CN116200347A - 一种gI、gE和TK三基因缺失株猫疱疹病毒疫苗及其应用 - Google Patents
一种gI、gE和TK三基因缺失株猫疱疹病毒疫苗及其应用 Download PDFInfo
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- CN116200347A CN116200347A CN202211138110.5A CN202211138110A CN116200347A CN 116200347 A CN116200347 A CN 116200347A CN 202211138110 A CN202211138110 A CN 202211138110A CN 116200347 A CN116200347 A CN 116200347A
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Abstract
本发明提供一种猫疱疹病毒gI、gE和TK基因缺失株及其制备方法和应用,通过CRISPR/Cas9介导的同源重组技术缺失猫疱疹病毒gI、gE和TK基因片段得到一株缺失毒株。是由亲本猫疱疹病毒FHV WX19株进行gE、gI和TK基因缺失后制备的,并保藏于中国典型培养物保藏中心;保藏地址为:中国武汉,武汉大学,邮编430072;保藏日期为:2022年7月26日,保藏编号为CCTCC NO:V202260。
Description
技术领域
本发明属于病毒学基因工程技术和预防兽医学技术领域,特别涉及疫苗弱毒株制备技术领域,具体涉及一种gI、gE和TK三基因缺失株猫疱疹病毒疫苗及其应用,并进一步公开其构建方法,以及该基因缺失毒株的应用。
背景技术
猫疱疹病毒Ⅰ型(Feline herpesvirus,FHV-1)属于疱疹病毒科,α-疱疹病毒亚科成员,是世界范围内猫科动物的重要病原。FHV-1主要感染幼猫,感染后可在上呼吸道黏膜细胞内复制并导致机体体温升高、精神沉郁、咳嗽、打喷嚏等临床症状甚至引起慢性鼻窦炎、角膜结膜炎等并发症,幼猫致死率可达50%。和其他疱疹病毒一样,疱疹病毒也具有潜伏感染的特点,感染猫康复后,FHV-1主要潜伏于三叉神经、视神经节内,可造成机体的持续性感染和潜伏性感染,当机体受刺激后还可再次激活引起猫的再次发病,给该病的诊断和防治带来极大的困扰。自从1958年美国科学家Crandell和Maurer首次从患病幼猫的气管内分离到该病毒后,全球各国陆续有该病的报道。近年来甚至有国内东北虎、华南虎感染FHV-1的相关报道。目前,FHV-1呈世界性流行,作为上呼吸道感染和眼部损伤的主要病原严重的危害宠物猫及野生猫科动物的健康。
FHV-1、猫杯状病毒(FCV)和猫泛白细胞减少症病毒(FPV)疫苗是猫最核心疫苗。据估计,在所有被诊断为猫的病毒性上呼吸道感染中,FHV-1约占50-75%。世界小动物兽医协会(WSAVA)建议是在小猫6-8周龄时接种预防FHV-1感染的疫苗,然后在2-4周后至少再接种一次。灭活和改良活病毒(MLV)猫疱疹病毒疫苗广泛使用,但与FCV和FPV相比,FHV-1感染的保护效果有限且持续时间较短。目前使用的FHV-1疫苗可以降低临床症状的严重程度,但它们本身并不能预防以前接种过疫苗猫的FHV-1感染,因此不能减少FHV-1潜伏期的形成。环境和生理压力因素的改变都可能导致潜伏期的病毒重新激活。
目前市面上FHV-1有灭活和改良的活疫苗。然而,这些疫苗只能减少疾病的临床症状,但不能预防感染,导致潜伏期建立或重新激活。此外,商业疫苗在猫鼻内给药时显示出残留毒力。因此,有必要构建一种安全的第二代疫苗,该疫苗可以通过粘膜给药并有效地防止临床症状、感染和潜伏期。
FHV-1的基因组大小为126-134kbp,总体G+C含量为45%-50%。FHV-1的基因组由两段独特的DNA组成,被称为长独特区(UL)和短独特区(US)。基因组的US区两侧有一对相同但倒置的序列,被称为内部重复短序列(IRS)和末端重复短序列(TRS)。FHV-1基因组可编码约70多种蛋白质,主要包括毒力蛋白、结构蛋白和复制酶等。在FHV-1基因组的UL区已经绘制了56个基因,主要编码帽状糖蛋白gB、gC、gK、gL、gM、gN、CIRC、核苷酸还原酶(RR)和胸苷酸激酶(TK)。US区主要编码膜糖蛋白gD、gE、gG、gI和丝氨酸/苏氨酸激酶PK。FHV-1的gE和gI基因序列位于US区。Sussman MD等人构建了一种缺乏gI基因的C端和gE基因的5'编码区的重组FHV-1,发现这种重组病毒复制缓慢。对小猫鼻腔或皮下注射重组病毒FHV-1,小猫只表现出轻微的临床症状,机体产生了强烈的免疫保护。Lee等人发现,缺失TK基因的重组病毒毒力降低,对猫的致病性减弱,发现TK基因与病毒毒力有关。
自1990年以来,FHV-1一直是研究猫科动物载体疫苗的首选对象。最近几年CRISPR/Cas9技术被广泛应用于病毒编辑、疫苗研制等方面。通过产生安全的基因缺失病毒,可以通过皮下和粘膜两种途径的组合给药,刺激更全面的免疫反应。基因缺失疫苗的另一个优点“标记”疫苗,可以区分疫苗病毒和流行的野病毒,以及疫苗接种与野病毒感染引起的免疫反应。基因缺失病毒已在伪狂犬病病毒(PRV)、牛疱疹病毒1(BHV-1))和马疱疹病毒1(EHV-1)广泛应用。此外,Kimman等人报道鼻内接种双缺失gE和PK或双缺失gE和TK重组病毒的猪排毒量减少。
FHV-1的gI和gE糖蛋白可以形成异源二聚体,在病毒的细胞间传播和整个宿主神经系统的感染传播中发挥作用。FHV-1的gI和gE复合体对病毒生长是非必须的,但当部分gI和gE基因缺失时,致病毒的毒力显著降低。幼猫经口鼻免疫FHV-1gI/gE缺失株,随后用高剂量野毒攻击只表现出轻微的临床症状。FHV-1TK基因编码胸腺激酶,对病毒复制不发挥作用。宿主细胞复制生长中,本身可以产生胸腺激酶,FHV-1在复制过程中可以利用这些激酶完成病毒增殖。缺少TK基因不会影响病毒在细胞中的复制增殖,但会影响FHV-1感染机体后神经嗜性过程,缺少TK基因会降低病毒的毒力。在一项研究中,鼻内接种FHV-1TK缺失重组病毒会导致打喷嚏,并且在野毒攻击后,在口腔、鼻腔和眼部分泌物中检测到病毒。结合提及的gI/gE基因,因此选择将gI、gE和TK基因同时缺失是一个更好的疫苗策略。但同时缺失FHV-1三种基因的疫苗株并没有相关的研究,而且利用传统的基因编辑技术(同源重组、细菌人工染色体和Fosmid文库)操作困难、重组效率低和筛选周期长。本发明利用CRISPR/Cas9介导的基因组编辑技术可以实现多个位点的同时编辑,大大提高了重组成功率和缩短试验周期。高效的sgRNA是影响CRISPR/Cas9基因编辑效率的重要因素。由于FHV-1基因组GC含量高,筛选有效的sgRNA比较困难。本发明前期通过大量的噬斑实验筛选高效切割gI、gE和TK基因片段的sgRNA组合,为提高筛选基因缺失株奠定了基础。
发明内容
本发明的提供一种猫疱疹病毒gI、gE和TK基因缺失株及其制备方法和应用,通过CRISPR/Cas9介导的同源重组技术缺失猫疱疹病毒gI、gE和TK基因片段得到一株缺失毒株。
本发明进一步提供用于编辑猫疱疹病毒的sgRNA组合,所述sgRNA组合包含靶序列为猫疱疹病毒的gI、gE和TK基因片段的sgRNA,包括SEQ ID NO:3-SEQ ID NO:10所示核苷酸序列。
本发明进一步提供包含所述sgRNA组合的生物材料,所述生物材料为表达盒、载体或转基因细胞。
本发明进一步对该猫疱疹病毒gI、gE和TK基因缺失株的生长特性进行鉴定,结果如下:猫疱疹病毒缺水株具有良好的遗传稳定性,生长特性与亲本毒株FHV WX19株相似。
本发明首先提供一种猫疱疹病毒基因缺失株,是由亲本猫疱疹病毒FHV WX19株进行gE、gI和TK基因缺失后制备的,并保藏于中国典型培养物保藏中心;保藏地址为:中国武汉,武汉大学,邮编430072;保藏日期为:2022年7月26日,保藏编号为CCTCCNO:V202260。
本发明的目的地提供一种重组猫疱疹病毒三基因缺失及应用,即将猫疱疹病毒的gI、gE和TK基因进行缺失来获得弱毒株,并制备疫苗。
所述的疫苗为活疫苗。
本发明还提供上述的猫疱疹病毒基因缺失株在制备中的应用;
本发明在一个方面提供一种猫疱疹病毒疫苗,由抗原和保护剂组成的,其中抗原包含有上述的猫疱疹病毒缺失毒株。
附图说明
图1:重组质粒示意图;
图2:纯化病毒绿色荧光鉴定图(×200)
图3:重组病毒一步生长曲线
图4:免疫组和攻毒组28日体温变化结果
图5:免疫组和攻毒组28日临床评分结果
图6:免疫组和对照组攻毒后14日体温变化结果
图7:免疫组和对照组攻毒后14日临床评分结果
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
本发明所使用的猫疱疹病毒FHV WX19流行毒株,由我实验室分离鉴定保存并进行保藏。
实施例1:sgRNA表达载体的构建
参考发表在GenBank的FHV全基因组序列,将缺失病毒基因组序列输入sgRNA在线设计网站(http//crispr.mit.edu),寻找有PAM(NGG)的sgRNA序列,选择脱靶率较低的sgRNA,将其克隆至lentiCRISPR V2载体(北京慕鸥生物科技有限公司,Addgene),sgRNA序列(SEQ ID NO:3-10)如表1所示。设计合成如表1所示:
表1 sgRNA引物
所述oligos的磷酸化修饰与退火体系包括:
oligo 1(100μM)1μ;
oligo 2(100μM)1μL;
去离子水补至10μL;
PCR仪95℃处理5min,按照每分钟降低5℃最后降至25℃
质粒酶切,反应体系包括:
lentiCRISPR V2质粒2μg;
BsmBI 2μL;
10X Buffer 2μL
去离子水补至40μL;
于37℃进行酶切3h,跑胶并回收。
上述oligos与酶切后的载体连接体系包括:
Oligos稀释1001μL;
lentiCRISPR V2 10ng;
10xNEB连接酶buffer 1μL;
NEB连接酶1μL;
去离子水补至10μL;
于室温下连接4-6h,进行转化,挑取克隆,测序正确的进行下一步。
实施例2:转移质粒pT-eGFP和pT-mCherry的构建
将FHV WX19株的gI/gE缺失位置的上下游同源片段和eGFP表达盒子(SEQ ID NO:1)依次插入质粒pMD19T中,构建同源重组转运载体pT-eGFP;将FHV WX19株的TK缺失位置的上下游同源片段和mCHerry表达盒子(SEQ ID NO:2)依次插入质粒pMD19T中,构建同源重组转运载体pT-mCherry。扩增引物序列如表2所示。
表2与转移质粒构建相关的引物序列
实施例3:重组病毒的构建
使用Lipofectamin 3000转染方法在CRFK细胞中将Cas9质粒sgRNA-gI/gE和sgRNA-TK,转运载体质粒pT-eGFP和pT-mCherry共转染18小时。共转染后,用FHV WX19株感染细胞(MOI=0.1)。细胞维持在37℃、5% CO2的培养箱中,当90%的CPE发生时,收集细胞并进行三次冻融。
实施例4:重组病毒的纯化与鉴定
首先,将CRFK细胞传代于六孔细胞板,待细胞汇合度达到90%-100%时,弃掉原细胞培养液并用PBS洗涤2次;同时,将病毒原液用EMEM进行倍比稀释,将稀释好的病毒以10-2-10-5均匀接种于细胞板上,在37℃温箱中孵育3h,弃掉病毒液;将2%的低熔点琼脂糖溶液(60℃保存)与2×细胞维持液按1:1的比例混匀后,加入各培养孔中,2ml/孔,冷却后凝固成覆盖层。通过荧光显微镜挑选双荧光空斑,经过7轮纯化后,获得重组的FHV△gIgE/TKeGFP-mCherry毒株(图2),并将其纯化后保藏于中国典型培养物保藏中心;保藏地址为:中国武汉,武汉大学,邮编430072;保藏日期为:2022年7月26日,保藏编号为CCTCC NO:V202260。
实施例5:重组病毒的生长特性的测定
FHV WX19株和FHV△gIgE/TK eGFP-mCherry株分别按照MOI=0.1比例接种铺满单层的CRFK细胞,置于培养箱内继续培养。每隔6h收取病毒液测定TCID50,并绘制一步生长曲线(图3)。结果显示FHV△gIgE/TK eGFP-mCherry和亲本株FHV WX19的生长曲线基本一致。
实施例6:重组病毒的安全性评价
6.1试验方法:选取3~4月的猫疱疹病毒阴性布偶猫12只;随机分为3组,每组4只幼猫,其中一组的幼猫滴鼻接种FHV△gIgE/TK eGFP-mCherry株(106.0TCID50/只),另一组的幼猫滴鼻接种FHV WX19株(106.0TCID50/只),第3组滴鼻接种1mL的EMEM作为对照组。接种后观察28天,每天测量体温,观察和记录临床症状。
6.2试验结果:如图4-5所示,FHV△gIgE/TK eGFP-mCherry株接种后,幼猫体温正常,仅一只幼猫出现打喷嚏症状,而FHV WX19接种幼猫后则出现高热、打喷嚏、流眼泪、死亡,表明gI、gE和缺失TK基因后的FHV△gIgE/TK eGFP-mCherry已充分致弱,对幼猫不致病。
实施例7:重组病毒的免疫保护力测定
7.1试验方法:选取3~4月的猫疱疹病毒阴性布偶猫8只;随机分为2组,每组4只幼猫,在0和28天滴鼻免疫106TCID50的病毒,在28天加强皮下免疫106TCID50,在49天用105TCID50 FHV WX19滴鼻感染病毒。攻毒后观察14天,每天测量体温,观察和记录临床症状。
7.2试验结果:如图6-7所示,FHV WX19攻毒后对照组所有幼猫出现高热、打喷嚏、流眼泪等临床症状,FHV△gIgE/TK eGFP-mCherry株接种组,幼猫体温正常,仅一只幼猫出现轻微打喷嚏症状,表明FHV△gIgE/TK eGFP-mCherry株对幼猫具有良好的保护能力。
目前,猫病毒性上呼吸道感染中FHV-1被诊断为约占50-75%,已成为猫上呼吸道传染病的重要传染源。针对FHV-1的疫苗效果并不理想,可以降低临床症状的严重程度,但保护效果有限且持续时间较短,并不能达到预防FHV-1的效果。疱疹病毒是一种理想的病毒载体,既有灭活疫苗安全的优良又有活疫苗的免疫效果好、成本低等优点,同时又能诱导免疫机体产生广泛的体液免疫和细胞免疫。与FHV-1毒力相关的基因有gI、gE、PK和TK。通过缺失相应的毒力基因,FHV-1的毒力会显著减弱。前期Yokoyama等研究通过缺失TK基因构建C7301d/TK,在通过不同途径多次接种重组C7301d/TK后,尽管靶器官有高滴度的病毒排出,但用亲本FHV-1株C7301攻毒后有显著的保护作用。Kruger等通过对FHV-1gIgE基因进行缺失构建FHVβ-galgIgE重组株,比较了滴鼻和皮下接种途径,并评估了它诱导保护性免疫和防止攻击后病毒脱落的能力。发现搞剂量FHVβ-galgIgE毒株皮下或滴鼻接种的猫只有轻微的临床症状,并用野毒攻击后产生了强大的免疫力。
本研究通过CRISPR/Cas9技术对FHV-1WX19株gI、gE和TK基因进行缺失后制备的FHV△gIgE/TK eGFP-mCherry。通过安全试验FHV△gIgE/TK eGFP-mCherry候选毒株具有良好的安全性;接种FHV△gIgE/TK eGFP-mCherry候选毒株幼猫未出现体温升高、排毒和死亡等症状,而野毒株感染后出现了体温升高、持续打喷嚏、流眼泪和死亡等明显的症状,表明三缺失基因FHV已充分致弱,对幼猫是安全的。通过免疫效力试验FHV△gIgE/TK eGFP-mCherry候选毒株具有良好的免疫效力;通过两次黏膜和一次皮下加强免疫,FHV△gIgE/TKeGFP-mCherry试验组的抗体水平明显升高,仅黏膜免疫后出现短暂的打喷嚏现象。在攻毒后14d内未出现死亡,FHV△gIgE/TK eGFP-mCherry组未出现排毒,仅试验组一只猫出现打喷嚏症状。通过安全试验和免疫效力试验FHV△gIgE/TK eGFP-mCherry毒株具有良好的安全性。
以上所述之实施例,只是本发明的较佳实施例而已,并非限制本发明的实施范围,故凡依本发明专利范围所述的构造、特征及原理所做的等效变化或修饰,均应包括于本发明申请专利范围内。
序列表
上海兽医研究所
一种猫疱疹病毒gI、gE和TK三基因缺失株及其应用10
eGFP表达盒子
1
1079
DNA
人工序列(Artificial Sequence)
1
TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCATGCATTAGTTATTAATAGTAA
TCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGG
TAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACG
TATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTAC
GGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG
ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACT
TTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTG
GCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACC
CCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGT
CGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTA
TATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGTCGCCACCAT
GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC
GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCT
ACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCC
CACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACA
TGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCAC
CATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGC
GACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACA
TCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGAC
AAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCA
GCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT
GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAG
AAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCAT
GGACGAGCTGTACAAGTACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGG
TACCGCGGGCCCGGGATCCACCGGATCTAGATAACTGATCATAATCAGCCATACCACATT
TGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAA
AATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC
AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGT
CCAAACTCATCAATGTATCTTAACGCGTAAATTGTAAGCGTTAATATTT
2
mCherry表达盒子
1591
DNA
人工序列(Artificial Sequence)
2
TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG
TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG
ACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA
TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCA
AGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC
ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT
GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGAT
TTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGG
GACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGT
ACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCGCT
ACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAG
TTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGA
GGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGAC
CAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTC
CAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCG
AGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGAC
CCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACC
AACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTC
CGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAA
GCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAA
GCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACA
ACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGG
CGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTG
CAGTCGACGGTACCGCGGGCCCGGGATCCACCGGATCTAGATAACTGATCATAATCAGC
CATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAAC
CTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTT
ACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGC
3
25
DNA
人工序列(Artificial Sequence)
3
CACCGAGACTACGCCAGTATAGCAT 4
25
DNA
人工序列(Artificial Sequence)
4
AAACATGCTATACTGGCGTAGTCTC 5
25
DNA
人工序列(Artificial Sequence)
5
CACCGGGCGGCGGCACATTCATCAG 625
DNA
人工序列(Artificial Sequence)
6
AAACCTGATGAATGTGCCGCCGCCC 7
25
DNA
人工序列(Artificial Sequence)
7
CACCGACACTCAAAAGAGTGAACGT 8
25
DNA
人工序列(Artificial Sequence)
8
AAACACGTTCACTCTTTTGAGTGTC9
26
DNA
人工序列(Artificial Sequence)
9
CACCGGTATGTTTCGTGGTGTCTCCG 10
26
DNA
人工序列(Artificial Sequence)
10
AAACCGGAGACACCACGAAACATACC
Claims (6)
1.一种gI、gE和TK三基因缺失株猫疱疹病毒,其特征在于,所述的gI、gE和TK三基因缺失株猫疱疹病毒是由亲本猫疱疹病毒FHV WX19株进行gI、gE和TK基因缺失后制备的FHV△gIgE/TK eGFP-mCherry,所述FHV△gIgE/TK eGFP-mCherry保藏于中国典型培养物保藏中心;保藏地址为:中国武汉,武汉大学,邮编430072;保藏日期为:2022年7月26日,保藏编号为CCTCC NO:V202260。
2.权利要求1所述的一种gI、gE和TK三基因缺失株猫疱疹病毒在制备治疗或者预防猫疱疹病毒Ⅰ型的药物中的应用。
3.如权利要求2所述的应用,其特征在于,所述药物为防治猫传染性鼻气管炎疾病的疫苗。
4.如权利要求2所述的应用,其特征在于,所述疫苗为全病毒灭活疫苗、减毒疫苗或基因工程疫苗。
5.一种免疫组合物,其特征在于包括:权利要求1所述一种gI、gE和TK三基因缺失株猫疱疹病毒疫苗;以及,医药学上可接受的载剂。
6.如权利要求5所述的免疫组合物,其特征在于:所述医药学上可接受的载剂包括MONTANIDE ISA 206、MONTANIDE ISA 201、MONTANIDE GEL 01 ST、氢氧化铝胶佐剂、明矾、弗氏佐剂、脂多糖、胆固醇、植物油、细胞因子之中的任意一种或者两种以上的组合;优选为氢氧化铝胶佐剂。
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CN117143924A (zh) * | 2023-09-11 | 2023-12-01 | 华中农业大学 | 共表达猫杯状病毒和猫细小病毒抗原蛋白的重组猫疱疹病毒及其活载体疫苗和应用 |
CN117143924B (zh) * | 2023-09-11 | 2024-03-22 | 华中农业大学 | 共表达猫杯状病毒和猫细小病毒抗原蛋白的重组猫疱疹病毒及其活载体疫苗和应用 |
CN117085119A (zh) * | 2023-10-07 | 2023-11-21 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 一株表达猫杯状病毒vp1基因重组猫疱疹病毒疫苗及其应用 |
CN117085119B (zh) * | 2023-10-07 | 2024-02-09 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 一株表达猫杯状病毒vp1基因重组猫疱疹病毒疫苗及其应用 |
CN117482218A (zh) * | 2023-12-05 | 2024-02-02 | 中国农业科学院北京畜牧兽医研究所 | 一种猫弓形虫病重组活载体疫苗及其应用 |
CN117482218B (zh) * | 2023-12-05 | 2024-07-30 | 中国农业科学院北京畜牧兽医研究所 | 一种猫弓形虫病重组活载体疫苗及其应用 |
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