CN109234315A - 一种表达传染性法氏囊病毒vp2基因的重组火鸡疱疹病毒株 - Google Patents
一种表达传染性法氏囊病毒vp2基因的重组火鸡疱疹病毒株 Download PDFInfo
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Abstract
本发明提供一种表达传染性法氏囊病毒嵌合型VP2基因重组火鸡疱疹病毒株,本发明所提供的重组3型火鸡疱疹病毒rHVT‑VP2,于2018年9月7日保藏在位于中国武汉、武汉大学的中国典型培养物保藏中心,保藏编号为CCTCC NO:V201845。本发明以表达EGFP标记基因的重组火鸡疱疹病毒rHVT‑EGFP为基础,利用同源重组原理,将携带CMV启动子的嵌合型VP2基因表达盒插入到火鸡疱疹病毒复制非必需区的US2区,插入的表达盒依次由CMV启动子、VP2基因、WPRE调控元件和TkployA组成,获得一株表达嵌合型VP2基因的重组火鸡疱疹病毒(rHVT‑VP2),该病毒免疫鸡后,能够提供针对传染性法氏囊病毒的完全保护力,可用于鸡传染性法氏囊病的预防。
Description
技术领域
本发明属于分子生物学和基因工程疫苗技术领域,具体涉及一种表达传染性法氏囊病毒VP2基因的重组火鸡疱疹病毒株,该重组病毒免疫家禽可用于传染性法氏囊病的预防。
背景技术
马立克氏病(Marek’s disease,MD)和鸡传染性法氏囊病(Infectious bursaldisease,IBD)是常见的两种影响家禽生产性能的免疫抑制病。其中,IBD是由鸡传染性法氏囊病毒(Infectious bursal disease virus,IBDV)引起的禽类急性、高度接触性免疫抑制性传染病,该病主要感染3周龄~12周龄青年鸡,可通过导致鸡法氏囊损伤、引起免疫抑制和雏鸡死亡等对家禽养殖造成巨大的经济损失。目前IBDV超强毒株(vvIBDV)和变异株的出现给该病的防控带来巨大难度。IBD的免疫预防已成为目前防控该病的主要措施,而商品化的传染性法氏囊病活疫苗分为弱毒、中等毒力和强毒三类。弱毒活疫苗对有母源抗体鸡群的免疫保护效果不理想。与弱毒疫苗相比,中等毒力和强毒疫苗的免疫保护力虽然高很多,但由于其具有一定的致病性,对法氏囊有一定的损伤。同时,由于受母源抗体干扰及IBDV的特点,导致传统疫苗在实际应用中形成不可避免的免疫空白期。因此,针对传染性法氏囊病新型疫苗的研究成为目前的热点之一。IBDV的VP2蛋白是主要的免疫保护性抗原,针对VP2蛋白的中和抗体可抵御IBDV强毒株的攻击,因此构建表达IBDV VP2基因的重组活载体疫苗具有潜在的应用价值。
影响外源基因表达水平的因素有很多,例如启动子的选择、密码子的偏嗜性、表达抗原的类型以及调控元件的作用等。稀有密码子的使用已经被证实为阻碍基因表达的一个重要因素,因此为了提高外源基因在不同物种体内的翻译和转录水平,常常对外源基因的密码子进行改造,使之符合该物种的密码子的偏嗜分布,从而提高表达效率。
鸡的组织相容性复合体又称为B复合体(Bcomplex),主要由B-F,B-L和B-G三个格局功能的基因位点组成。其中B-F所编码的抗原在结构和功能上分别类似于哺乳动物的MHCI类抗原,并且存在于所有的细胞内。MHCI类分子与胞内抗原蛋白结合,抗原表位被递呈至细胞表面,同时也可以刺激MHCⅡ分子的抗原递呈反应。据报道,在外源基因的5’端添加MHCⅠss,3’端添加MITD,可以提高抗原递呈的效率,同时可以刺激CD4+和CD8+T细胞的多样化反应。
除了启动子的强弱可影响抗原的表达效率以外,其他调控元件也可有效的提高抗原基因的表达水平,在抗原基因的3’端添加土拨鼠转录后调控元件后,能够无物种特异性的增强基因表达水平。但目前传染性法氏囊病毒VP2基因还存在表达量低下的问题。
发明内容
本发明的目的是提供一种表达IBDV的VP2基因重组火鸡疱疹病毒株,该病毒株可用于家禽中诱导针对IBDV的保护性免疫应答。
本发明首先提供一种在火鸡疱疹病毒(HVT)中插入外源基因的方法,是将外源基因插入火鸡疱疹病毒的插入位点US2区。
更具体的,所述的插入位点位于HVT基因组US2区的140276nt-140285nt之间。
本发明再一方面提供一种重组火鸡疱疹病毒,是通过在火鸡疱疹病毒的US2区插入表达IBDV嵌合型VP2基因所构建;
所述的表达IBDV嵌合型VP2基因(M-VP2基因),是在VP2基因的5′端添加Gallus的MHCI的信号肽序列(MHCIss),3′端添加MHCI的跨膜区和胞内区MTTD的核苷酸序列组成。
本发明再一个方面提供一种重组火鸡疱疹病毒,由CMV启动子负责嵌合型M-VP2基因的起始转录,WPRE元件负责增强外源基因表达水平,TkployA终止子为M-VP2基因mRNA转录提供终止信号,同时mRNA 3’端添加polyA尾巴。
所提供的重组火鸡疱疹病毒,其一种为重组火鸡疱疹病毒(rHVT-VP2)株,于2018年9月7日保藏在位于中国武汉、武汉大学的中国典型培养物保藏中心,保藏编号为CCTCCNO:V201845。
本发明的rHVT-VP2株用于表达IBDV嵌合型VP2蛋白,
本发明的rHVT-VP2株的另一方面的用途是用于制备疫苗;
本发明以火鸡疱疹病毒(HVT)FC-126疫苗毒株为载体,以其复制非必需区US2为插入位点,首先利用同源重组原理在该位点插入EGFP基因,构建表达EGFP标记基因的重组病毒rHVT-EGFP。免疫保护评价结果表明,本发明的rHVT-VP2株免疫鸡群后,针对IBDV强毒株SNJ93的攻击,可以提供100%攻毒保护。
附图说明
图1:转移的构建模式图;
图2:重组病毒rHVT-VP2的构建示意图;
图3:M-VP2基因及其表达盒PCR电泳图;
图4:转移载体pFR-EGFP转染DF1的荧光图;
图5:Western-Blot试验鉴定转移载体pFR-VP2的蛋白表达;
图6:重组病毒rHVT-EGFP的荧光图;
图7:PCR鉴定重组病毒rHVT-VP2
图8:Western-Blot试验鉴定重组病毒rHVT-VP2中VP2蛋白的表达
图9:IFA鉴定重组病毒rHVT-VP2。
具体实施方式
本发明选择IBDV F52/70株的完整的VP2基因,在其5’端添加Gallus的MHCI的信号肽(MHCIss)序列和3’端添加MHCI的跨膜区和胞内区MTTD的核苷酸序列组成。合成后的嵌合型M-VP2基因插入pcDNA3.4载体中,构建基因表达盒,并通过PCR方法将该基因表达盒从pcDNA3.4载体中扩增出来,连接到含同源臂的转移载体pFR中,构建表达嵌合型M-VP2基因的转移载体质粒pFR-M-VP2(如图1所示)。再次,用上述转移载体质粒pFR-M-VP2替换重组病毒rHVT-EGFP的标记基因EGFP,筛选出表达嵌合型VP2基因重组病毒rHVT-VP2(如图2所示)。
下面结合实施例对本发明进行详细的描述。
实施例1
1实验材料
1.1毒株、菌株和质粒
HVT FC-126疫苗株由扬州大学农业部畜禽传染病重点开放实验室保存。
IBDV强毒株F52/70的VP2基因(GenBank中的登录号:HG974565.1),及其5’端添加Gallus的MHCI的信号肽(MHCIss)序列和3’端添加MHCI的跨膜区和胞内区MTTD的核苷酸序列偶联后由华大基因科技有限公司合成,并连接到质粒pCU19质粒中,连接成功的质粒命名为pCU19-M-VP2。
感受态细胞Trans-T1购自北京全式金生物技术有限公司;鸡胚成纤维细胞系(DF-1)由青岛易邦生物工程有限公司保藏。
质粒:pEASY-T3,pEASY-Blunt,pEASY-M1购自北京全式金生物科技有限公司;PCR2.1、pT-EGFP由本扬州大学农业部畜禽传染病重点开放实验室保存,其中pT-EGFP包含表达绿色荧光蛋白(GFP)的表达盒,表达盒两端分别有NotⅠ和AvrⅡ酶切位点。
2实验方法
2.1中间转移载体pFR的构建
2.1.1同源臂引物的设计
选择HVT的US2区插入外源基因,参照GenBank中登录的HVT FC126株(登录号:AF291866)的全基因序列,用Primer Premier 5.0引物设计软件设计两对引物,分别PCR扩增Fwd、Rev同源臂。Fwd同源臂上游引入KpnⅠ酶切位点,下游引入SpeⅠ和AvrⅡ酶切位点。Rev同源臂上游引入NotⅠ酶切位点,下游引入ApaⅠ酶切位点。引物序列如下:
Fwd-F:5'-GAAggtacc TTCTAAATGGAAAGAAAACAAGGCG-3'
Fwd-R:5'-AAA actagtcctaggACGTTCCCCAGCTGCAGGGGCTT-3'
Rev-F:AAAgcggccgcGAGCCGATAATTTGATATACGC
Rev-R:TATTgggcccGGAGCGAGAGATGAAAGAATACT
引物用超纯水稀释至10μM,-20℃保存备用。
2.1.2转移载体pFR的构建
以HVT FC-126株感染CEF病变细胞的总DNA为模板,分别以Fwd-F/Fwd-R和Rev-F/Rev-R为引物扩增Fwd、Rev同源臂,回收目的片段。回收产物与pEASY TM-T3克隆载体进行连接转化。测序鉴定正确的克隆,分别命名为pT-Fwd和pT-Rev,提取质粒,置于-20℃冰箱保存备用。
将质粒pT-Fwd和pCR2.1用KpnⅠ、SpeⅠ进行酶切,1%凝胶电泳后切胶回收pT-Fwd载体中的Fwd片段和线性化pCR2.1载体。将回收后的Fwd片段和线性化载体pCR2.1连接,构建质粒p-F。将质粒pT-Rev和p-F用NotⅠ和ApaⅠ进行酶切,1%凝胶电泳后切胶回收pT-Rev载体中的Rev片段和线性化载体p-F,将回收后的Rev片段和线性化载体p-F连接,构建质粒p-FR。
2.2真核表达载体pT-cmv-VP2的构建
2.2.1引物设计
参照GenBank中登录的IBDV强毒株F52/70株的基因序列、MHCIss和MITD序列,人工合成嵌合型M-VP2抗原序列,在该序列的两端合成引物M-VP2-F/M-VP2-R。参考载体pcDNA3.4载体序列,用Primer Premier 5.0软件设计引物CMV-PA-F/CMV-PA-R,该扩增片段包含完整的基因表达盒,由CMV启动子、M-VP2基因、WPRE元件和TkpolyA终止子组成。设计的两对引物分别用于PCR扩增嵌合型VP2基因和其表达盒(如图3所示)。扩增pcDNA3.4载体表达盒引物的5’端引入NotⅠ位点,引物3’端引入AvrⅡ位点。
CMV-PA-F:5’-GTATAGCGGCCGC GCTTCGCGATGTACGGGCCAGATA-3’
CMV-PA-R:5’-GAAATCCTAGG GTGGGGATACCCCCTAGAGCCCCA-3’
M-VP2-F:5’-ATGGGGCCGTGCGGGGCGCTGG-3’
M-VP2-R:5’-TTAGATGGCGGGGTTGCTCC-3’
引物用超纯水稀释至10μM,-20℃保存备用。
2.2.2嵌合型M-VP2基因的扩增
以质粒pCU19-M-VP2为模版,M-VP2-F/M-VP2-R为引物,进行PCR扩增,反应结束后进行1%琼脂糖凝胶电泳(如图3所示),回收目的条带,回收产物放置-20℃冰箱保存备用;其中M-VP2基因的核苷酸序列为SEQ ID NO:1。
2.2.3 PCR产物连接与阳性克隆的鉴定
将上述回收的目的片段定向连接到真核表达载体pcDNA3.4中,PCR鉴定阳性克隆送测序,测序正确的克隆命名为pc-M-VP2。
2.2.4克隆载体pT-cmv-M-VP2的构建
以质粒pc-M-VP2为模板,以CMV-PA-F/CMV-PA-R为引物,PCR扩增VP2基因表达盒。反应结束后,PCR产物进行1%琼脂糖凝胶电泳。回收大小约为2700bp左右目的条带(如图3所示)。回收产物连接pEASYTM-T3克隆载体,鉴定正确的阳性克隆命名为pT-cmv-M-VP2。
2.3转移载体pFR-EGFP和pFR-M-VP2的构建
质粒pT-EGFP、pT-cmv-M-VP2和p-FR用NotⅠ、AvrⅡ进行双酶切。1%凝胶电泳后,回收NotⅠ-EGFP-AvrⅡ、NotⅠ-M-VP2-AvrⅡ片段,与同样经NotⅠ、AvrⅡ酶切的线性化载体p-FR进行连接。构建的阳性克隆分别命名为pFR-EGFP、pFR-M-VP2。
2.4转移载体的鉴定表达
2.4.1载体pFR-EGFP的鉴定
将构建成功的转移载体pFR-EGFP瞬时转染到长至80%的DF1细胞培养皿中,继续培养18~24h后,荧光显微镜下观察绿色荧光蛋白的表达情况(如图4所示)。
2.4.2载体pFR-VP2的鉴定
①Western-blot试验
转移载体pFR-M-VP2转染DF1细胞,继续培养24h后收集细胞。Western-blot试验鉴定蛋白表达,其中一抗选用传染性法氏囊病毒的阳性血清,二抗选择HRP标记的羊抗鸡IgG(结果如图5所示)。
②间接免疫荧光试验(IFA)
转移载体pFR-M-VP2瞬时转染于铺有DF1细胞的96孔板中,24h后进行间接免疫荧光试验,其中一抗选用传染性法氏囊病毒的阳性血清,二抗选用异硫氰酸荧光素(FITC)标记羊抗鸡IgG,倒置荧光显微镜下观察转染细胞的荧光情况。
2.5表达EGFP的重组火鸡疱疹病毒(rHVT-EGFP)构建
2.5.1 HVT FC-126株基因组的提取
按常规方法制备CEF,HVT FC-126毒株接种于长满单层CEF的细胞瓶中,37℃、5%CO2细胞培养箱中培养60~84h,待80%的细胞产生典型蚀斑病变后,收集细胞,用于提取病毒DNA。具体操作步骤如下:弃掉培养液,PBS洗三次,每T75细胞瓶加入5ml PK(20mM Tris-Hcl,pH 8.0;0.5%SDS;150mM NaCl;2mM EDTA,pH 8.0;0.2mg/mL胰蛋白酶)溶液,37℃培养箱静置2h;细胞裂解物转移至50ml离心管中,加入1/2体积的饱和酚(使蛋白变性),轻轻颠倒混匀,作用1min;加入1/2体积的的氯仿/异戊醇(24:1)溶液,轻轻混匀,8000r/min离心15min;吸取上清于新离心管中,再次加入等体积的氯仿/异戊醇抽提杂蛋白,8000r/min离心5min;吸取上清于新离心管中,加入2倍体积预冷的无水乙醇和1/10体积的3M醋酸钠(pH5.2),-20℃静置30min;4500r/min离心5min,弃上清,加入10ml预冷的70%乙醇清洗DNA沉淀物,4500r/min离心10min,弃上清,瞬离吸干上清;自然干燥后,加适量TE缓冲液溶解DNA;紫外分光光度计测定DNA的浓度及纯度,4℃保存备用。
2.5.2表达EGFP的重组病毒拯救
转染前制备次代CEF细胞,细胞生长于加入M199培养基(含3%类胎牛血清)的60mm培养皿中,待细胞长至铺满单层的80%时,配制转染体系,进行转染。采用磷酸钙共沉淀法拯救重组病毒,配制体系如下:
上述体系混匀后,从管底缓慢加入2M CaCl2 31μl;缓慢加入2×HBSP溶液(10mg/ml Herpes;0.74mg/ml KCl;16mg/ml NaCl;0.25mg/ml Na2HPO4;2mg/ml葡萄糖,pH值调至6.96)250μl,轻轻颠倒混匀,室温静置30min;向制备次代CEF的60mm培养皿中加入上述混合物,37℃,5%CO2条件下继续培养。转染细胞培养4h后,配制甘油休克液,对细胞进行休克。配制2.5ml体系如下:
取出细胞培养皿,弃掉培养基,用M199培养基洗涤细胞2~3次;加甘油休克液静置1min;再次用M199培养基清洗细胞2~3次;加适量3%M199培养基(含3%类胎牛血清);37℃,5%CO2条件下培养5~7d,荧光显微镜下观察,挑选带有绿色荧光的病毒蚀斑。
2.5.3重组病毒rHVT-EGFP的纯化
荧光显微镜下观察转染细胞,用记号笔标注出带有绿色荧光的病毒蚀斑,胰酶消化法挑取标记的病变细胞,接种于次代CEF单层细胞上,37℃,5%CO2条件下培养3~4d;重复上述步骤,至所有蚀斑均显示荧光,完全纯化的重组病毒命名为rHVT-EGFP(如图6所示)。
2.6表达IBDV嵌合型VP2基因重组火鸡疱疹病毒的构建
将重组病毒rHVT-EGFP接种于原代CEF细胞上,待80%细胞出现特征性病变时,提取细胞基因组,提取步骤同上。依据同源重组原理,将转移载体pFR-M-VP2与rHVT-EGFP基因组磷酸钙法转染次代CEF细胞,转染方法同上。若rHVT-EGFP基因组DNA与转移载体在细胞内发生同源重组,即转移载体中M-VP2基因表达盒将重组病毒rHVT-EGFP基因组中的EGFP表达盒替换,产生新的重组病毒蚀斑将不含有绿色荧光。筛选不带荧光的病毒蚀斑,采用有限稀释法不断筛选纯化,直至培养皿中所有蚀斑均不带有荧光,拯救成功重组病毒命名为rHVT-VP2。于2018年9月7日保藏在位于中国武汉、武汉大学的中国典型培养物保藏中心,保藏编号为CCTCC NO:V201845。
2.7重组病毒的传代及鉴定
2.7.1 PCR鉴定
rHVT-VP2在CEF连续传20代,每隔5代次,提取重组病毒rHVT-VP2的基因组,以该基因组为模板,以M-VP2-F/M-VP2-R、CMV-PA-F/CMV-PA-R、Fwd-F/Rev-R为引物进行PCR,分别可扩增出1300bp、2300bp、4000bp左右的目的条带,表明嵌合型M-VP2基因成功插入到HVT基因组中,并未出现基因丢失现象(如图7所示)。
2.7.2 Western-Blot试验鉴定
重组病毒rHVT-VP2接种原代CEF细胞,3~4d后收集病变细胞制备蛋白样品。western-blot检测蛋白表达情况,试验中用传染性法氏囊病毒阳性血清作为一抗,用HRP标记的羊抗鸡IgG作为二抗。试验中设置阴性对照(用rHVT-EGFP细胞毒制备蛋白样品)。增强型化学发光法(ECL)显色,结果表明,由接种rHVT-VP2的CEF制备的蛋白样品可以扩增出预期的特异性条带(如图8所示)。
2.7.3间接免疫荧光试验(IFA)鉴定
将重组病毒rHVT-VP2接种于长满单层CEF的96孔细胞培养板中,37℃,5%CO2条件下培养,待细胞出现典型蚀斑后,将培养液弃掉,用80%丙酮固定10min,PBS洗3次,每个空中加入50μL(1:100稀释)Flag单抗,37℃恒温培养箱中孵育1h,用PBS洗3次,每孔加50μLFITC标记抗鼠IgG荧光抗体,放置37℃恒温培养箱中孵育1h,用PBS洗3次,在倒置荧光显微镜下观察,rHVT-VP2感染孔出现特异性绿色荧光,而HVT感染孔无荧光,表明rHVT-VP2病毒株中HA基因能够正确的表达(如图9所示)。
2.8重组病毒对IBDV强毒的免疫保护评价
将40只1日龄SPF鸡随机分成两组,分别为rHVT-VP2组、攻毒对照组;其中rHVT-VP2组于1日龄时通过颈部皮下接种5000PFU保藏编号为CCTCC NO:V201845的重组疫苗,攻毒对照组接种无菌PBS。各组于28日龄时,用IBDV超强毒SNJ93株经滴鼻、点眼途径进行攻毒,攻毒剂量为100LD50,攻毒后每天观察并统计各组鸡的发病和死亡情况,从表中可以看出rHVT-VP2组鸡的攻毒保护率可达100%,而攻毒对照组的保护率为0%。
组别 | 攻毒用毒株 | 发病率 | 死亡率 | 攻毒保护率 |
rHVT-VP2组 | SNJ93 | 0/20 | 0/20 | 100% |
攻毒对照组 | SNJ93 | 20/20 | 14/20 | 0% |
每组随机挑选10羽SPF鸡,从1日龄开始,每隔7天采血,分离血清用Synbiotics公司的传染性法氏囊病抗体检测ELISA试剂盒,检测抗体效价;检测主要步骤为:①血清样品做100倍稀释,②酶标仪所用波长为405-410nm,③效价计算公式为log10Titer=1.172(log10S/P)+3.614;④S/P值>0.299;Titer值大于998.8,判定阳性。通过抗体效价的监测来揭示鸡群免疫重组疫苗后外源基因在体内的表达水平,抗体检测结果见下表所示。
免疫评价结果表明,鸡群免疫表达IBDV VP2基因的重组火鸡疱疹病毒后,可以对IBDV强毒株的攻击提供良好的免疫保护。
SEQUENCE LISTING
<110> 青岛易邦生物工程有限公司
<120> 一种表达传染性法氏囊病毒VP2基因的重组火鸡疱疹病毒株
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1356
<212> DNA
<213> 1
<400> 1
atggggccgtgcggggcgctgggcctggggctgctgctcgccgccgtgtgcggggcggcggcccccatgaacacacagat
gacaaacctgcaagatcaaacccaacagattgttccgttcatacggagccttctgatgccaacaaccggaccggcgtc
cattccggacgacaccctggagaagcacactctcaggtcagagacctcgacctacaatttgactgtgggggacacagggt
cagggctaattgtctttttccctggattccctggctcaattgtgggtgctcactacacactgcagagcaatgggaactac
aagttcgatcagatgctcctgactgcccagaacctaccggccagctacaactactgcagactagtgagtcggagtctcac
agtgaggtcaagcacactccctggtggcgtttatgcactaaacggcaccataaacgccgtgaccttccaaggaagcctga
gtgaactgacagatgttagctacaatgggttgatgtctgcaacagccaacatcaacgacaaaattgggaatgtcctggta
ggggaaggggtcactgtcctcagcctacccacatcatatgatcttgggtatgtgaggcttggtgaccccattcccgctat
agggcttgacccaaaaatggtagctacatgcgacagcagtgacaggcccagagtctacaccataactgcagccgatgatt
accaattctcatcacagtaccaaccaggtggggtaacaatcacactgttctcagccaacattgatgctatcacaagcctc
agcattgggggagagctcgtgtttcaaacaagcgtccaaggccttgtactgggcgccaccatctaccttataggctttga
tgggactgcggtaatcaccagagctgtggccgcagataatgggctgacggccggcaccgacaatcttatgccattcaatc
ttgtcattccaaccaatgagataacccagccaatcacatccatcaaactggagatagtgacctccaaaagtggtggtcag
gcaggggatcagatgtcatggtcggcaagtgggagcctagcagtgacgatccatggtggcaactatccaggggccctccg
tcccgtcacactagtagcctacgaaagagtggcaacaggatccgtcgttacggtcgctggggtgagtaacttcgagctga
ttccaaatcctgaactagcaaagaacctggttacagaatacggccgatttgacccaggagccatgaactacacaaaattg
atactgagtgagagggaccgtcttggcatcaagaccgtctggccaacaagggagtacactgattttcgtgagtacttcat
ggaggtggccgacctcaactctcccctgaagattgcaggagcatttggcttcaaagacataatccgggctataaggtaca
aagatgtggagccgccacagcccaacctggtgcccatcgtggcgggggtggccgtcgccattgtggccattgccatcatg
gttggtgttggattcatcatctacagacgccatgcagggaagaaggggaagggctacaacatcgcgcccgggagca
accccgccatctaa
<210> 2
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<212> DNA
<213> 2
<400> 2
TTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACAT
AACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATA
GTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGT
GTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCT
TATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATC
AATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA
CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGG
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AGACACCGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGCCCTTTCGTCGATCGCAGCGATGACAAACCTGCAAGATC
AAACCCAACAGATTGTTCCGTTCATACGGAGCCTTCTGATGCCAACAACCGGACCGGCGTCCATTCCGGACGACACCCTG
GAGAAGCACACTCTCAGGTCAGAGACCTCGACCTACAATTTGACTGTGGGGGACACAGGGTCAGGGCTAATTGTCTTTTT
CCCTGGATTCCCTGGCTCAATTGTGGGTGCTCACTACACACTGCAGAGCAATGGGAACTACAAGTTCGATCAGATGCTCC
TGACTGCCCAGAACCTACCGGCCAGCTACAACTACTGCAGACTAGTGAGTCGGAGTCTCACAGTGAGGTCAAGCACACTC
CCTGGTGGCGTTTATGCACTAAACGGCACCATAAACGCCGTGACCTTCCAAGGAAGCCTGAGTGAACTGACAGATGTTAG
CTACAATGGGTTGATGTCTGCAACAGCCAACATCAACGACAAAATTGGGAATGTCCTGGTAGGGGAAGGGGTCACTGTCC
TCAGCCTACCCACATCATATGATCTTGGGTATGTGAGGCTTGGTGACCCCATTCCCGCTATAGGGCTTGACCCAAAAATG
GTAGCTACATGCGACAGCAGTGACAGGCCCAGAGTCTACACCATAACTGCAGCCGATGATTACCAATTCTCATCACAGTA
CCAACCAGGTGGGGTAACAATCACACTGTTCTCAGCCAACATTGATGCTATCACAAGCCTCAGCATTGGGGGAGAGCTCG
TGTTTCAAACAAGCGTCCAAGGCCTTGTACTGGGCGCCACCATCTACCTTATAGGCTTTGATGGGACTGCGGTAATCACC
AGAGCTGTAGCCGCAGATAATGGGCTGACGGCCGGCACCGACAATCTTATGCCATTCAATCTTGTCATTCCAACCAATGA
GATAACCCAGCCAATCACATCCATCAAACTGGAGATAGTGACCTCCAAAAGTGGTGGTCAGGCAGGGGATCAGATGTCAT
GGTCGGCAAGTGGGAGCCTAGCAGTGACGATCCATGGTGGCAACTATCCAGGGGCCCTCCGTCCCGTCACACTAGTAGCC
TACGAAAGAGTGGCAACAGGATCCGTCGTTACGGTCGCTGGGGTGAGTAACTTCGAGCTGATTCCAAATCCTGAACTAGC
AAAGAACCTGGTTACAGAATACGGCCGATTTGACCCAGGAGCCATGAACTACACAAAATTGATACTGAGTGAGAGGGACC
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CGGATTACAAGGACGATGACGATAAGGAATTCTAGTAATGAGTTTAAACGGGGGAGGCTAACTGAAACACGGAAGGAGAC
AATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAA
CGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTC
CTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAG
CAG
Claims (6)
1.一种在火鸡疱疹病毒中插入外源基因的方法,其特征在于,所述的方法是将外源基因插入火鸡疱疹病毒的插入位点US2区。
2.如权利要求1所述的方法,其特征在于,所述的插入位点位于HVT基因组US2区的140276nt-140285nt之间。
3.一种重组火鸡疱疹病毒,其特征在于,所述的重组火鸡疱疹病毒是通过在火鸡疱疹病毒的US2区插入表达IBDV嵌合型VP2基因所构建;所述的表达IBDV嵌合型VP2基因,是在VP2基因的5′端添加Gallus的MHCI的信号肽序列,3′端添加MHCI的跨膜区和胞内区MTTD的核苷酸序列组成。
4.如权利要求4所述的重组火鸡疱疹病毒,其一种为重组火鸡疱疹病毒(rHVT-VP2)株,于2018年9月7日保藏在位于中国武汉、武汉大学的中国典型培养物保藏中心,保藏编号为CCTCC NO:V201845。
5.权利要求4或5所述的重组火鸡疱疹病毒在表达IBDV嵌合型VP2蛋白中的应用。
6.权利要求4或5所述的重组火鸡疱疹病毒在制备疫苗中的应用。
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