CN114214291A - 表达禽腺病毒血清8b型纤突蛋白的禽腺病毒血清4型重组病毒及其构建方法和应用 - Google Patents
表达禽腺病毒血清8b型纤突蛋白的禽腺病毒血清4型重组病毒及其构建方法和应用 Download PDFInfo
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Abstract
本发明属于动物基因工程疫苗领域,具体涉及表达禽腺病毒血清8b型(FAdV‑8b)纤突蛋白(Fiber)的禽腺病毒血清4型(FAdV‑4)重组病毒及其构建方法和应用。所述重组病毒是利用FAdV‑8b的Fiber基因替换FAdV‑4 Fiber1基因得到,该重组病毒可用于制备防控鸡肝炎‑心包积液综合征和/或鸡包涵体肝炎的二联疫苗,利用本发明中的重组病毒制备的二联疫苗可以达到打一针疫苗同时预防两种疫病的效果。由此类推,利用本发明中的FAdV‑4作为载体插入外源基因可以达到一针防控两种甚至多种疾病的目的。
Description
技术领域
本发明属于动物基因工程疫苗领域,具体涉及表达禽腺病毒血清8b型纤突蛋白的禽腺病毒血清4型重组病毒及其构建方法和应用。
背景技术
我国是世界养殖大国,养殖业在国民经济中占据重要地位,畜禽疫病是严重阻碍养殖业可持续发展的重要瓶颈之一。禽腺病毒(Fowl aviadenovirus,FAdV)属于腺病毒科、禽腺病毒属,是无囊膜的双链DNA病毒。禽腺病毒属目前包括5个种共12个血清型,即A(FAdV-1)、B(FAdV-5)、C(FAdV-4、-10)、D(FAdV-2、-3、-9、-11)和E(FAdV-6、-7、-8a、-8b)。禽腺病毒在养殖场广泛存在,临床上与禽腺病毒感染相关的疫病主要包括由FAdV-4引起的肝炎-心包积液综合征(Hepatitis-hydropericardium syndrome,HHS)及FAdV-8b和FAdV-11引起的包涵体肝炎(Inclusion body hepatitis,IBH)。HHS主要影响3~6周龄肉鸡,发病鸡以心包充满浅黄色透明液体,肝脏褪色、肿大、密布出血点和坏死为特征,死亡率高达20%~90%;也可感染10~20周龄蛋鸡和种母鸡;其它禽类如鹌鹑、鸽子等感染的案例也有报道。IBH主要影响3~5周龄的鸡,以肝坏死和肝细胞中出现嗜酸性或嗜碱性核内包涵体为主要特征,死亡率可达到10%。自2015年起由FAdV-4新基因型引起的HHS在我国河南、山东、浙江、安徽、吉林、河北、辽宁、江苏、山西和湖北等省区鸡群中大面积流行,给我国养禽业造成了巨大的经济损失。近年来,HHS和IBH临床病例在世界各地不断增多,混合感染也很普遍,给世界养禽业造成了很大损失。
禽腺病毒病毒粒子呈二十面体对称,其衣壳由六邻体(Hexon)、五邻体(Penton)和纤突蛋白(Fiber)构成。禽腺病毒的Fiber是组成病毒衣壳的主要蛋白,突出于病毒粒子表面,构成病毒衣壳的顶端。研究表明Fiber蛋白在介导病毒感染、病毒与细胞相互作用和诱导机体产生病毒中和抗体等方面发挥关键作用。在FAdV-4的基因组中存在两个Fiber蛋白基因Fiber1和Fiber2。Fiber1基因与毒力无关,与病毒的感染有关【Liu R,Zhang Y,Guo H,Li N,Wang B,Tian K,Wang Z,Yang X,Li Y,Wang H,Zhang Y,Fu J,Zhao J.Theincreased virulence of hypervirulent fowl adenovirus 4 is independent offiber-1 and penton.Res Vet Sci.2020 Aug;131:31-37.doi:10.1016/j.rvsc.2020.04.005.;Zou X,Rong Y,Guo X,Hou W,Yan B,Hung T,Lu Z.Fiber1,but notfiber2,is the essential fiber gene for fowl adenovirus 4(FAdV-4).J GenVirol.2021 Mar;102(3).doi:10.1099/jgv.0.001559;Wang W,Liu Q,Li T,Geng T,ChenH,Xie Q,Shao H,Wan Z,Qin A,Ye J.Fiber-1,Not Fiber-2,Directly Mediates theInfection of the Pathogenic Serotype 4 Fowl Adenovirus via Its Shaft and KnobDomains.J Virol.2020 Aug 17;94(17):e00954-20.doi:10.1128/JVI.00954-20.】;而Fiber2不参与病毒的感染过程,但可以诱导针对FAdV-4的中和抗体【Schachner A,MarekA,Jaskulska B,Bilic I,Hess M.Recombinant FAdV-4 fiber-2 protein protectschickens against hepatitis-hydropericardium syndrome(HHS).Vaccine.2014 Feb19;32(9):1086-92.doi:10.1016/j.vaccine.2013.12.056.】。FAdV-8b只有一个Fiber蛋白,既参与病毒对细胞的感染过程,同时又诱导机体产生针对FAdV-8b的中和抗体,前期研究证明FAdV-8b Fiber亚单位疫苗能提供针对FAdV-8b的良好保护【Gupta A,Ahmed KA,Ayalew LE,Popowich S,Kurukulasuriya S,Goonewardene K,Gunawardana T,Karunarathna R,Ojkic D,Tikoo SK,Willson P,Gomis S.Immunogenicity andprotective efficacy of virus-like particles and recombinant fiber proteins inbroiler-breeder vaccination against fowl adenovirus(FAdV)-8b.Vaccine.2017 May9;35(20):2716-2722.doi:10.1016/j.vaccine.2017.03.075.】。
目前国内外防控肝炎-心包积液综合征和包涵体肝炎主要使用传统的灭活疫苗,有用病毒感染鸡的肝脏匀浆液制备的灭活疫苗,或者用细胞增殖的病毒制备的灭活疫苗。这种传统的制备疫苗工艺存在工艺繁琐、生产成本高、容易散播潜在病原,而且还存在接种一次疫苗只能防控一种疫病等缺陷。临床上不同血清型的禽腺病毒常出现混合感染,但国内外目前尚未有用于防控FAdV-4和FAdV-8b混合感染的多联疫苗。传统的多联疫苗的制备需要经过分别培养多种病原,然后将每种病原分别浓缩、灭活,再按照一定比例混合、与疫苗佐剂进行乳化等一系列繁琐的工艺,疫苗生产成本高。因此,研发针对FAdV-4和FAdV-8b中国流行株的高效、廉价多联疫苗具有现实意义。
发明人所在团队和国内其他研究团队的前期研究表明,在中国流行的FAdV-4新基因型基因组中存在1966bp的自然缺失,且该缺失区可以作为外源基因的插入位点。具体的,在缺失区插入绿色荧光蛋白基因构建重组病毒,根据重组病毒与对照(缺失区未插入外源病毒)对鸡的致病性和在细胞上的生长能力可以确定,该缺失区可以作为外源基因插入位点。同时,我们将1966bp的自然缺失区进行人工补充后,并不影响重组病毒的复制和致病性,更进一步说明该缺失区可以作为外源基因的插入位点【Zhang Y,Liu R,Tian K,WangZ,Yang X,Gao D,Zhang Y,Fu J,Wang H,Zhao J.Fiber2 and hexon genes are closelyassociated with the virulence of the emerging and highly pathogenic fowladenovirus 4.Emerg Microbes Infect.2018Dec 5;7(1):199.doi:10.1038/s41426-018-0203-1;Pan Q,Wang J,Gao Y,Cui H,Liu C,Qi X,Zhang Y,Wang Y,Wang X.The NaturalLarge Genomic Deletion Is Unrelated to the Increased Virulence of the NovelGenotype Fowl Adenovirus 4Recently Emerged in China.Viruses.2018 Sep 13;10(9):494.doi:10.3390/v10090494.】。
发明内容
本发明提供了一种表达禽腺病毒血清8b型(FAdV-8b)纤突蛋白(Fiber蛋白)的禽腺病毒血清4型(FAdV-4)重组病毒,其构建方法与现有技术不同,具体为:运用FAdV-4反向遗传技术平台,以FAdV-4作为载体,构建其全长基因组感染性克隆,在大肠杆菌中利用同源重组技术将FAdV-4的Fiber1基因替换为FAdV-8b的Fiber基因,构建表达FAdV-8bFiber蛋白的FAdV-4重组病毒,本发明为防控FAdV-4和FAdV-8b的混合感染提供安全、有效的二联疫苗,为新型重组疫苗研发提供高效、快速的操作平台。
为实现上述目的,本发明提供的一种表达FAdV-8bFiber蛋白的FAdV-4重组病毒rHNJZ-Fiber/FAdV-8b,所述FAdV-4重组病毒rHNJZ-Fiber/FAdV-8b是用FAdV-8b的Fiber基因替换FAdV-4基因组的Fiber1基因得到,其中,所述FAdV-8b的Fiber基因序列如SEQ IDNO:1所示。所述FAdV-8b的Fiber基因序列扩增自FAdV-8b分离株SDQD2021,该毒株由河南农业大学禽病研究所分离、鉴定及保存。
本发明还提供了一种制备上述表达FAdV-8b Fiber蛋白的FAdV-4重组病毒的方法,包括以下步骤:
1、构建FAdV-4的基因组感染性克隆
将FAdV-4的全长基因组利用核酸外切酶联合重组酶技术一步克隆到含有抗性筛选标记的载体中,得到FAdV-4的基因组感染性克隆。
其中,所述FAdV-4为中国流行株CH/HNJZ/2015。
具体的,以p15A-cm-tetR-tetO-ccdB-hyg质粒DNA为模板,以含有FAdV-4基因组左右两端反向末端重复序列和上、下游引物扩增包含FAdV-4基因组左右两端反向末端重复序列的骨架载体,扩增的骨架载体片段两端含有酶切位点。
提取FAdV-4的病毒基因组,将病毒基因组DNA和上述制备的包含FAdV-4基因组左右两端反向末端重复序列的骨架载体用T4 DNA聚合酶处理。转化筛选,得到含有FAdV-4的基因组感染性克隆。
2、含有双选择标记筛选表达盒的FAdV-4感染性克隆的构建
以含有双选择标记筛选表达盒的表达载体为模板,利用带有FAdV-4的Fiber1基因两侧同源臂的特异性引物进行PCR扩增筛选表达盒,利用Redαβ重组酶介导的同源重组技术,将FAdV-4感染性克隆的Fiber1基因精确替换为筛选表达盒,筛选得到含有筛选表达盒的FAdV-4感染性克隆。
其中,所述双选择标记筛选表达盒为氨苄青霉素抗性(amp)筛选标记和大肠杆菌自杀基因(ccdB)表达盒。
其中,带有FAdV-4的Fiber1基因两侧同源臂的特异性引物:
Fiber1-ampccdB-F:
5’-TATTTTTAACCAATATCTTCTAGGCTCCGCCATTTAATTAATTTGTTTATTTTTCTAAA-3’;
Fiber1-ampccdB-R:
5’-TTCGGAATGTCTTCTTTTAGGGGCCCGGAGCATTTAATTAATTTGTTCAAAAAAAAGCC-3’。
3、构建含有FAdV-8b Fiber基因的FAdV-4感染性克隆
以FAdV-8b病毒基因组为模板,利用带有FAdV-4的Fiber1基因两侧同源臂的特异性引物扩增含有FAdV-8b的Fiber基因片段,然后利用Redαβ重组酶介导的同源重组技术,将FAdV-4感染性克隆的Fiber1基因精确替换为FAdV-8b的Fiber基因,筛选得到含有FAdV-8bFiber基因的FAdV-4感染性克隆。
其中,带有FAdV-4的Fiber1基因两侧同源臂的特异性引物:
8bFiber-F:
5’-CGTTTATTTTTAACCAATATCTTCTAGGCTCCGCCATATGGCGACCTCGACTCCTCACG-3’;
8bFiber-R:
5’-CGTTTTCGGAATGTCTTCTTTTAGGGGCCCGGAGCATTCAAGGAGCGTTGGCGGTGCTT-3’。
4、制备表达FAdV-8b Fiber蛋白的FAdV-4重组病毒
将含有FAdV-8b Fiber基因的FAdV-4感染性克隆用限制性内切酶线性化去除基因组两端的载体序列后,转染鸡胚肝癌细胞系LMH,拯救出表达FAdV-8b Fiber蛋白的FAdV-4重组病毒。
本发明还提供了一种上述表达FAdV-8b Fiber的FAdV-4重组病毒在防治鸡肝炎—心包积液综合征和包涵体肝炎中的应用。
本发明的原理:
本发明利用Red/ET重组技术构建FAdV-4的感染性克隆,在此基础上将FAdV-4基因组中Fiber1基因替换为双选择标记筛选表达盒,然后用FAdV-8b Fiber基因替代双选择标记筛选表达盒,获得在FAdV-4基因组中Fiber1基因替换为FAdV-8bFiber基因的感染性克隆。在此基础上,提供一种表达FAdV-8bFiber基因的FAdV-4重组病毒及其应用。本发明利用大肠杆菌提取含有FAdV-8b Fiber基因的FAdV-4感染性克隆重组质粒,经PmeI限制性内切酶线性化后,转染鸡胚肝癌细胞系(LMH细胞)快速拯救出表达FAdV-8b Fiber蛋白的FAdV-4重组病毒。
本发明的有益效果在于:
1.本发明利用FAdV-4感染性克隆操作平台构建重组病毒,利用氨苄青霉素抗性基因(amp)和大肠杆菌自杀基因(ccdB)双选择标记筛选插入外源基因的重组质粒,所得重组质粒经PmeI限制性内切酶线性化后,转染鸡胚肝癌细胞系(LMH细胞)可以快速拯救出重组病毒。
2.本发明中的FAdV-4感染性克隆重组外源基因后,经PmeI限制性内切酶线性化即可实现对载体序列的全部剔除,无功能性外源序列插入,对病毒基因组也无影响,不存在遗传物质发生跨物种转移的可能。
3.利用本发明中的表达FAdV-8b Fiber蛋白的FAdV-4重组病毒制备的灭活疫苗可以达到同时预防肝炎-心包积液综合征和包涵体肝炎的效果。由此类推,利用本发明中的FAdV-4人工染色体重组外源基因可以达到一针防控两种甚至多种疾病的目的。
4.利用本发明中的表达FAdV-8b Fiber蛋白的FAdV-4重组病毒制备的灭活疫苗对1周龄雏鸡进行1次免疫,机体可在2周后产生抗FAdV-4和FAdV-8b的特异性抗体,一次免疫即可防控两种疫病。
5.传统的二联疫苗的生产需要分别培养两种病原,然后将两种病原分别浓缩至少1倍,再将两种病原分别灭活后取等量进行混合,最后与疫苗佐剂按照一定比例混合、乳化等诸多步骤。而利用本发明中的表达FAdV-8b Fiber蛋白的FAdV-4重组病毒制备灭活疫苗则只需要培养一种病毒,且不需要经过浓缩即可用于制备二联疫苗,生产工艺大大简化,成本降低至少一半。
综上所述,本发明以FAdV-4作为载体,构建其感染性克隆,在大肠杆菌中利用同源重组技术重组FAdV-8b Fiber基因,构建表达FAdV-8b Fiber蛋白的FAdV-4重组病毒。以重组病毒作为种毒制备的灭活疫苗具有安全性高,保存运输方便;不受母源抗体影响,一次免疫即可获得针对FAdV-4和FAdV-8b的特异性抗体,且抗体可以在机体内持续存在。本发明所包含的FAdV-4的反向遗传技术操作平台可为制备防控鸡肝炎-心包积液综合征和鸡包涵体肝炎的安全、高效、廉价二联疫苗,为新型重组疫苗高效、快速研发提供技术支撑。
附图说明
图1为重组质粒p15A-cm-HNJZ的EcoRI酶切鉴定图。图中,M.1kbDNA Marker;泳道1~3为正确重组质粒EcoRI酶切图谱。
图2为重组质粒p15A-cm-HNJZ-Fiber/FAdV-8b的XbaI酶切鉴定图。图中,M.1kbDNA Marker;3~7.正确重组质粒XbaI酶切图谱。
图3为重组病毒rHNJZ-Fiber/FAdV-8b感染LMH细胞病变情况图。图中,A.未转染的LMH细胞对照;B.p15A-cm-HNJZ-Fiber/FAdV-8b转染的LMH细胞。
图4为重组病毒rHNJZ-Fiber/FAdV-8b的PCR鉴定结果图。图中,M.250bp DNALadder;1.重组病毒rHNJZ-Fiber/FAdV-8b的PCR扩增产物;2.FAdV-4亲本株CH/HNJZ/2015的PCR扩增产物;3.LMH细胞基因组的PCR扩增产物。
图5为重组病毒rHNJZ-Fiber/FAdV-8b的Western blot鉴定结果图。图中,M.蛋白质相对分子质量标准(15~180kDa);1~3.第2、5和10代rHNJZ-Fiber/FAdV-8b感染的LMH细胞裂解液;4.FAdV-4亲本株CH/HNJZ/2015感染的LMH细胞裂解液;5.未感染LMH细胞裂解液;6.FAdV-8b感染LMH细胞裂解液。
图6为重组病毒rHNJZ-Fiber/FAdV-8b和CH/HNJZ/2015的复制动态对比图。图中,HNJZ表示CH/HNJZ/2015。
图7为不同代次重组病毒rHNJZ-Fiber/FAdV-8b的间接免疫荧光鉴定情况图。图中,1~3.第2、5和10代rHNJZ-Fiber/FAdV-8b感染的LMH细胞;4.CH/HNJZ/2015感染的LMH细胞;5.LMH细胞对照;6.FAdV-8b感染LMH细胞。
图8为重组病毒rHNJZ-Fiber/FAdV-8b制备的灭活疫苗诱导的抗FAdV-4Fiber2蛋白和FAdV-8bFiber蛋白抗体动态图。
图9为重组病毒rHNJZ-Fiber/FAdV-8b制备的二联灭活疫苗对FAdV-4强毒株(A)和FAdV-8b毒株(B)攻毒的保护率。
保藏信息:
FAdV-4中国流行强毒株CH/HNJZ/2015:
保藏时间:2016年12月14日;
保藏单位名称:中国微生物菌种保藏管理委员会普通微生物中心;
保藏编号:CGMCC NO:13385;
保藏单位地址:北京市朝阳区北辰西路1号院3号;
分类命名:禽腺病毒4型。
FAdV-8b中国流行株SDQD2021:
保藏时间:2021年12月13日;
保藏单位名称:中国微生物菌种保藏管理委员会普通微生物中心;
保藏编号:CGMCC NO:45057;
保藏单位地址:北京市朝阳区北辰西路1号院3号;
分类命名:禽腺病毒血清型8b型。
具体实施方式
下面通过具体实施方式对本发明进行更加详细的说明,以便于对本发明技术方案的理解,但并不用于对本发明保护范围的限制。
表达FAdV-8b Fiber的FAdV-4重组病毒的构建方法,包括以下步骤:
1.构建FAdV-4中国流行株CH/HNJZ/2015的基因组感染性克隆p15A-cm-HNJZ
1.1.含有FAdV-4同源臂的p15A-cm骨架载体的构建
以p15A-cm-tetR-tetO-ccdB-hyg质粒为模板,以含有FAdV-4基因组左右两端反向末端重复序列的上、下游引物扩增包含FAdV-4中国流行株CH/HNJZ/2015基因组左右两端反向末端重复序列的骨架载体,扩增的骨架载体片段两端含有PmeI酶切位点,扩增用引物为:
上游引物FAdV4-1为:
5’-CGCGCTGCGCGCGGCGGTTGTAAGTGTGTCAAAAGACGCGGTTATATAAGATGATGGTTTAAACAGATCCGAAAACCCCAAGTTACG-3’;
下游引物FAdV4-2为:
5’-CGCGCTGCGCGCGGCGGTTGTAAGTGTGTCAAAAGACGCGGTTATATAAGATGATGGTTTAAACAGATCCTTTCTCCTCTTTAGATC-3’。
1.2.构建FAdV-4中国流行株CH/HNJZ/2015的基因组感染性克隆p15A-cm-HNJZ
利用商品化的QIAamp DNA blood Mini Kit从FAdV-4中国流行株CH/HNJZ/2015感染的LMH细胞裂解物中提取病毒基因组,将病毒基因组DNA和步骤1.1中制备的含有FAdV-4同源臂的p15A-cm骨架载体用T4 DNA聚合酶处理。聚合体系为200ng的病毒基因组DNA、2μg的线性化p15A-cm骨架载体、2μL的10×NEB Buffer 2.1、0.2μL的T4 DNA聚合酶、加入双蒸水补足体系至20μL,反应程序为25℃1h、75℃20min、50℃30min;将反应体系电转化至10%L-阿拉伯糖诱导的GB05-dir感受态细胞中,复苏1h后涂布至带有氯霉素抗性的LB平板,37℃过夜培养;从平板上挑取单菌落扩大培养,提取质粒后用EcoRI限制性内切酶对重组质粒进行酶切鉴定,鉴定结果如图1所示,将正确的重组克隆命名为p15A-cm-HNJZ。
2.含有氨苄青霉素抗性筛选标记(amp)和大肠杆菌自杀基因(ccdB)的FAdV-4感染性克隆的构建
为了快速高效筛选含有外源基因的重组体,以自行构建的p15A-ampccdB质粒DNA为模板,利用带有FAdV-4Fiber1基因两侧基因组序列同源臂的引物进行PCR扩增amp-ccdB表达盒。
所述p15A-ampccdB质粒是在市售p15A质粒中的NdeI和EcoRI位点之间插入amp-ccdB序列。
带有FAdV-4Fiber1基因两侧基因组序列同源臂的引物为:
Fiber1-ampccdB-F:
5’-TATTTTTAACCAATATCTTCTAGGCTCCGCCATTTAATTAATTTGTTTATTTTTCTAAA-3’;
Fiber1-ampccdB-R:
5’-TTCGGAATGTCTTCTTTTAGGGGCCCGGAGCATTTAATTAATTTGTTCAAAAAAAAGCC-3’。
然后利用Redαβ重组酶介导的同源重组技术,将FAdV-4感染性克隆p15A-cm-HNJZ的Fiber1基因精确替换为amp-ccdB表达盒,筛选得到含有氨苄青霉素抗性筛选标记(amp)和大肠杆菌自杀基因(ccdB)的FAdV-4感染性克隆p15A-cm-HNJZ-ΔFiber1-amp-ccdB。
3.含有FAdV-8bFiber基因的重组质粒p15A-cm-HNJZ-Fiber/FAdV-8b的构建
3.1.带有FAdV-4Fiber1两侧同源臂的FAdV-8bFiber基因的扩增
提取FAdV-8b中国流行株SDQD2021病毒基因组,以基因组DNA为扩增模板,利用带有FAdV-4的Fiber1基因两侧同源臂的特异性引物扩增FAdV-8bFiber基因。扩增用引物为:
8bFiber-F:
5’-CGTTTATTTTTAACCAATATCTTCTAGGCTCCGCCATATGGCGACCTCGACTCCTCACG-3’;
8bFiber-R:
5’-CGTTTTCGGAATGTCTTCTTTTAGGGGCCCGGAGCATTCAAGGAGCGTTGGCGGTGCTT-3’。将PCR产物进行琼脂糖凝胶电泳,利用Qiagen公司的QIAquick胶回收试剂盒纯化扩增的FAdV-8b的Fiber基因。
3.2.p15A-cm-HNJZ-Fiber/FAdV-8b感染性克隆构建
使用PacI限制性内切酶37℃过夜酶切p15A-cm-HNJZ-ΔFiber1-amp-ccdB质粒,通过T4 DNA聚合酶将线性化的p15A-cm-HNJZ-ΔFiber1-amp-ccdB载体与FAdV-8bFiber基因片段进行聚合反应。聚合体系为200ng的FAdV-8bFiber基因、2μg的线性化p15A-cm-HNJZ-ΔFiber1-amp-ccdB、2μL的10×NEB Buffer 2.1、0.2μL的T4 DNA聚合酶、加入双蒸水补足体系至20μL,反应程序为25℃1h、75℃20min、50℃30min;将反应体系电转化至10%L-阿拉伯糖诱导的GB05-dir感受态细胞中,复苏1h后涂布至带有氯霉素抗性的LB平板,37℃过夜培养;从平板上挑取单菌落扩大培养,提取质粒后用XbaI限制性内切酶对重组质粒进行酶切鉴定,鉴定结果如图2所示。将正确的重组克隆命名为p15A-cm-HNJZ-Fiber/FAdV-8b。
4.制备表达FAdV-8bFiber蛋白的FAdV-4重组病毒
4.1.重组病毒rHNJZ-Fiber/FAdV-8b的拯救
提取重组质粒p15A-cm-HNJZ-Fiber/FAdV-8b,将重组质粒用PmeI限制性内切酶线性化,并进行酚-氯仿抽提和乙醇沉淀;将LMH细胞(购自美国标准生物品收藏中心)培养于6孔板内,每孔细胞数量为2×106个,过夜培养至细胞丰度约为80%时,将5μg线性化的p15A-cm-HNJZ-Fiber/FAdV-8b按Lipofectamine 3000试剂盒说明书转染至LMH细胞内,转染后6h弃去转染液,加入含有2%FBS的DMEM/F12维持液,培养至细胞出现葡萄串样病变,细胞病变情况如图3所示。收获病变的细胞,冻融三次,10000rpm离心1min收取上清中的重组病毒,命名为rHNJZ-Fiber/FAdV-8b。
4.2.重组病毒的鉴定
4.2.1.重组病毒的PCR鉴定
用酚-氯仿抽提法提取病毒基因组DNA,以FAdV-4中国流行强毒株CH/HNJZ/2015Fiber1基因两侧的特异性引物:Fiber1-F和Fiber1-R:进行PCR扩增,将PCR产物进行琼脂糖凝胶电泳和序列测定。
Fiber1-F:5’-CAATATCTTCTAGGCTCCGCC-3’;
Fiber1-R:5’-CTTTTAGGGGCCCGGAGCAT-3’。
结果显示PCR能够扩增出1572bp的FAdV-8b-Fiber条带,如图4所示。而没有替换FAdV-8bFiber基因的FAdV-4亲本毒株只能扩增出1296bp的FAdV-4Fiber1基因片段。通过序列测定分析,结果显示CH/HNJZ/2015基因组中的Fiber1基因被FAdV-8b Fiber基因成功替换。
5.2.2.重组病毒的Western blot分析
为了检验重组病毒rHNJZ-Fiber/FAdV-8b的稳定性和FAdV-8b Fiber蛋白的表达,将LMH细胞铺至6孔细胞板中,使用第2、5、10代rHNJZ-Fiber/FAdV-8b分别感染LMH细胞,待细胞病变至80%时,使用预冷的细胞裂解液收取蛋白,同时设立CH/HNJZ/2015感染LMH细胞裂解液为阴性对照、LMH细胞裂解液为空白对照、FAdV-8b感染LMH细胞裂解液为阳性对照进行SDS-PAGE,然后将蛋白电转印至硝酸纤维素膜上,以小鼠抗FAdV-8b抗体为一抗,HRP标记兔抗鼠IgG抗体为二抗进行Western blot,采用化学发光法显色后拍摄图像。结果显示在第2、5、10代的rHNJZ-Fiber/FAdV-8b感染LMH细胞裂解液中均可以检测到分子量约为60kDa的蛋白条带,如图5所示。该蛋白条带与预测的FAdV-8b Fiber蛋白分子量相符,证明重组病毒rHNJZ-Fiber/FAdV-8b可以正确并稳定地表达FAdV-8b Fiber蛋白。
5.3.重组病毒和亲本毒在LMH细胞上的生长特性比较
将LMH细胞铺于6孔细胞培养板中,每孔细胞数量为2×106个,按MOI=0.001接种FAdV-4流行株CH/HNJZ/2015和第10代重组病毒,分别于感染后12、24、36、48、60、72、84和96h收获病毒液,按照常规方法测定两种病毒不同时间点的半数组织感染量(TCID50),以收毒时间为横坐标,Log(TCID50/100μL)为纵坐标绘制病毒一步生长曲线。
结果显示,重组病毒rHNJZ-Fiber/FAdV-8b的病毒滴度达到105.6TCID50/100μL,与其亲本株CH/HNJZ/2015在LMH细胞上保持相似的高滴度生长特性和复制动态,如图6所示。5.4.重组病毒的遗传稳定性分析
将LMH细胞铺于6孔细胞培养板中,每孔细胞数量为2×106个,按MOI=0.01接种FAdV-4流行株CH/HNJZ/2015和第2、5和10代重组病毒,同时设置空白LMH细胞对照和FAdV-8b感染的LMH细胞阳性对照。待细胞病变至50%后弃去培养基,无菌PBS洗涤细胞2遍,加入预冷的无水乙醇于-20℃固定细胞30min,加入1%TritonX-100透化15min,PBST洗涤细胞3遍,加入小鼠抗FAdV-8bFiber蛋白抗体(FAdV-8b Fiber基因(如SEQ ID NO:1所示)连接原核表达载体构建pET32a-Fiber,转化大肠杆菌BL21(DE3),IPTG诱导FAdV-8b Fiber蛋白表达,组氨酸层析柱纯化蛋白后测定蛋白浓度,以每只小鼠100ug的剂量免疫Balb/c小鼠,每隔一周免疫一次,第三次免疫两周后采集小鼠尾静脉血分离血清制备),置于37℃孵育1h,PBST洗涤细胞3遍,加入FITC标记羊抗鼠IgG抗体,置于37℃孵育1h,PBST洗涤细胞3遍,在荧光显微镜下观察并记录结果。间接免疫荧光结果表明,不同代次的重组病毒均可以稳定表达FAdV-8b的Fiber蛋白,如图7所示。
6.利用重组病毒rHNJZ-Fiber/FAdV-8b制备的灭活二联疫苗对雏鸡免疫效果评价
6.1.重组病毒rHNJZ-Fiber/FAdV-8b灭活二联疫苗制备
将按照上述1~5所示方法培养的重组病毒rHNJZ-Fiber/FAdV-8b(105.6TCID50/100μL)100mL置于500mL灭菌锥形瓶中,无菌操作加入甲醛至终浓度为0.1%,37℃振摇24小时。取等量高压蒸汽灭菌过的ADJ501佐剂,使用组织匀浆机以每分钟500转的速度搅拌,将灭活的病毒缓慢滴入佐剂中,混合完成后再搅拌10min即可完成乳化,成品4℃保存。
6.2疫苗稳定性检测
取1mL6.1制备的样品加入1.5mL的EP管中,以3000rpm离心15min,未见分层现象。
6.3.重组二联灭活疫苗接种
将购自北京梅里亚的SPF种蛋在无菌孵化箱孵化的140只7日龄SPF雏鸡,分为2组,每组20只,接种6.1制备的疫苗。第1组按照0.2mL/只剂量(相当于105.0TCID50/只)免疫rHNJZ-Fiber/FAdV-8b灭活疫苗,第2组为不免疫不攻毒对照组。
6.4.重组病毒rHNJZ-Fiber/FAdV-8b灭活疫苗刺激机体产生的抗体水平检测和免疫效力评价
6.4.1所用试剂
包被液(25mmol/L碳酸盐缓冲液):Na2CO3:1.59g,NaHCO3:2.93g,用ddH2O定容至1000mL(pH9.6)。
10倍洗涤液:NaCl:80g,KCl:2g,Na2HPO4·12H2O:29g,KH2PO4:2g,Tween-20:5mL,用ddH2O定容至1000mL(pH=7.4)。
封闭液:5g脱脂乳溶于100mL洗涤液。
底物液:3,3’,5,5’-四甲基联苯胺(TMB)购自北京索莱宝科技有限公司终止液(2mol/L H2SO4溶液):用移液管取98%硫酸10.87mL于100mL容量瓶中,将移液管用双蒸水冲洗3次,冲洗液转移到容量瓶中,缓慢加水至容量瓶中,直至液位达到100mL刻度。盖上容量瓶盖,轻轻摇动溶液。
6.4.2间接ELISA操作步骤
(1)包被抗原的制备:将FAdV-4Fiber2基因(如SEQ ID NO:2所示)和FAdV-8bFiber基因(如SEQ ID NO:1所示)分别连接原核表达载体构建pET32a-Fiber2和pET32a-Fiber,转化大肠杆菌BL21(DE3),IPTG诱导FAdV-4Fiber2和FAdV-8b Fiber蛋白表达,组氨酸层析柱纯化蛋白后测定蛋白浓度。
(2)包被:用包被液稀释抗原至工作浓度2μg/mL,每孔100μL加入到酶标板中,2~8℃温育15小时。
(3)洗板:弃包被液,PBST洗涤液,200μL/孔,洗涤3次,每次3min。
(4)封闭:拍干酶标板,加入封闭液,200μL/孔,37℃孵育2h,封闭非特异性结合位点。
(5)洗板:弃封闭液,同步骤(4)。
(6)加待检血清:拍干酶标板,加入待检血清,100μL/孔,设阴性对照,37℃孵育1.5h。
(7)洗板:弃待检样品液,同步骤(4)。
(8)加酶标抗体:拍干酶标板后加入HRP酶标兔抗鸡IgY(购自Proteintech公司),体积比为1:8000倍稀释,100μL/孔,37℃放置1h。
(9)洗板:弃酶标抗体,同步骤(3)。
(10)显色:每孔加入底物显色液TMB 100μL,室温避光显色10min。
(11)终止反应:每孔加入100μL终止液终止反应。
(12)测定OD450nm值:酶标仪测定波长为450nm时OD值。若FAdV-4抗体OD450nm值≥0.384判定为阳性,OD450nm值<0.384判定为阴性。若FAdV-8b抗体OD450nm值≥0.45判定为阳性,OD450nm值<0.45判定为阴性。
6.4.3免疫重组病毒rHNJZ-Fiber/FAdV-8b灭活疫苗血清抗体检测结果
在6.3分组接种重组二联灭活疫苗后,于免疫后每周采血分离血清。
按照6.4.2所述的间接ELISA操作步骤检测血清抗体,检测结果如图8所示,免疫重组病毒rHNJZ-Fiber/FAdV-8b灭活疫苗的鸡只产生了抗FAdV-4和抗FAdV-8b特异性抗体,而不免疫不攻毒对照组未检测到FAdV-4和FAdV-8b特异性抗体。
6.4.4免疫重组病毒rHNJZ-Fiber/FAdV-8b灭活疫苗对FAdV-4和8b型流行毒株攻毒的保护
上述6.3中的第1组雏鸡,在重组病毒rHNJZ-Fiber/FAdV-8b灭活疫苗免疫后第3周(即28日龄),分别用2×105TCID50剂量的FAdV-4流行毒株CH/HNJZ/2015和FAdV-8b流行毒株CH/SDQD/2021通过肌肉注射途径攻毒。攻毒后观察鸡的精神状态,统计死亡情况。攻毒后1~3周每周采血,分离血清。第三周采血后,处死全部鸡只,并统计组织器官病变情况。结果显示FAdV-4攻毒对照组死亡率达到100%,FAdV-8b攻毒对照组死亡率达到30%,而免疫重组病毒rHNJZ-Fiber/FAdV-8b灭活疫苗对FAdV-4和FAdV-8b流行毒株攻毒的保护率均为100%,如图9所示。
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。
SEQUENCE LISTING
<110> 河南农业大学
<120> 表达禽腺病毒血清8b型纤突蛋白的禽腺病毒血清4型重组病毒及其构建方法
和应用
<130> 无
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1572
<212> DNA
<213> Fowl aviadeno virus
<400> 1
atggcgacct cgactcctca cgccttctcc tttggccaaa tcggctcccg aaaacgccct 60
gcgggtggcg atggcgagcg agacgcctcg aaagtgccga aaatgcagac ccccgctccg 120
agcgcgaccg ccaacggaaa tgacgagctg gacctggtct accccttttg gctccaaaac 180
ggctctaccg gaggaggagg aggaggcggc ggttccggtg gaaacccgtc cctcaacccg 240
ccgtttttgg accccaacgg acccctggcc gtccaaaaca acctcctgaa ggtcaatacc 300
gcggccccca tcaccgtcgc caataaggcc ctgacactcg cctatgaacc ggatagtctc 360
gagctcacta accaacagca actggcggtc aaaatcgacc ccgaagggcc tctgaaagcc 420
acgaccgagg gaatacagct gtcggtcgac cctacgacgt tggaggttga tgacgtcgac 480
tgggagttaa ccgtgaaact cgaccccgac ggccccctgg attcctcagc cacaggaatc 540
acggtcagag tcgatgagac cttgctcatc gaggatgttg gatccggaca gggcaaagaa 600
ctcggagtca atctcaaccc caccggaccg attacggccg atgaccaggg tctggactta 660
gaaatagaca accagacgct caaggtcaac agtgttaccg gcgggggcgt cctagctgta 720
caactcaaat cccaaggtgg tcttaccgca cagactgacg gtatccaagt gaacactcag 780
aacagcatca ccgttacaaa cggagctctg gacgtgaaag tagccgccaa cggacctttg 840
gagtcaaccg acaccgggct cacactcaac tatgaccccg gagacttcac agttaatgcg 900
ggcacgttga gcattatcag ggatccggct ctcgtggcca atgcgtacct cacatccggc 960
gcctccaccc ttcagcaatt tacagctaag agtgaaaatt ccagtcaatt ttctttccca 1020
tgcgcatact atctgcaaca atggctttcc gacgggttgg ttttgagctc gctctatctg 1080
aagctcgaca gagcacagtt cacgaacatg ccaacgggtg caaactatca gaacgccagg 1140
tactttacct tctgggttgg agcgggcact tcatttaatc tttctaccct taccgaaccc 1200
actattacac ccaacaccac acaatggaat gcattcgccc ctgcccttga ttactcaggt 1260
gctcctccct tcatctacga cgcgtcttcc gtagttacga tttattttga acccaccagt 1320
ggtcgactgg aaagctatct ccccgtcctt accgataact ggagccaaac ctacaacccc 1380
ggcaccgtca ccctgtgtgt aaaaacggta agggttcaat tgagatcaca aggaaccttc 1440
agcactctag tctgttacaa tttccgctgt cagaacacgg gcatttttaa cagcaacgct 1500
acagcgggaa ccatgacact tggacctatc ttcttcagtt gtcccgccct aagcaccgcc 1560
aacgctcctt ga 1572
<210> 2
<211> 1440
<212> DNA
<213> Fowl aviadeno virus
<400> 2
atgctccggg cccctaaaag aagacattcc gaaaacggga agcccgagac cgaagcggga 60
ccttccccgg ctccaatcaa gcgcgccaaa cgcatggtga gagcatccca gcttgacctg 120
gtttatcctt tcgattacgt ggccgacccc gtcggagggc tcaacccgcc ttttttggga 180
ggctcaggac ccctagtgga ccagggcgga cagcttacgc tcaacgtcac cgatcccatc 240
atcatcaaga acagatcggt ggacttggcc cacgacccca gtctcgatgt caacgcccaa 300
ggtcaactgg cggtggccgt tgaccccgaa ggggccctgg acatcacccc cgatggactg 360
gacgtcaagg tcgacggagt gaccgtaatg gtcaacgatg actgggaact ggccgtaaaa 420
gtcgacccgt ccggcggatt ggattccacc gcgggtggac tgggggtcag cgtggacgac 480
accttgctcg tggatcaggg agaactgggc gtacacctca accaacaagg acccatcact 540
gccgatagca gtggtatcga cctcgagatc aatcctaaca tgttcacggt caacacctcg 600
accggaagcg gagtgctgga actcaaccta aaagcgcagg gaggcatcca agccgacagt 660
tcgggagtgg gcgtttccgt ggatgaaagc ctacagattg tcaacaacac tctggaagtg 720
aaaccggatc ccagcggacc gcttacggtc tccgccaatg gcctagggct gaagtacgac 780
actaataccc tagcggtgac cgcgggcgct ttaaccgtgg tcggaggggg gagcgtctcc 840
acacccatcg ctacttttgt ctcgggaagt cccagcctca acacctacaa tgccacgacc 900
gtcaattcca gcgcgaacgc cttctcttgc gcctactacc ttcaacagtg gaacatacag 960
gggctccttg ttacctccct ctacttgaaa ttggacagcg ccaccatggg gaatcgccct 1020
ggggacctca actccgccaa tgccaaatgg ttcacctttt gggtgtccgc ctatctccag 1080
caatgcaacc cctccgggat tcaagcggga acggtcagcc cctccaccgc caccctcacg 1140
gactttgaac ccatggccaa taggagcgtg accagcccat ggacgtactc ggccaatgga 1200
tactatgaac catccatcgg ggaattccaa gtgttcagcc cggtggtaac aggtgcctgg 1260
aacccgggaa acatagggat ccgcgtcctc cccgtgccgg tttcggcctc cggagagcga 1320
tacacccttc tatgctatag tctgcagtgc acgaacgcga gcatttttaa tccaaacaac 1380
agcggaacca tgatcgtggg acccgtgctc tacagctgtc cagcggcctc cctcccgtaa 1440
Claims (10)
1.表达禽腺病毒血清8b型纤突蛋白的禽腺病毒血清4型重组病毒,其特征在于,所述重组病毒是用FAdV-8b的Fiber基因替换FAdV-4基因组的Fiber1基因后得到。
2.根据权利要求1所述的表达禽腺病毒血清8b型纤突蛋白的禽腺病毒血清4型重组病毒,其特征在于,FAdV-4为FAdV-4中国流行株CH/HNJZ/2015,所述FAdV-8b的Fiber基因序列如SEQ ID NO:1所示。
3.权利要求1所述的表达禽腺病毒血清8b型纤突蛋白的禽腺病毒血清4型重组病毒的构建方法,其特征在于,利用Red/ET重组技术构建FAdV-4的感染性克隆,将FAdV-4基因组中Fiber1基因替换为双选择标记筛选表达盒,然后用FAdV-8b的Fiber基因替代筛选表达盒,获得将FAdV-4基因组中Fiber1基因替换为FAdV-8bFiber基因的感染性克隆,转化宿主菌,提取含有FAdV-8b Fiber基因的FAdV-4感染性克隆重组质粒,经限制性内切酶线性化后,转染鸡胚肝癌细胞系快速拯救出表达禽腺病毒血清8b型纤突蛋白的禽腺病毒血清4型重组病毒。
4.根据权利要求3所述的构建方法,其特征在于,包括以下步骤:
(1)构建FAdV-4的基因组感染性克隆:
将FAdV-4的全长基因组利用核酸外切酶联合重组酶技术一步克隆到含有抗性筛选标记的载体中,得到FAdV-4的基因组感染性克隆;
(2)含有双选择标记筛选表达盒的FAdV-4感染性克隆的构建:
以含有双选择标记筛选表达盒的表达载体为模板,利用带有FAdV-4的Fiber1基因两侧同源臂的特异性引物进行PCR扩增筛选表达盒,利用Redαβ重组酶介导的同源重组技术,将FAdV-4感染性克隆的Fiber1基因替换为筛选表达盒,筛选得到含有筛选表达盒的FAdV-4感染性克隆;
(3)构建含有FAdV-8b Fiber基因的FAdV-4感染性克隆:
以FAdV-8b病毒基因组为模板,利用带有FAdV-4的Fiber1基因两侧同源臂的特异性引物扩增含有FAdV-8b的Fiber基因片段,利用Redαβ重组酶介导的同源重组技术,将FAdV-4感染性克隆的Fiber1基因替换为FAdV-8b的Fiber基因,筛选得到含有FAdV-8b Fiber基因的FAdV-4感染性克隆;
(4)制备表达FAdV-8b Fiber蛋白的FAdV-4重组病毒:
将含有FAdV-8b Fiber基因的FAdV-4感染性克隆用限制性内切酶线性化去除基因组两端的载体序列后,转染鸡胚肝癌细胞系,拯救出表达FAdV-8b Fiber蛋白的FAdV-4重组病毒。
5.根据权利要求4所述的构建方法,其特征在于,步骤(1)为:
以p15A-cm-tetR-tetO-ccdB-hyg质粒DNA为模板,以含有FAdV-4基因组左右两端反向末端重复序列的上、下游引物扩增包含FAdV-4基因组左右两端反向末端重复序列的骨架载体,扩增的骨架载体片段两端含有酶切位点;
提取FAdV-4的病毒基因组,将病毒基因组DNA和所述包含FAdV-4基因组左右两端反向末端重复序列的骨架载体用T4 DNA聚合酶处理;转化筛选,得到含有FAdV-4的基因组感染性克隆。
6.根据权利要求4所述的构建方法,其特征在于,所述FAdV-4为中国流行株CH/HNJZ/2015,所述FAdV-8b的Fiber基因序列如SEQ ID NO:1所示。
7.根据权利要求4所述的构建方法,其特征在于,所述双选择标记筛选表达盒为氨苄青霉素抗性筛选标记和大肠杆菌自杀基因表达盒。
8.根据权利要求4所述的构建方法,其特征在于,步骤(2)中带有FAdV-4的Fiber1基因两侧同源臂的特异性引物:
Fiber1-ampccdB-F:
5’-TATTTTTAACCAATATCTTCTAGGCTCCGCCATTTAATTAATTTGTTTATTTTTCTAAA-3’;
Fiber1-ampccdB-R:
5’-TTCGGAATGTCTTCTTTTAGGGGCCCGGAGCATTTAATTAATTTGTTCAAAAAAAAGCC-3’。
9.根据权利要求6所述的构建方法,其特征在于,步骤(3)中带有FAdV-4的Fiber1基因两侧同源臂的特异性引物:
8bFiber-F:
5’-CGTTTATTTTTAACCAATATCTTCTAGGCTCCGCCATATGGCGACCTCGACTCCTCACG-3’;
8bFiber-R:
5’-CGTTTTCGGAATGTCTTCTTTTAGGGGCCCGGAGCATTCAAGGAGCGTTGGCGGTGCTT-3’。
10.权利要求1所述的重组病毒在制备防制鸡肝炎—心包积液综合征和/或包涵体肝炎疫苗中的应用。
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