CN116327911A - 一种鲤疱疹病毒ii型dna疫苗及其制备方法和应用 - Google Patents
一种鲤疱疹病毒ii型dna疫苗及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域,尤其涉及一种鲤疱疹病毒II型DNA疫苗及其制备方法和应用。本发明提供了一种鲤疱疹病毒II型DNA疫苗,其包含载体和ORF66基因的核酸分子,所述ORF66基因的核酸序列如SEQ ID NO:1所示,所述载体为pVAX1。本发明制备得到的鲤疱疹病毒II型DNA疫苗更加安全可靠,具有很好的免疫保护性,可以抑制鲤疱疹病毒II型对鲫鱼的感染,降低鲫鱼养殖过程中因病害导致的经济损失。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及一种鲤疱疹病毒II型DNA疫苗及其制备方法和应用。
背景技术
鲫鱼(Carassiu sauratus)是我国重要的淡水养殖品种之一。鲤疱疹病毒Ⅱ型(cyprinid herpesvirus 2,CyHV-2)所引发的鲫疱疹病毒病(也称鲫造血器官坏死病)已成为严重危害我国鲫鱼养殖业健康发展的重要疾病之一。鲤疱疹病毒Ⅱ型能感染鲫及其变种,常于水温15~25℃发病,在全球多个国家和地区都有流行,致死率高达90%。近年来,我国江苏、北京、广州、武汉等地均陆续暴发了由CyHV-2感染导致的异育银鲫大规模死亡病例,造成了重大的经济损失。免疫接种是预防水生动物疾病发生、保障水产养殖业持续发展的有效途径。然而,目前针对CyHV-2尚无有效的商业化疫苗或治疗药物,因此研制安全有效的鲤疱疹病毒Ⅱ型保护性疫苗对于防控鲫疱疹病毒病的暴发和流行具有重要意义。
DNA疫苗也称核酸疫苗、基因疫苗,是将含有编码保护性抗原蛋白的基因序列和表达所必需调控元件的质粒DNA导入动物组织后,被宿主细胞摄取、表达、加工并呈递给免疫系统,诱发机体产生特异性体液免疫和细胞免疫应答,形成对该抗原的免疫保护作用。DNA疫苗制作相对简单、成本低廉、容易运输储存、不存在毒力返强现象、免疫途径多样,具有广阔的应用前景。
用于构建DNA疫苗的表达载体必须兼具安全性和有效性,即表达载体不会整合到宿主细胞的基因组中,并且其所携带的抗原基因能在动物细胞内获得适量表达并激发宿主产生免疫应答。鱼用DNA疫苗常用的质粒载体有pcDNA3.1(+)、pET-32a、pEGFP-N1等。pVAX1载体是由美国食品和药品管理委员会(FDA)推荐的唯一可以应用于人体实验的质粒载体。其具有以下优点:首先其仅保留真核表达最基本的DNA序列,以最大限度减少染色体整合的可能性。其次,因氨苄青霉素在部分机体中可诱发过敏反应,其采用卡那霉素抗性筛选基因作为替代,以最大程度地减少抗性筛选基因和人类基因组发生重组的可能性。基于此,哺乳动物中使用pVAX1载体构建核酸疫苗的应用较为广泛。然而,目前pVAX1载体在鱼用DNA疫苗开发中的应用报道较为少见。
本实验室前期对ORF66蛋白进行了原核表达,并制备了其特异性多抗,发现ORF66蛋白具有较好的免疫原性。本发明首次将ORF66蛋白编码基因构建至pVAX1真核表达载体,制备了一种鲤疱疹病毒II型DNA疫苗。本发明提供了一种鲤疱疹病毒II型DNA疫苗的制备方法,并对其实际应用效果进行了检测。
发明内容
有鉴于现有技术的上述缺陷,本发明所要解决的技术问题是如何预防鲫鱼患上鲫疱疹病毒病。
为实现上述目的,本发明提供了一种鲤疱疹病毒II型DNA疫苗,其包含载体和ORF66基因的核酸分子。
优选地,所述ORF66基因的核酸序列如SEQ ID NO:1所示。
优选地,所述载体为pVAX1。
在本发明的较佳实施方式中,发明提供了一种鲤疱疹病毒II型DNA疫苗的制备方法,其包括以下步骤:以CyHV-2的DNA为模板,以pVAX-ORF66-F/pVAX-ORF66-R为引物进行PCR扩增,将PCR扩增产物与pVAX1连接,构建质粒载体疫苗;
所述pVAX-ORF66-F如SEQ ID NO:2所示,
所述pVAX-ORF66-R如SEQ ID NO:3所示。
具体地,一种鲤疱疹病毒II型DNA疫苗的制备方法,其包括以下步骤:
(3)采用病毒DNA提取试剂盒提取CyHV-2感染组织中病毒DNA,以其作为模板,以pVAX-ORF66-F/pVAX-ORF66-R为引物进行PCR扩增,得到PCR扩增产物;
(4)用DNA胶回收试剂盒对(1)中所述PCR扩增产物进行切胶纯化回收,然后分别将回收的片段和pVAX1载体用Hind III和Bam HI限制性内切酶37℃酶切3h,酶切片段纯化回收,在T4 DNA连接酶作用下于16℃连接过夜,连接产物转化DH5α大肠杆菌感受态细胞,菌液PCR筛选阳性克隆并进行双向序列测定,测序正确的重组质粒即为pVAX-ORF66。
优选地,所述PCR反应体系如下:ddH2O 30.5μL、10×LA Buffer II(Mg2+plus)5μL、dNTP Mixture(2.5mmol/L)8μL、上下游引物(10μmol/L)各2μL、LA Taq Polymerase(5U/μL)0.5μL、DNA模板2μL,总体积为50μL。
优选地,所述PCR反应条件为:95℃预变性5min;95℃变性30s,57℃退火30s,72℃延伸2min,共35个循环;72℃延伸10min;4℃保温。
在本发明的另一较佳实施方式中,本发明提供一种药物组合物,其包含鲤疱疹病毒II型DNA疫苗。
在本发明的又一较佳实施方式中,本发明提供了鲤疱疹病毒II型DNA疫苗用于制备治疗鲫疱疹病毒病药物的应用。
本发明制备得到的鲤疱疹病毒II型DNA疫苗更加安全可靠,具有很好的免疫保护性,可以抑制鲤疱疹病毒II型对鲫鱼的感染,降低鲫鱼养殖过程中因病害导致的经济损失。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例的鲤疱疹病毒II型ORF66基因编码区的扩增结果示意图;
图2是本发明的一个较佳实施例的构建的重组质粒pVAX-ORF66限制性内切酶双酶切的结果示意图;
图3是本发明的一个较佳实施例的利用间接免疫荧光法检测ORF66蛋白在细胞中过表达的结果示意图;
图4是本发明的一个较佳实施例的利用RT-PCR检测ORF66在鲫鱼体内转录表达的结果示意图;
图5是本发明的一个较佳实施例的利用免疫印迹法检测ORF66蛋白在鲫鱼体内表达的结果示意图;
图6是本发明的一个较佳实施例的不同实验组攻毒后存活率曲线图;
图7是本发明的一个较佳实施例的应用的pVAX1空载体的结构示意图谱;
图8是本发明的一个较佳实施例的制备的重组质粒pVAX-ORF66的结构示意图谱。
具体实施方式
以下介绍本发明的多个优选实施例,使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现,本发明的保护范围并非仅限于文中提到的实施例。
(1)CyHV-2ORF66基因的扩增
首先利用Primer Express 5.0和BioEdit 7.0软件,根据GenBank中CyHV-2ORF66基因序列保守区设计引物pVAX-ORF66-F(SEQ ID NO:2)和pVAX-ORF66-R(SEQ ID NO:3),并引入限制性内切酶酶切位点(以斜体下划线标注),引物序列如下:
pVAX-ORF66-F:ATTAAGCTTGCCACCATGACCTCCCTCCAGCAAGCTA;
pVAX-ORF66-R:CCGGGATCCTTAATAAATCATGAGATCGTCTAGGTCGTG。
采用病毒DNA提取试剂盒(购自TaKaRa公司)提取CyHV-2感染组织中病毒DNA,以其作为模板进行PCR扩增,PCR反应体系如下:ddH2O 30.5μL、10×LA Buffer II(Mg2+plus)5μL、dNTP Mixture(2.5mmol/L)8μL、上下游引物(10μmol/L)各2μL、LA Taq Polymerase(5U/μL)0.5μL、DNA模板2μL,总体积为50μL。
PCR反应条件为:95℃预变性5min;95℃变性30s,57℃退火30s,72℃延伸2min,共35个循环;72℃延伸10min;4℃保温。
PCR扩增产物经1%琼脂糖凝胶电泳分析,扩增结果如图1所示。
(2)重组质粒pVAX-ORF66的构建
用DNA胶回收试剂盒对(1)中所述PCR产物进行切胶纯化回收,然后分别将回收的片段和pVAX1载体(购于Invitrogen公司,图谱如图7所示)用Hind III和Bam HI限制性内切酶37℃酶切3h,酶切片段纯化回收,在T4 DNA连接酶作用下于16℃连接过夜。连接产物转化DH5α大肠杆菌感受态细胞,菌液PCR筛选阳性克隆并送生工生物工程(上海)股份有限公司进行双向序列测定,将测序正确的重组质粒命名为pVAX-ORF66(图谱如图8所示)。
(3)重组质粒pVAX-ORF66和空载体pVAX1的大量制备
将50μL测序正确的单克隆菌液接种到5mL含50μg/mL卡那霉素的LB液体培养基中,37℃、220转/分钟,摇床培养8h。取200μL菌液接种到200mL含50μg/mL卡那霉素的LB液体培养基中,37℃、220转/分钟,摇床培养过夜,用质粒提取试剂盒(购于基因生物技术国际贸易(上海)有限公司)提取所述重组质粒pVAX-ORF66和所述空载体质粒pVAX1,具体步骤如下:
将培养过夜的菌液置于50mL离心管中,4℃、6000×g离心10min收集菌体,去上清,重复以上操作直至所有菌液离心完毕;
在菌体沉淀中加入16mL RES充分悬浮;
加入16mL Buffer LYS,上下颠倒混匀,室温静置5min;
期间加入12mL Buffer EQU平衡润湿过滤器和离心柱;
加入16mL Buffer NEU至裂解物中,立即颠倒混匀,直至无色;
将裂解物转移至过滤器中,待其在重力作用下全部滤出,弃滤液;
沿过滤器边缘加入5mL Buffer EQU洗一次,弃滤液;
去掉过滤器,加入8mL Buffer WASH至离心柱,弃滤液;
加入5mL Buffer ELU至离心柱,用15mL离心管收集洗脱液;
加入3.5mL异丙醇,涡旋,4℃、10000×g,离心30min,去上清;
加入2mL 70%乙醇洗一次,4℃、10000×g,离心5min,去上清;
室温10-15min晾干;
加入0.8~1mL TE缓冲液溶解,用分光光度计测定质粒浓度。
(4)重组质粒pVAX-ORF66的双酶切鉴定
将pVAX-ORF66重组质粒用Hind III和Bam HI限制性内切酶37℃酶切3h,凝胶电泳显示,重组质粒经酶切后获得了大小分别约3000bp和1200bp的特异性片段(结果如图2所示),与目标片段大小一致,进一步表明所构建的重组质粒阅读框正确无误。
(5)重组质粒pVAX-ORF66和空载体pVAX1转染GFB细胞
具体步骤如下:转染前一天,铺24孔细胞培养板,第二天待细胞密度达到约70%-90%时转染,转染时每孔制备以下混合液:
A管:将800ng DNA溶于50μL Opti-MEM培养基中;
B管:将2μL Lipofectamine 2000(购于Invitrogen公司)溶于50μL Opti-MEM培养基中,混匀,室温放置5min;
然后将AB两管混合,室温放置20min;期间将细胞用Opti-MEM洗两次,将混合物加入对应孔中,轻轻混匀,培养4-6h后换成含10%胎牛血清的完全培养基。
(6)间接免疫荧光
所述重组质粒pVAX-ORF66和空载体pVAX1转染GFB细胞36h后,用PBS洗细胞2次,每次3min,每孔加入400μL 4%多聚甲醛,室温固定30min;
吸除固定液,PBS洗细胞3次,每孔加入400μL含0.2% Triton X-100的PBS通透15min;
吸除通透液,PBS洗3次,加入4%牛血清白蛋白封闭液,37℃封闭2h;
吸除封闭液,加入用封闭液稀释的一抗(本实验室制备,兔抗CyHV-2ORF66蛋白抗体,以体积比1:1000稀释),37℃孵育2h;
吸除一抗,PBS洗3次,每次3min,加入荧光二抗(Alexa Fluor 488标记的驴抗兔,购于Invitrogen公司,以体积比1:2000稀释),37℃孵育1h;
吸除二抗,PBS洗3次,每次3min,加入DAPI染核液(购于Roche公司,以体积比1:2000稀释),室温染色10min,PBS洗3次,每次3min;
每孔加入500μL PBS,置于倒置荧光显微镜下观察并拍照。
结果如图3A和3B所示,pVAX-ORF66转染细胞出现明显的绿色荧光,而空载体pVAX1转染细胞则无明显荧光信号。
(7)免疫印迹实验
所述重组质粒pVAX-ORF66和所述空载体pVAX1转染GFB细胞24h后,弃上清,用PBS洗两次,每次3min,加入RIPA裂解液(购于碧云天生物技术有限公司),用枪吹打数下,使裂解液和细胞充分接触,冰上孵育10min,将细胞裂解液转移至1.5mL EP管中,12000×g,离心5min,取20μL离心上清和5μL的5×蛋白上样缓冲液混匀,95℃煮5min,进行SDS-PAGE凝胶电泳。
裁剪与分离胶大小完全吻合的滤纸和PVDF膜,在转移缓冲液中平衡15min;
凝胶电泳结束后,拆卸凝胶夹层,分离胶于适量转移缓冲液中平衡10min;
组装转印夹层,在恒压下转移蛋白20V,40min;
转膜完毕,将膜取出,放入杂交盒,加10mL 5% PBST-牛血清白蛋白封闭液,37℃孵育2h;
倒掉封闭液,加入本实验室制备的CyHV-2ORF66蛋白抗体(以体积比1:1000稀释),37℃孵育2h;
PBST洗3次,每次10min,加入HRP标记的山羊抗兔IgG抗体(二抗,购于上海翊圣生物科技有限公司,以体积比1:5000稀释),37℃孵育1h;
倒掉二抗,PBST洗3次,每次10min;
加入ECL发色底物(购自天根生化科技(北京)有限公司),条带在5-10min内显色。
免疫印迹结果如图3C所示,pVAX-ORF66转染细胞中,ORF66蛋白的表达水平显著提高,表明本发明的DNA疫苗可以在真核细胞内高水平表达。
(8)重组质粒pVAX-ORF66在鲫鱼体内的转录检测
实验鲫鱼(约10g)在实验开展前于水族箱中充气暂养两周,在其背鳍基部注射20μg pVAX-ORF66(溶于100μL PBS),同时设置注射空载体pVAX1和PBS的鲫鱼作为对照组。
免疫三天后,采用Trizol法分别提取实验组和对照组注射部位肌肉总RNA,提取过程中用DNase I(购于Invitrogen公司)处理彻底除去pVAX-ORF66的干扰。
用经PCR检测无pVAX-ORF66污染的总RNA为模板,用反转录试剂盒(购于Invitrogen公司)进行cDNA合成,具体步骤如下:
在PCR反应管中依次加入1μg上述提取的所述RNA、1μL随机六聚体引物、补充无RNA酶的水至总体积12μL;于65℃反应5min,立即置于冰上,然后加入4μL 5×ReactionBuffer、1μL RiboLock RNase Inhibitor、2μL 10mM dNTP Mix和1μL RevertAid M-MuLVRT,25℃反应5min,之后42℃保温45min获得cDNA。
PCR检测pVAX-ORF66在鱼体的转录,扩增条件与(1)中相同,结果如图(4)中所示,所扩增的片段与ORF66基因大小相符,说明pVAX-ORF66在鱼体内能够正常转录。
(9)重组质粒pVAX-ORF66在鲫鱼体内的蛋白表达检测
实验鲫鱼(约10g)于水族箱中暂养两周,在其背鳍基部注射20μg pVAX-ORF66(溶于100μL PBS),同时设置注射空载体pVAX1和PBS作为对照组。
免疫三天后,分别取实验组和对照组注射部位肌肉,用RIPA裂解液裂解细胞,然后4℃、13000×g,离心5min,取20μL离心上清进行免疫印迹检测,其步骤同(7)中所述。结果显示,实验组鲫鱼组织样品中存在分子量大小约为45kD的条带,与ORF66蛋白大小一致,而对照组中无此条带(图5)。表明pVAX-ORF66在鱼体内能够正常进行ORF66的表达。
(10)重组质粒pVAX-ORF66的免疫保护效果
实验鲫鱼(约20g)于水族箱中充气暂养两周,在其背鳍基部注射20μg/尾pVAX-ORF66(溶于100μL PBS),同时设置注射空载体pVAX1和PBS作为对照组。
免疫28天后,对各组鲫鱼进行攻毒,腹腔注射106TCID50/mL病毒液,连续观察42天,每天观察记录各组鱼的死亡情况。取死亡鱼鳃、肝、脾和肾进行CyHV-2的PCR检测,并计算相对免疫保护率(Relative percentage survival,RPS)。相对免疫保护率RPS=[1-(免疫组死亡率/对照组死亡率)]×100%。结果如图6所示,pVAX-ORF66组的相对免疫保护率达60%,显著高于其他两组,表明本发明制备的DNA疫苗具有较好的免疫保护效果,可以作为抗CyHV-2的核酸疫苗。
本实施例通过基因克隆技术将CyHV-2核衣壳蛋白ORF66克隆至真核表达载体pVAX1,通过一系列的试验验证,表明本发明的DNA疫苗具有很好的免疫保护性、成本低廉且安全性高,可以抑制鲤疱疹病毒II型对鲫鱼的感染。本发明的DNA疫苗可以显著降低鲫鱼养殖过程中因病毒性疾病导致的经济损失,增加渔民收入,同时减少药物使用,提升水产品质量,保护环境,具有广阔的应用前景。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (9)
1.一种鲤疱疹病毒II型DNA疫苗,其包含载体和ORF66基因的核酸分子。
2.根据权利要求1所述的鲤疱疹病毒II型DNA疫苗,其特征在于,所述ORF66基因的核酸序列如SEQ ID NO:1所示。
3.根据权利要求1所述的鲤疱疹病毒II型DNA疫苗,其特征在于,所述载体为pVAX1。
4.权利要求1-3任一所述的鲤疱疹病毒II型DNA疫苗的制备方法,其特征在于,包括以下步骤:以CyHV-2的DNA为模板,以pVAX-ORF66-F/pVAX-ORF66-R为引物进行PCR扩增,将PCR扩增产物与pVAX1连接,构建质粒载体疫苗;
所述pVAX-ORF66-F如SEQ ID NO:2所示,
所述pVAX-ORF66-R如SEQ ID NO:3所示。
5.根据权利要求4所述的鲤疱疹病毒II型DNA疫苗的制备方法,其特征在于,包括以下步骤:
(1)采用病毒DNA提取试剂盒提取CyHV-2感染组织中病毒DNA,以其作为模板,以pVAX-ORF66-F/pVAX-ORF66-R为引物进行PCR扩增,得到PCR扩增产物;
(2)用DNA胶回收试剂盒对(1)中所述PCR扩增产物进行切胶纯化回收,然后分别将回收的片段和pVAX1载体用Hind III和Bam HI限制性内切酶37℃酶切3h,酶切片段纯化回收,在T4 DNA连接酶作用下于16℃连接过夜,连接产物转化DH5α大肠杆菌感受态细胞,菌液PCR筛选阳性克隆并进行双向序列测定,测序正确的重组质粒即为pVAX-ORF66。
6.根据权利要求5所述的鲤疱疹病毒II型DNA疫苗的制备方法,其特征在于,所述PCR反应体系如下:ddH2O 30.5μL、10×LA Buffer II(Mg2+plus)5μL、dNTP Mixture(2.5mmol/L)8μL、上下游引物(10μmol/L)各2μL、LA Taq Polymerase(5U/μL)0.5μL、DNA模板2μL,总体积为50μL。
7.根据权利要求5所述的鲤疱疹病毒II型DNA疫苗的制备方法,其特征在于,所述PCR反应条件为:95℃预变性5min;95℃变性30s,57℃退火30s,72℃延伸2min,共35个循环;72℃延伸10min;4℃保温。
8.一种药物组合物,其包含权利要求1-3所述的鲤疱疹病毒II型DNA疫苗。
9.权利要求1-3所述的鲤疱疹病毒II型DNA疫苗用于制备治疗鲫疱疹病毒病药物的应用。
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