln Tyr
195 200 205
Arg Tyr Tyr Leu Ser Cys Ile Arg Asn Leu Asn Pro Pro Thr Tyr Thr
210 215 220
Gly Gln Ser Gln Gln Ile Thr Asp Ser Ile Gln Thr Gly Leu His Ser
225 230 235 240
Asp Ile Met Phe Tyr Thr Ile Glu Asn Ala Val Pro Ile His Leu Leu
245 250 255
Arg Thr Gly Asp Glu Phe Ser Thr Gly Ile Tyr His Phe Asp Thr Lys
260 265 270
Pro Leu Lys Leu Thr His Ser Trp Gln Thr Asn Arg Ser Leu Gly Leu
275 280 285
Pro Pro Lys Leu Leu Thr Glu Pro Thr Thr Glu Gly Asp Gln His Pro
290 295 300
Gly Thr Leu Pro Ala Ala Asn Thr Arg Lys Gly Tyr His Gln Thr Ile
305 310 315 320
Asn Asn Ser Tyr Thr Glu Ala Thr Ala Ile Arg Pro Ala Gln Val Gly
325 330 335
Tyr Asn Thr Pro Tyr Met Asn Phe Glu Tyr Ser Asn Gly Gly Pro Phe
340 345 350
Leu Thr Pro Ile Val Pro Thr Ala Asp Thr Gln Tyr Tyr Asp Asp Glu
355 360 365
Pro Asn Gly Ala Ile Arg Phe Thr Met Gly Tyr Gln His Gly His Leu
370 375 380
Thr Thr Ser Ser Gln Glu Leu Glu Arg Tyr Thr Phe Asn Pro Gln Ser
385 390 395 400
Lys Cys Gly Arg Ala Pro Lys Gln Gln Phe Asn Gln Gln Ala Pro Leu
405 410 415
Asn Leu Glu Asn Thr Asn Asn Gly Thr Leu Leu Pro Ser Asp Pro Ile
420 425 430
Gly Gly Lys Ser Asn Met His Phe Met Asn Thr Leu Asn Thr Tyr Gly
435 440 445
Pro Leu Thr Ala Leu Asn Asn Thr Ala Pro Val Phe Pro Asn Gly Gln
450 455 460
Ile Trp Asp Lys Glu Leu Asp Thr Asp Leu Lys Pro Arg Leu His Val
465 470 475 480
Thr Ala Pro Phe Val Cys Lys Asn Asn Pro Pro Gly Gln Leu Phe Val
485 490 495
Lys Ile Ala Pro Asn Leu Thr Asp Asp Phe Asn Ala Asp Ser Pro Gln
500 505 510
Gln Pro Arg Ile Ile Thr Tyr Ser Asn Phe Trp Trp Lys Gly Thr Leu
515 520 525
Thr Phe Thr Ala Lys Met Arg Ser Ser Asn Met Trp Asn Pro Ile Gln
530 535 540
Gln His Thr Thr Thr Ala Glu Asn Ile Gly Lys Tyr Ile Pro Thr Asn
545 550 555 560
Ile Gly Gly Ile Lys Met Phe Pro Glu Tyr Ser Gln Leu Ile Pro Arg
565 570 575
Lys Leu Tyr
Claims (7)
1.一种猪细小病毒空衣壳抗原的制备方法,其特征在于,包括:将猪细小病毒空衣壳蛋白VP2基因或优化的猪细小病毒空衣壳蛋白VP2基因克隆到杆状病毒运载载体中,构建得到转移表达载体;将所构建的转移表达载体与家蚕杆状病毒BmNPV DNA进行共转染,获得重组家蚕杆状病毒;用重组家蚕杆状病毒感染家蚕;培养被感染的家蚕表达重组猪细小病毒空衣壳蛋白;收获并纯化所表达的重组猪细小病毒空衣壳蛋白,即得;所述的猪细小病毒空衣壳蛋白基因VP2的核苷酸序列为SEQ ID NO:1所示;所述优化的猪细小病毒空衣壳蛋白VP2基因的核苷酸序列为SEQ ID NO:2所示。
2.按照权利要求1所述的制备方法,其特征在于:所述的杆状病毒运载载体选自AcRP23-lacZ,AcRP6-SC,AcUWl-lacZ,BacPAK6,Bac to Pac,Bacmid,BlucBacII(pETL),p2Bac,p2Blue,p89B310,pAc360,pAc373,pAcAB3,pAcAB4,PAcAS3,pAcC129,pAcC4,DZI,pAcGP67,pAcIEl,pAcJPl,pAcMLF2,pAcMLF7,pAcMLF8,pAcMPl,pAcMP2,pAcRP23,pAcRP25,pAcRW4,pAcsMAG,pAcUWl,pAcUW21,pAcUW2A,pAcUW2B,pAcUW3,pAcUW31,pAcUW41,pAcUW42,pAcUW43,pAcUW51,pAcVC2,pAcVC3,pAcYMl,pAcJcC5,pBacl、pBac2,pBlueBacIII,pBlueBacHis,pEV55,mXIV,pIEINeo,pJVETL,pJVNhel,pJVP10,pJVrsMAG,pMBac,pP10,pPAKl,pPBac,pSHONEX1.1,pSYN XIV VI+,pSYNVI+wp,pSYNXIV VI-,pVL1391,pVL1392,pVL1393,pVL941,pVL945,pVL985,pVTBac,pBM030或pUAC-5。
3.按照权利要求1所述的制备方法,其特征在于:所述家蚕杆状病毒BmNPV为家蚕杆状病毒亲本株Bm-NPV-ZJ8。
4.按照权利要求1所述的制备方法,其特征在于:所述的感染是将重组家蚕杆状病毒通过口食来感染1-5龄的家蚕幼虫或透过表皮来感染1-5龄的家蚕幼虫或蛹体。
5.按照权利要求4所述的制备方法,其特征在于:所述的透过表皮来感染1-5龄的家蚕幼虫或蛹体是将重组家蚕杆状病毒穿刺接种1-5龄的家蚕幼虫或蛹体,在感染3-6天后收集含猪细小病毒抗原的家蚕幼虫或蛹的体液或组织匀浆;其中,所述的蛹体为1-2天的早期嫩蛹。
6.优化的猪细小病毒空衣壳蛋白VP2基因,其特征在于:其核苷酸序列为SEQ IDNO:2所示。
7.权利要求6所述的优化的猪细小病毒空衣壳蛋白VP2基因在制备预防或治疗猪细小病毒疫苗中的用途。
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CN103908664B (zh) * | 2013-01-09 | 2016-06-29 | 普莱柯生物工程股份有限公司 | 一种疫苗组合物及其制备方法 |
US20140234354A1 (en) * | 2013-02-15 | 2014-08-21 | Boehringer Ingelheim Vetmedica, Inc. | Porcine parvovirus 5b, methods of use and vaccine |
CN103387996B (zh) * | 2013-08-19 | 2015-07-15 | 长春西诺生物科技有限公司 | 犬细小病毒病毒样颗粒、其制备方法及应用 |
CN105263953B (zh) * | 2014-01-15 | 2020-01-07 | 勃林格殷格翰动物保健美国有限公司 | 猪细小病毒5a、使用方法及疫苗 |
CN106148358A (zh) * | 2016-07-15 | 2016-11-23 | 河南省农业科学院 | 一种利用大肠杆菌表达系统制备猪细小病毒病毒样颗粒亚单位疫苗的方法及应用 |
CN107868131A (zh) * | 2017-10-13 | 2018-04-03 | 长春西诺生物科技有限公司 | 一种猪细小病毒病亚单位疫苗及其制备方法 |
CN108776225A (zh) * | 2018-05-23 | 2018-11-09 | 中国农业科学院兰州兽医研究所 | 猪细小病毒VLPs抗体检测试剂盒及其制备方法、应用 |
KR102117811B1 (ko) * | 2018-12-05 | 2020-06-02 | 대한민국(농림축산식품부 농림축산검역본부장) | 재조합 돼지파보바이러스 항원 단백질 및 이의 용도 |
CN110283236B (zh) * | 2019-06-19 | 2021-05-04 | 扬州优邦生物药品有限公司 | 一种猪细小病毒病毒样颗粒的纯化方法及其应用 |
CN114480439A (zh) * | 2022-02-23 | 2022-05-13 | 成都史纪生物制药有限公司 | 一种猪细小病毒vp2蛋白基因及其应用 |
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