CN117143924B - 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 - Google Patents
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 Download PDFInfo
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Abstract
本发明公开了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,属于动物生物制品和病毒学技术领域,本发明公开了该重组猫疱疹病毒的构建方法,包括以下步骤:利用同源重组和PCR扩增方法,获得TKhm1‑VP2‑TKhm2和gIhm1‑VP1‑gEhm2同源重组片段;将同源重组片段转染至CRFK稳转细胞系,将转染细胞感染rWH/2020‑△TK/gI/gE‑EGFP+‑mCherry+毒株即可。本发明构建的重组猫疱疹病毒的活载体疫苗可防控猫细小、猫杯状病毒病和猫传染性鼻气管炎,能大大减少猫只免疫次数,降低免疫成本,对提升养猫业经济效益和宠物福利意义重大,具有良好的应用前景。
Description
技术领域
本发明涉及动物生物制品和病毒学技术领域,特别是涉及一种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。
背景技术
猫杯状病毒(FCV)在猫科动物中具有较高的感染性和传播性,会引起口腔溃疡、口腔黏膜感染、鼻结膜炎等症状,除了引起呼吸系统症状的FCV毒株,能够引起患猫跛行、流产、皮肤水肿、皮肤溃烂等全身多系统、器官病变(Virulent system disease,VSD)甚至死亡的强毒株FCV-VSD也被相继报道,强毒株感染的猫死亡率在50%~100%。FCV基因组分别编码3个开放阅读框(Open reading frame,ORFs),基因组的组成从5'末端到3'末端分别是ployA、ORF1、ORF2,ORF3、VPg,ORF1位于基因组5'端,主要编码FCV的非结构蛋白,主要包括病毒复制、增殖必须的蛋白酶。ORF2编码的VP1,是FCV的主要衣壳蛋白。VP1作为FCV最重要的结构蛋白,在病毒基因组复制、病毒感染、宿主识别中都发挥重要的作用,VP1可以被宿主的免疫系统识别,诱导动物机体产生中和抗体,是目前FCV疫苗研发、治疗制剂、检测方法建立的重要靶标。
猫泛白细胞减少症(Feline panleukopenia,FPL),又称为猫瘟热、猫细小病毒病,是由猫细小病毒(Feline parvovirus,FPV)感染引起的,以动物淋巴细胞受损、血液中白细胞数量大量减少为特征的急性、高度接触性传染病。目前FPV在全球范围内皆有流行,对家养宠物、野生动物、经济动物养殖业都有严重危害。FPV基因组主要编码两种结构蛋白VP1和VP2。其中VP2占88~90﹪,而VP1仅占10~12﹪。VP2是FPV主要的结构性蛋白和功能性蛋白,在FPV的感染过程中有识别宿主的重要功能,VP2蛋白含有FPV主要的抗原位点siteA和siteB,大多数识别site B的单克隆抗体都对FPV具有有效的中和活性,VP2是FPV重要的保护性抗原蛋白,也是FPV疫苗、治疗制剂研发的重要靶标。
猫传染性鼻气管炎又称为猫鼻支,是由猫疱疹病毒I型(FHV-1)引起的一种以急性上呼吸道症状为特征的高度接触性传染病,具体症状包括:角膜结膜炎、上呼吸道感染和流产,但以打喷嚏、过度流涎、眼鼻分泌物增多等上呼吸道症状为主。本病临床常见于猫,猫的发病率高达100%,病死率在不同年龄段的猫中差异很大,成年猫一般不引起死亡,但幼仔猫的死亡率可达50%。患病动物可终生带毒排毒,且在一定条件刺激下反复感染。
目前,国内尚无自主研发的FHV-1、FCV和FPV疫苗,主要依赖于接种进口的“妙三多”防控FHV-1、FCV和FPV,但面临进口难、成本高、免疫效果不佳,不能有效控制当前FHV-1、FCV和FPV的流行等问题。因此,急需开发一款自主研发的疫苗以精准防控FHV-1、FCV和FPV的流行。研究表明,以猫疱疹病毒为载体插入其它病毒免疫原的重组弱毒活疫苗安全性良好,除体液免疫外,能够激起较强的细胞免疫和黏膜免疫,尤其是激活的细胞免疫能够增强机体对外源抗原的免疫反应,因此其免疫保护效果通常优于灭活疫苗。同时,此类重组猫疱疹病毒活载体疫苗属联苗产品,具备一针防多病的优点,可以大大简化免疫程序,进而降低免疫成本及免疫应激反应。
目前,大多数研究往往是采用在猫疱疹病毒载体中插入一种抗原基因,如插入猫杯状病毒、猫白血病病毒、猫艾滋病病毒或狂犬病毒的抗原单基因,这些重组病毒因外源抗原免疫原性及插入位点的差异,表现出针对上述疾病不同的免疫保护效果。同时,少有在猫疱疹病毒载体中插入多种抗原基因的研究报道,原因在于,插入多种抗原基因通常需要替换猫疱疹病毒的多个基因,而猫疱疹病毒基因组较大,操纵较为困难。对此,本发明通过CRISPR Cas9介导的同源重组法,快速将免疫原性良好的猫杯状病毒和猫细小病毒的抗原蛋白分别插入到猫疱疹病毒的gI/gE和TK位置,以开发FHV-1、FCV和FPV三联重组猫疱疹病毒活载体疫苗产品。
发明内容
本发明的目的是提供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,以解决上述现有技术存在的问题,该活载体疫苗缺失了猫疱疹病毒I型的主要毒力基因TK、gI、gE基因,同时稳定表达猫杯状病毒主要抗原蛋白VP1及猫细小病毒VP2抗原蛋白,该疫苗在免疫动物体内,可以诱导良好的体液免疫与细胞免疫应答,适用于作为猫瘟、猫杯状病毒病和猫传染性鼻气管炎的三联活载体疫苗。
为实现上述目的,本发明提供了如下方案:
本发明提供一种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的构建方法,包括以下步骤:
分别获取FCV病毒的VP1基因、FPV病毒的VP2基因以及FHV-1病毒的TK左右侧同源臂TKhm1、TKhm2和gI、gE同源臂gIhm1、gEhm2,利用同源重组和PCR扩增方法,获得TKhm1-VP2-TKhm2和gIhm1-VP1-gEhm2片段;
依据CMV、polyA基因的sgRNA引物,利用CRISPR基因编辑方法构建CMV、polyA基因的sgRNA表达载体,转染、收取慢病毒、感染CRFK细胞,获得CRFK稳转细胞系;
将TKhm1-VP2-TKhm2和gIhm1-VP1-gEhm2片段转染至CRFK稳转细胞系,得到转染细胞,将转染细胞感染rWH/2020-△TK/gI/gE-EGFP+-mCherry+毒株,得到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。
进一步地,所述TKhm1-VP2-TKhm2片段的核苷酸序列如SEQ ID NO:1所示,所述gIhm1-VP1-gEhm2片段的核苷酸序列如SEQ ID NO:2所示。
进一步地,所述sgRNA引物的核苷酸序列如SEQ ID NO:3-6所示。
本发明还提供一种所述构建方法获得的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。
本发明还提供一种所述的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在制备防治猫杯状病毒病的疫苗中的应用。
本发明还提供一种所述的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在制备防治猫细小病毒病的疫苗中的应用。
本发明还提供一种所述的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在制备防治猫传染性鼻气管炎的疫苗中的应用。
本发明还提供一种同时防治猫杯状病毒、猫细小病毒和猫疱疹病毒的活载体疫苗,所述活载体疫苗包含所述的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。
进一步地,所述活载体疫苗还包含保护剂。
进一步地,将所述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接种细胞,收取病毒液,将病毒液与保护剂混合后,冷冻干燥,得到所述活载体疫苗。
本发明公开了以下技术效果:
本发明构建的重组猫疱疹病毒的活载体疫苗,是以缺失TK、gI和gE基因的猫疱疹病毒为活载体,构建表达猫杯状病毒与猫细小病毒的主要免疫保护性抗原基因的重组活载体疫苗,在免疫动物体内,该重组活载体疫苗能诱导良好的体液免疫与细胞免疫应答,可以弥补常规灭活疫苗不能有效激活细胞免疫的缺陷。本发明获得的活载体疫苗(rWH2020-ΔTK/gI/gE-VP1-VP2)具有猫疱疹病毒I型弱毒疫苗株的特征,可作为防控猫细小、猫杯状病毒病和猫传染性鼻气管炎三联活载体疫苗,能大大减少猫只免疫次数,降低免疫成本,对提升养猫业经济效益和宠物福利意义重大,具有良好的应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2的构建示意图;
图2为重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2感染CRFK细胞后细胞病变图;图中A、B、C分别是绿色荧光、红色荧光、明场视野下重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2感染CRFK细胞后细胞病变结果;
图3为重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2的PCR鉴定结果;图中A是对TK基因进行扩增,M:DL5000 DNAMarker,泳道1是重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2扩增条带,泳道2是亲本毒株WH2020扩增条带,泳道3是阴性对照;图中B是对gI/gE基因进行扩增,M:DL5000 DNAMarker,泳道1是重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2扩增条带,泳道2是亲本毒株WH2020扩增条带,泳道3是阴性对照;
图4为重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2遗传稳定鉴定图,图中A、B和C分别是使用FHV-1特异性引物TK-F/R、gI/gE-F/R、gB-F/R扩增条带结果;A中M:DL5000 DNAMarker,泳道1-10分别是1-10代rWH2020-ΔTK/gI/gE-VP1-VP2经引物TK-F/R扩增条带,泳道11是亲本毒株WH2020扩增条带,泳道12是阴性对照;B中M:DL5000 DNAMarker,泳道1-10分别是1-10代rWH2020-ΔTK/gI/gE-VP1-VP2经引物gI/gE-F/R扩增条带,泳道11是亲本毒株WH2020扩增条带,泳道12是阴性对照;C中M:DL2000 DNAMarker,泳道1-10分别是1-10代rWH2020-ΔTK/gI/gE-VP1-VP2经引物gB-F/R扩增条带,泳道11是亲本毒株WH2020扩增条带,泳道12是阴性对照;
图5为重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2表达VP1蛋白和VP2蛋白的荧光图;
图6为幼猫二次免疫后21d的特异性抗体检测结果,A为gB特异性抗体,B为VP1特异性抗体,C为VP2特异性抗体。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1构建稳定表达猫杯状病毒VP1蛋白和猫细小病毒VP2蛋白的rWH2020-ΔTK/gI/gE-VP1-VP2
1、毒株
FHV-1/WH/2020株(以下简称WH2020株),为发明人前期申请专利(CN113308441A)公开的猫疱疹病毒I型FHV-1/WH/2020,其保藏编号为:CCTCC NO:V202126;
FPVWH1株,为本实验室前期申请专利(CN113337478A)公开的猫细小病毒FPV WH1,其保藏编号为:CCTCC NO:V202127;
FCV F9株(GenBank accession no.M86379.1)的VP1由南京金斯瑞合成于载体pMD18T上,构成质粒pMD18T-VP1用作VP1扩增模板。
CRFK细胞与293T细胞,以含10%胎牛血清的DMEM培养液培养(gbico公司)。
2、重组载体构建
以WH2020株为模板PCR扩增TK左右侧同源臂TKhm1、TKhm2;gI、gE同源臂gIhm1、gEhm2;以质粒pMD18T-VP1和FPVWH1株为模板扩增VP1、VP2;引物分别为TKhm1-F(CATGCCTGCAGGTCGACGATGGCTCACGCCAATAATCC)、TKhm1-R(TGAACTGCTCCATCACTCATCGTCTGATCTGTGTATGATG);TKhm2-F(CACCTAGAAAATTATATTAAACATTAGTGGTGTTCCCT) 、 TKhm2-R(CCCGGGGATCCTCTAGAGATCCGTCCATGTCTTGTGG) ; gIhm1-F(CATGCCTGCAGGTCGACGATGGATCCGAAGATAACAGCTG)、gIhm1-R(TTAGCGCAGGTTGAGCACATATTAAGTATTATGCTGTGGTCC);gEhm2-F(GTCCCATGACTAAGTTATGACCCGTCGACGAGTTCTA) 、 gEhm2-R(CCCGGGGATCCTCTAGAGATCTCGCCTTCATAAGCCATC); VP1-F( ACCACAGCATAATACTTAATATGTGCTCAACCTGCG ) 、 VP1-R(TGCTAGAACTCGTCGACGGGTCATAACTTAGTCATGGGACTCC);VP2-F(CATCATACACAGATCAGACGATGAGTGATGGAGCAGTTCA)、VP2-R(ATAGGGAACACCACTAATGTTTAATATAATTTTCTAGGTGCTAG)。扩增体系为:2×phanta MIX 25μL,上游引物2μL,下游引物2μL,模板2μL,ddH2O 19μL。将上述试剂充分混匀,按下列条件进行扩增:95℃变性5min后进入循环,循环参数:95℃15s,55℃15s,72℃1min,35个循环后72℃延伸5min,16℃2min。各扩增产物序列如下:
VP1氨基酸序列:
MCSTCANVLKYYDWDPHFKLVINPNNFLSVGFCSNPLMCCYPELLPEFGTVWDCDRSPLEIYLESILGDDEWASTFDAVDPVVPPMHWGAAGKIFQPHPGVLMHHLIGKVAAGWDPDLPLIRLEADDGSITAPEQGTMVGGVIAEPSAQMSTAADMATGKSVDSEWEAFFSFHTSVNWSTSETQGKILFKQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPVQSTSMLQYPHVLFDARQVEPVIFCLPDLRSTLYHLMSDTDTTSLVIMVYNDLINPYANDANSSGCIVTVETKPGPDFKFHLLKPPGSMLTHGSIPSDLIPKTSSLWIGNRYWSDITDFVIRPFVFQANRHFDFNQETAGWSTPRFRPISVTITEQNGAKLGIGVATDYIVPGIPDGWPDTTIPGELIPAGDYAITNGTGNDITTATGYDTADIIKNNTNFRGMYICGSLQRAWGDKKISNTAFITTATLDGDNNNKINPCNTIDQSKIVVFQDNHVGKKAQTSDDTLALLGYTGIGEQAIGSDRDRVVRISTLPETGARGGNHPIFYKNSIKLGYVIRSIDVFNSQILHTSRQLSLNHYLLPPDSFAVYRIIDSNGSWFDIGIDSDGFSFVGVSGFGKLEFPLSASYMGIQLAKIRLASNIRSPMTKL;
VP1核苷酸序列:
ATGTGCTCAACCTGCGCTAACGTGCTTAAATATTATGATTGGGACCCCCATTTCAAATTGGTAATCAACCCCAACAACTTCCTCTCTGTTGGCTTTTGTAGTAACCCTTTAATGTGTTGCTACCCAGAACTCCTTCCGGAATTTGGAACTGTTTGGGATTGCGATCGGTCACCACTTGAAATTTACCTAGAATCAATACTTGGTGATGATGAATGGGCATCCACTTTTGACGCTGTTGACCCAGTCGTTCCCCCAATGCACTGGGGTGCTGCTGGAAAAATTTTCCAGCCACACCCCGGTGTTCTCATGCACCATCTCATTGGTAAGGTTGCTGCAGGTTGGGACCCCGATCTGCCTCTAATTCGACTCGAGGCGGATGACGGGTCAATCACAGCACCCGAGCAAGGAACAATGGTTGGCGGCGTCATCGCTGAACCCAGCGCCCAGATGTCAACAGCTGCTGATATGGCCACCGGGAAAAGCGTTGATTCTGAGTGGGAGGCATTCTTCTCCTTTCACACCAGCGTCAATTGGAGTACATCTGAAACCCAAGGAAAGATTCTCTTCAAACAATCCTTAGGCCCTTTGCTCAACCCATATCTAGAACACCTTGCTAAGCTATATGTTGCGTGGTCTGGGTCGATTGAGGTTAGGTTCTCTATCTCTGGCTCTGGTGTCTTTGGTGGGAAGCTCGCAGCTATTGTTGTACCTCCTGGGGTTGATCCAGTGCAGAGTACTTCGATGCTACAATACCCCCATGTTTTGTTTGATGCTCGTCAGGTGGAACCAGTTATCTTCTGTCTTCCTGATCTAAGAAGCACCCTGTACCACCTTATGTCTGACACTGACACTACATCCTTGGTCATTATGGTGTACAATGATCTCATCAATCCCTATGCCAATGATGCCAACTCTTCTGGGTGTATTGTCACTGTCGAGACAAAACCTGGCCCTGACTTCAAGTTTCACCTCCTTAAGCCACCCGGATCTATGCTAACCCATGGCTCTATCCCTTCTGATTTAATTCCCAAAACATCTTCGCTCTGGATCGGTAACCGCTACTGGTCAGACATAACTGATTTTGTGATTCGGCCGTTTGTCTTCCAAGCAAATCGTCATTTTGACTTTAATCAAGAGACCGCAGGGTGGAGCACACCACGGTTTCGGCCTATATCTGTTACCATTACTGAACAGAACGGAGCAAAATTGGGCATTGGGGTGGCAACAGATTACATAGTGCCTGGAATCCCTGATGGCTGGCCTGACACCACAATTCCTGGGGAGTTGATACCAGCTGGTGATTACGCAATCACCAATGGTACTGGCAATGACATCACCACGGCTACAGGATATGACACTGCTGATATAATTAAGAACAATACCAACTTTAGGGGCATGTACATATGTGGTTCGCTCCAGCGTGCCTGGGGTGATAAGAAAATTTCCAACACTGCCTTTATCACCACTGCCACCCTAGATGGTGACAACAACAACAAGATCAATCCCTGTAATACCATAGACCAGTCAAAGATCGTCGTGTTTCAAGACAACCATGTTGGAAAGAAAGCGCAAACCTCAGACGATACATTGGCCCTGCTTGGTTACACTGGCATTGGTGAGCAGGCCATCGGGTCTGATAGGGACCGGGTTGTGCGCATCAGCACTCTCCCTGAAACTGGTGCTCGAGGCGGTAACCACCCAATTTTCTACAAGAACTCCATTAAATTGGGATATGTAATTAGGTCTATTGATGTCTTTAATTCACAAATCTTGCACACTTCCAGACAGTTATCGCTAAATCATTACCTACTCCCACCTGATTCTTTTGCCGTCTATAGAATAATTGACTCAAATGGCTCGTGGTTTGATATTGGAATTGATAGTGATGGGTTCTCTTTTGTTGGTGTTTCTGGCTTTGGTAAATTAGAATTTCCCCTTTCTGCCTCCTACATGGGAATACAATTGGCAAAGATCCGGCTTGCCTCTAACATTAGGAGTCCCATGACTAAGTTATGA;
VP2氨基酸序列:
MSDGAVQPDGGQPAVRNERATGSGNGSGGGGGGGSGGVGISTGTFNNQTEFKFLENGWVEITANSSRLVHLNMPESENYKRVVVNNMDKTAVKGNMALDDIHVQIVTPWSLVDANAWGVWFNPGDWQLIVNTMSELHLVSFEQEIFNVVLKTVSESATQPPTKVYNNDLTASLMVALDSNNTMPFTPAAMRSETLGFYPWKPTIPTPWRYYFQWDRTLIPSHTGTSGTPTNVYHGTDPDDVQFYTIENSVPVHLLRTGDEFATGTFFFDCKPCRLTHTWQTNRALGLPPFLNSLPQSEGATNFGDIGVQQDKRRGVTQMGNTDYITEATIMRPAEVGYSAPYYSFEASTQGPFKTPIAAGRGGAQTDENQAADGDPRYAFGRQHGQKTTTTGETPERFTYIAHQDTGRYPEGDWIQNINFNLPVTNDNVLLPTDPIGGKTGINYTNIFNTYGPLTALNNVPPVYPNGQIWDKEFDTDLKPRLHVNAPFVCQNNCPGQLFVKVAPNLTNEYDPDASANMSRIVTYSDFWWKGKLVFKAKLRASHTWNPIQQMSINVDNQFNYVPNNIGAMKIVYEKSQLAPRKLY;
VP2核苷酸序列:
ATGAGTGATGGAGCAGTTCAACCAGACGGTGGTCAACCTGCTGTCAGAAATGAAAGAGCTACAGGATCTGGGAACGGGTCTGGAGGCGGGGGTGGTGGTGGTTCTGGGGGTGTGGGGATTTCTACGGGTACTTTCAATAATCAGACGGAATTTAAATTTTTGGAAAACGGATGGGTGGAAATCACAGCAAACTCAAGCAGACTTGTACATTTAAATATGCCAGAAAGTGAAAATTATAAAAGAGTAGTTGTAAATAATATGGATAAAACTGCAGTTAAAGGAAACATGGCTTTAGATGATATTCATGTACAAATTGTAACACCTTGGTCATTGGTTGATGCAAATGCTTGGGGAGTTTGGTTTAATCCAGGAGATTGGCAACTAATTGTTAATACTATGAGTGAGTTGCATTTAGTTAGTTTTGAACAAGAAATTTTTAATGTTGTTTTAAAGACTGTTTCAGAATCTGCTACTCAGCCACCAACTAAAGTCTATAATAATGATTTAACTGCATCATTGATGGTTGCATTAGATAGTAATAATACTATGCCATTTACTCCAGCAGCTATGAGATCTGAGACATTAGGTTTTTATCCATGGAAACCAACCATACCAACTCCATGGAGATATTATTTTCAATGGGATAGAACATTAATACCATCTCATACTGGAACTAGTGGCACACCAACAAATGTATATCATGGTACAGATCCAGATGATGTTCAATTTTATACTATTGAAAATTCTGTGCCAGTGCACTTACTAAGAACAGGTGATGAATTTGCTACAGGAACATTTTTTTTTGATTGTAAACCATGTAGACTAACACATACATGGCAAACAAATAGAGCATTGGGCTTACCACCATTTCTAAATTCTTTGCCTCAATCTGAAGGAGCTACTAACTTTGGTGATATAGGAGTTCAACAAGATAAAAGACGTGGTGTAACTCAAATGGGAAATACAGACTATATTACTGAAGCTACTATTATGAGACCAGCTGAGGTTGGTTATAGTGCACCATATTATTCTTTTGAAGCATCTACACAAGGGCCATTTAAAACACCTATTGCAGCAGGACGGGGGGGAGCGCAAACAGATGAAAATCAAGCAGCAGATGGTGATCCAAGATATGCATTTGGTAGACAACATGGTCAAAAAACTACTACAACAGGAGAAACACCCGAGAGATTTACATATATAGCACATCAAGATACAGGAAGATATCCAGAAGGAGATTGGATTCAAAATATTAACTTTAACCTTCCTGTAACAAATGATAATGTATTGCTACCAACAGATCCAATTGGAGGTAAAACAGGAATTAACTATACTAATATATTTAATACTTATGGTCCTTTAACTGCATTAAATAATGTACCACCAGTTTATCCAAATGGTCAAATTTGGGATAAAGAATTTGATACTGACTTAAAACCAAGACTTCATGTAAATGCACCATTTGTTTGTCAAAATAATTGTCCTGGTCAATTATTTGTAAAAGTTGCGCCTAATTTAACAAATGAATATGATCCTGATGCATCTGCTAATATGTCAAGAATTGTAACTTACTCAGATTTTTGGTGGAAAGGTAAATTAGTATTTAAAGCTAAACTAAGAGCATCTCATACTTGGAATCCAATTCAACAAATGAGTATTAATGTAGATAACCAATTTAACTATGTACCAAATAATATTGGAGCTATGAAAATTGTATATGAAAAATCTCAACTAGCACCTAGAAAATTATATTAA;
TKhm1核苷酸序列:
GGCTCACGCCAATAATCCACTGGAAATGTGGGAGTAATAAAATTTCTAGTGTCCGATAAAATCAGATCATCCCCGATGTTAGTTATTCGCATCGAGTCGAATACGTATTCGACTCGTCCAGTCATGATGACCCGTCTTCGACAACTTCTCCTCTCCAACAAATGATACCAGTGATTAATCTCTTTGATCCTAAATAAGGTTTTGATATATGATCTATATAGACTCATTACCCATCACATATAGGGCATATTAATACTCACGTGGTTGCGTAGCGGAGTGATCGGGAAAGAGAGATGCTACCCATTTTAAAGGGTTACTTTCGCCGCTTCCTATTGGAGTTAAAGTGTTTTTTTTATCCTTCACAGATACACACGATGACATCGGTACGTGGTTCGTATGTTTATAGACCCCAGACCCAGACCTTGGGTATTTTGATTTTTTCTTGGAGCGGATTACACCACCACTCTCGATTGGATTGTATTCGGCGAGCTTGGTAATATTAGCAGCCAGAACAGAAATGTTACTCGTAAGCATCTGCGGGGGTAAACTAGTAACTCCCAAAATTCTCAGACCGCGCTGCGCGATAAATGCCAATATGGGTATTAGAGTGAGCTTCTCCCCCCCTGGTGGTAGAATCTTGGTTATTAAACCCACAGAATCTGTTAATTGTTTCAAACCCTCACGACGTTGAATGTCTTTACTAGTTGTATCCATATTTTTTGAAAAACGACACGTTTTCAGCTCAATTAGAAAACATATACCACCCCCTTCTCCCTCAAATTGTATAGTACATACACAATCAGGTCGGCGACGACCCAAGTTAACCTCACATGCTAGGTACACGCCCTTAGCCTTTTTAAGAGACTCTGCGGATACAGAGCCGCCCAATAAACACTCGAGTCGGTCGGTATATACTCCACTCGCAGAGGTCGAGGATATATCGCGCTTGAGGACAGCATAAAAGCGATTGTGGCATCGAATTCCAGCCCGGAGCCTCAATCCGACACTGCGTCGTTGTTCACGTTTCATCATACACAGATCAGACG;
TKhm2核苷酸序列:
ACATTAGTGGTGTTCCCTATTACCCCCCTGTGGTGAATGTGTGGAGGTCAGGGGATAATTGTATAATGACCATCGTTTCATGAATAAAATAACCGTGTGTGATGTGGATGTATTCATTAATTGAATTTCTCTTCCGGTTTTAGATCTTTATAAGCGTAAAACTGGTGTTTTAAATCCAAGAGCCGGGTTCTTTGGAGGTTGGTCACATCATCGCCACAGCCCGTGGATTCAAGCAATCTATGATGTGTTTGATAATATACCTATCGATATTCCTGATCATTGTATCGAGGATGTTGACTGGTTTACCGATGATGGATAGACCTGATGAAGGTGGGCTGGCTCGACGAACAGTTGGTGAAGTAGAAGGGGAGTTTTCTTATAGGGACGATGTTGATGTAGCAGACGTGAGAAACTTATTTATCATGTTACCAAAAAATGGGAGCGATATATTTCTATTCATATTCGATAGACGCAGTCAACGTCAACGCGGTACTATGTTTTTATTCCCCAAGGCTGGGTTTGTACAACCAACACCCGCGAAGGTTCGCGATGAAGCGGCGGCCGCCCCATTTGGGTTTATATCCCCTGTATATCCACTATCGAGTCTTTTATTTAATCCATACAATGGGAGATATCTGACGACACGCCATCTGATTGCCTTTGAGGTAACCCCGGAATCCTCTCTTCATGATTGGTATTTTGCACGATCACCAACAACTGCTACTCAGACACAGCCATTAGGACATATAACTAACCCCCCCCGACGATCGCCAAAAGACAAACCGACCACCTCCGGCCATACAGATTTAATTATACGCTATTGCGCATTGGAGTTGGATTTTTTCCAGGACACAAGACGACAGCGTGATGGAATATATTTACCTAATTACGAGGCCGTATGGCCATTGGCAATGAATTTTTTGGAGGGGATGTGGATATGGAGTAATCGTACTTTAGTCAATGTAACGATCGGTGTTGGCTTTATGGGGTTTTCTTTAACCTCCATCTCTTATCCACCCTTGGAGATTATCGTCACACCTCACTACACCAATGCAAGAATGATAACACGATTTAAATCTAGTCTAGTATTAGATCCACCGGGACCTTCGGAAGGCCCATTGTATAAAGTATATGTTTTAGGCTATGGTAACAATAGGATCAATGGGAGCTTTTATAAGACCATGCGTACGATAGCATCATACCCAGAACAAAGCCTAGATTATCGTTACCACCTTTCCATGGCACATATGGAAACGGCCTTATTTTTATCACACGCTACACCACAAGACATGGACGG;
gIhm1核苷酸序列:
GGATCCGAAGATAACAGCTGAAGTACGTTATGTAACATCAATGGATTCATGTGGGATGGTGGCATTGATATCAGAGCCGGATATAGACGCTACTATTCGAACCATACAACTATCTCAAAAAAAAACATATAACGCGACTATAAGTTGGTTTAAGGTAACCCAGGGTTGTGAATACCCTATGTTTCTTATGGATATGAGACTTTGTGATCCTAAACGGGAATTTGGAATATGTGCTTTACGGTCGCCTTCATATTGGTTGGAACCTTTAACAAAGTATATGTTCCTAACAGACGATGAACTGGGTTTGATTATGATGGCCCCGGCCCAATTTAATCAAGGACAATATCGAAGAGTTATAACCATCGATGGTTCCATGTTTTATACAGATTTTATGGTACAACTATCTCCAACGCCATGTTGGTTCGCAAAACCCGATAGATACGAAGAGATTCTACATGAATGGTGTCGAAATGTTAAAACTATTGGCCTTGATGGAGCTCGTGATTACCACTATTATTGGGTACCCTATAACCCACAACCTCACCATAAAGCCGTACTCTTATATTGGTATCGGACTCATGGCCGAGAACCCCCAGTAAGATTCCAAGAGGCCATTCGATATGATCGTCCCGCCATACCGTCTGGGAGTGAGGATTCGAAACGGTCCAACGACTCTAGAGGAGAATCGAGTGGACCCAATTGGATAGACATTGAAAATTACACTCCTAAAAATAATGTGCCTATTATAATATCTGACGATGACGTTCCTACAGCCCCTCCCAAGGGCATGAATAATCAGTCAGTAGTGATACCCGCAATCGTACTAAGTTGTCTTATAATAGCACTGATTCTAGGAGTGATATATTATATTTTGAGGGTAAAGAGGTCTCGATCAACTGCATATCAACAACTTCCTATAATACATACAACTCACCATCCTTAAGTCCACATTCCAATCGAGTTGGTAGGGAAGATATGAAGTGGGCGGTACCAACCATCATAAAATAGGTTGGAGTCTGGACCAACGTTCACTCTTTTGAGTGTAAAGGACCACAGCATAATACTTAAT;
gEhm2核苷酸序列:
CCCGTCGACGAGTTCTAGCACCACGGGAATTGGAAGCTGCTCGTAAACTCCGTGAGATTTTCAACGCAGAGTACGTCGCACCTACGTTCACACTAGTCGATCCGGGGGATACGTCAAACGCGTATATTGTATGTAGGACCCCGGTGACCGAAGTAGTCTCTTCCATATCAAGAGGTATCGACAATAGAAAATCGGTAGATTCTTCATTTATTCGAATCGTCAGTAAATTAATCATTAGGAATGCTATTCACATGGGACTATCCGTCCTATGTGCATTTATATCCTATAATAAACCATGATAAATTTTATGTGGATATTTTATTAATCCTCCAAACCGTATGGGGGAGCACTTTTATAGAAATCTACCATAAAGAGTATATCCGTTAAATACCCGGGTTTGATTATATGTTTGTCAGTTGGTAGTTGAACTTCCATCGCCATCTAAGATGGACCATCAAACATCACTTATTAATGCCACAGATGATAATTGCTTAGACACGGATTCTAGTATAAACTTGCCAAGCATAGATAAATGCGAAATTGATGACAATTCAATTGCGGATGAAACTCTATCCGACAAGGGCTCCCCGGTCGCTATACCGCTATGCGCCACCATCGAGATCCCGCGTGGGAATGCGGACCGGCAGTCCCCAAGCCACGACGTACGAGGGGCCAATAGGACAAATTACGACTCCGATACCGGCTGTTATTATAGCGAGAGTGACAACGAGACGGCGACGCTGTTCATAAATAGAATAGGCAAACGCGAGACGGCCAAGAGACGGCGACGGAGGCGGTGTCTGGTGGCACTGGCCGTCTCAGGGGTGGCGACACTATGCGTGCTATCGGGATTATTAGGTGCGCTGCTGTGGCGGCTGATGGATGCCCCCGGGACGCGCCGGTGACGGGCTCGTTCAATAAACATAGCATACGTTATGACATGGTCTACCGCGTCTTATATGGGGACGATTGTTTTAGATTGGGTTTTCAGCGAGGCGCGTACAATATTGTACAGGGGAGTCTCCACGAACCCTAGGTTTTGGGTCGTAGATCACCACGGGGAGGGGATAGGGTACGAGTACATAAATCTTGTTGCTGGGATCGATCGTGTGATAAAGAATTTTGAGACCTTAGATGGCTTATGAAGGCGAG。
使用同源重组分别将扩增得到TKhm1、VP2、TKhm2;gIhm1、VP1、gEhm2片段分别克隆到pMD18T载体上,分别命名为重组质粒pMD18T-TK-VP2和pMD18T-gI/gE-VP1,重组体系为2×Uniclone Seamless Cloning Mix 5μL,线性化载体0.03pmol,插入片段0.02pmol,ddH2O补足至10μL。以上述两个重组质粒为模板,分别扩增TKhm1-VP2-TKhm2和gIhm1-VP1-gEhm2片段用于同源重组,引物分别为TK-F1(GGCTCACGCCAATAATCC)、TK-R1(CCGTCCATGTCTTGTGG);gI-F1(GGATCCGAAGATAACAGCTG)、gE-R1(CTCGCCTTCATAAGCCATC);扩增体系为:2×phantaMIX 25μL,上游引物2μL,下游引物2μL,模板2μL,ddH2O 19μL。将上述试剂充分混匀,按下列条件进行扩增:95℃变性5min后进入循环,循环参数:95℃15s,55℃15s,72℃2min10s,35个循环后72℃延伸5min,16℃2min。各扩增产物序列如下:
TKhm1-VP2-TKhm2核苷酸序列(SEQ ID NO:1):
GGCTCACGCCAATAATCCACTGGAAATGTGGGAGTAATAAAATTTCTAGTGTCCGATAAAATCAGATCATCCCCGATGTTAGTTATTCGCATCGAGTCGAATACGTATTCGACTCGTCCAGTCATGATGACCCGTCTTCGACAACTTCTCCTCTCCAACAAATGATACCAGTGATTAATCTCTTTGATCCTAAATAAGGTTTTGATATATGATCTATATAGACTCATTACCCATCACATATAGGGCATATTAATACTCACGTGGTTGCGTAGCGGAGTGATCGGGAAAGAGAGATGCTACCCATTTTAAAGGGTTACTTTCGCCGCTTCCTATTGGAGTTAAAGTGTTTTTTTTATCCTTCACAGATACACACGATGACATCGGTACGTGGTTCGTATGTTTATAGACCCCAGACCCAGACCTTGGGTATTTTGATTTTTTCTTGGAGCGGATTACACCACCACTCTCGATTGGATTGTATTCGGCGAGCTTGGTAATATTAGCAGCCAGAACAGAAATGTTACTCGTAAGCATCTGCGGGGGTAAACTAGTAACTCCCAAAATTCTCAGACCGCGCTGCGCGATAAATGCCAATATGGGTATTAGAGTGAGCTTCTCCCCCCCTGGTGGTAGAATCTTGGTTATTAAACCCACAGAATCTGTTAATTGTTTCAAACCCTCACGACGTTGAATGTCTTTACTAGTTGTATCCATATTTTTTGAAAAACGACACGTTTTCAGCTCAATTAGAAAACATATACCACCCCCTTCTCCCTCAAATTGTATAGTACATACACAATCAGGTCGGCGACGACCCAAGTTAACCTCACATGCTAGGTACACGCCCTTAGCCTTTTTAAGAGACTCTGCGGATACAGAGCCGCCCAATAAACACTCGAGTCGGTCGGTATATACTCCACTCGCAGAGGTCGAGGATATATCGCGCTTGAGGACAGCATAAAAGCGATTGTGGCATCGAATTCCAGCCCGGAGCCTCAATCCGACACTGCGTCGTTGTTCACGTTTCATCATACACAGATCAGACGATGAGTGATGGAGCAGTTCAACCAGACGGTGGTCAACCTGCTGTCAGAAATGAAAGAGCTACAGGATCTGGGAACGGGTCTGGAGGCGGGGGTGGTGGTGGTTCTGGGGGTGTGGGGATTTCTACGGGTACTTTCAATAATCAGACGGAATTTAAATTTTTGGAAAACGGATGGGTGGAAATCACAGCAAACTCAAGCAGACTTGTACATTTAAATATGCCAGAAAGTGAAAATTATAAAAGAGTAGTTGTAAATAATATGGATAAAACTGCAGTTAAAGGAAACATGGCTTTAGATGATATTCATGTACAAATTGTAACACCTTGGTCATTGGTTGATGCAAATGCTTGGGGAGTTTGGTTTAATCCAGGAGATTGGCAACTAATTGTTAATACTATGAGTGAGTTGCATTTAGTTAGTTTTGAACAAGAAATTTTTAATGTTGTTTTAAAGACTGTTTCAGAATCTGCTACTCAGCCACCAACTAAAGTCTATAATAATGATTTAACTGCATCATTGATGGTTGCATTAGATAGTAATAATACTATGCCATTTACTCCAGCAGCTATGAGATCTGAGACATTAGGTTTTTATCCATGGAAACCAACCATACCAACTCCATGGAGATATTATTTTCAATGGGATAGAACATTAATACCATCTCATACTGGAACTAGTGGCACACCAACAAATGTATATCATGGTACAGATCCAGATGATGTTCAATTTTATACTATTGAAAATTCTGTGCCAGTGCACTTACTAAGAACAGGTGATGAATTTGCTACAGGAACATTTTTTTTTGATTGTAAACCATGTAGACTAACACATACATGGCAAACAAATAGAGCATTGGGCTTACCACCATTTCTAAATTCTTTGCCTCAATCTGAAGGAGCTACTAACTTTGGTGATATAGGAGTTCAACAAGATAAAAGACGTGGTGTAACTCAAATGGGAAATACAGACTATATTACTGAAGCTACTATTATGAGACCAGCTGAGGTTGGTTATAGTGCACCATATTATTCTTTTGAAGCATCTACACAAGGGCCATTTAAAACACCTATTGCAGCAGGACGGGGGGGAGCGCAAACAGATGAAAATCAAGCAGCAGATGGTGATCCAAGATATGCATTTGGTAGACAACATGGTCAAAAAACTACTACAACAGGAGAAACACCCGAGAGATTTACATATATAGCACATCAAGATACAGGAAGATATCCAGAAGGAGATTGGATTCAAAATATTAACTTTAACCTTCCTGTAACAAATGATAATGTATTGCTACCAACAGATCCAATTGGAGGTAAAACAGGAATTAACTATACTAATATATTTAATACTTATGGTCCTTTAACTGCATTAAATAATGTACCACCAGTTTATCCAAATGGTCAAATTTGGGATAAAGAATTTGATACTGACTTAAAACCAAGACTTCATGTAAATGCACCATTTGTTTGTCAAAATAATTGTCCTGGTCAATTATTTGTAAAAGTTGCGCCTAATTTAACAAATGAATATGATCCTGATGCATCTGCTAATATGTCAAGAATTGTAACTTACTCAGATTTTTGGTGGAAAGGTAAATTAGTATTTAAAGCTAAACTAAGAGCATCTCATACTTGGAATCCAATTCAACAAATGAGTATTAATGTAGATAACCAATTTAACTATGTACCAAATAATATTGGAGCTATGAAAATTGTATATGAAAAATCTCAACTAGCACCTAGAAAATTATATTAAACATTAGTGGTGTTCCCTATTACCCCCCTGTGGTGAATGTGTGGAGGTCAGGGGATAATTGTATAATGACCATCGTTTCATGAATAAAATAACCGTGTGTGATGTGGATGTATTCATTAATTGAATTTCTCTTCCGGTTTTAGATCTTTATAAGCGTAAAACTGGTGTTTTAAATCCAAGAGCCGGGTTCTTTGGAGGTTGGTCACATCATCGCCACAGCCCGTGGATTCAAGCAATCTATGATGTGTTTGATAATATACCTATCGATATTCCTGATCATTGTATCGAGGATGTTGACTGGTTTACCGATGATGGATAGACCTGATGAAGGTGGGCTGGCTCGACGAACAGTTGGTGAAGTAGAAGGGGAGTTTTCTTATAGGGACGATGTTGATGTAGCAGACGTGAGAAACTTATTTATCATGTTACCAAAAAATGGGAGCGATATATTTCTATTCATATTCGATAGACGCAGTCAACGTCAACGCGGTACTATGTTTTTATTCCCCAAGGCTGGGTTTGTACAACCAACACCCGCGAAGGTTCGCGATGAAGCGGCGGCCGCCCCATTTGGGTTTATATCCCCTGTATATCCACTATCGAGTCTTTTATTTAATCCATACAATGGGAGATATCTGACGACACGCCATCTGATTGCCTTTGAGGTAACCCCGGAATCCTCTCTTCATGATTGGTATTTTGCACGATCACCAACAACTGCTACTCAGACACAGCCATTAGGACATATAACTAACCCCCCCCGACGATCGCCAAAAGACAAACCGACCACCTCCGGCCATACAGATTTAATTATACGCTATTGCGCATTGGAGTTGGATTTTTTCCAGGACACAAGACGACAGCGTGATGGAATATATTTACCTAATTACGAGGCCGTATGGCCATTGGCAATGAATTTTTTGGAGGGGATGTGGATATGGAGTAATCGTACTTTAGTCAATGTAACGATCGGTGTTGGCTTTATGGGGTTTTCTTTAACCTCCATCTCTTATCCACCCTTGGAGATTATCGTCACACCTCACTACACCAATGCAAGAATGATAACACGATTTAAATCTAGTCTAGTATTAGATCCACCGGGACCTTCGGAAGGCCCATTGTATAAAGTATATGTTTTAGGCTATGGTAACAATAGGATCAATGGGAGCTTTTATAAGACCATGCGTACGATAGCATCATACCCAGAACAAAGCCTAGATTATCGTTACCACCTTTCCATGGCACATATGGAAACGGCCTTATTTTTATCACACGCTACACCACAAGACATGGACGG;
gIhm1-VP1-gEhm2核苷酸序列(SEQ ID NO:2):
GGATCCGAAGATAACAGCTGAAGTACGTTATGTAACATCAATGGATTCATGTGGGATGGTGGCATTGATATCAGAGCCGGATATAGACGCTACTATTCGAACCATACAACTATCTCAAAAAAAAACATATAACGCGACTATAAGTTGGTTTAAGGTAACCCAGGGTTGTGAATACCCTATGTTTCTTATGGATATGAGACTTTGTGATCCTAAACGGGAATTTGGAATATGTGCTTTACGGTCGCCTTCATATTGGTTGGAACCTTTAACAAAGTATATGTTCCTAACAGACGATGAACTGGGTTTGATTATGATGGCCCCGGCCCAATTTAATCAAGGACAATATCGAAGAGTTATAACCATCGATGGTTCCATGTTTTATACAGATTTTATGGTACAACTATCTCCAACGCCATGTTGGTTCGCAAAACCCGATAGATACGAAGAGATTCTACATGAATGGTGTCGAAATGTTAAAACTATTGGCCTTGATGGAGCTCGTGATTACCACTATTATTGGGTACCCTATAACCCACAACCTCACCATAAAGCCGTACTCTTATATTGGTATCGGACTCATGGCCGAGAACCCCCAGTAAGATTCCAAGAGGCCATTCGATATGATCGTCCCGCCATACCGTCTGGGAGTGAGGATTCGAAACGGTCCAACGACTCTAGAGGAGAATCGAGTGGACCCAATTGGATAGACATTGAAAATTACACTCCTAAAAATAATGTGCCTATTATAATATCTGACGATGACGTTCCTACAGCCCCTCCCAAGGGCATGAATAATCAGTCAGTAGTGATACCCGCAATCGTACTAAGTTGTCTTATAATAGCACTGATTCTAGGAGTGATATATTATATTTTGAGGGTAAAGAGGTCTCGATCAACTGCATATCAACAACTTCCTATAATACATACAACTCACCATCCTTAAGTCCACATTCCAATCGAGTTGGTAGGGAAGATATGAAGTGGGCGGTACCAACCATCATAAAATAGGTTGGAGTCTGGACCAACGTTCACTCTTTTGAGTGTAAAGGACCACAGCATAATACTTAATATGTGCTCAACCTGCGCTAACGTGCTTAAATATTATGATTGGGACCCCCATTTCAAATTGGTAATCAACCCCAACAACTTCCTCTCTGTTGGCTTTTGTAGTAACCCTTTAATGTGTTGCTACCCAGAACTCCTTCCGGAATTTGGAACTGTTTGGGATTGCGATCGGTCACCACTTGAAATTTACCTAGAATCAATACTTGGTGATGATGAATGGGCATCCACTTTTGACGCTGTTGACCCAGTCGTTCCCCCAATGCACTGGGGTGCTGCTGGAAAAATTTTCCAGCCACACCCCGGTGTTCTCATGCACCATCTCATTGGTAAGGTTGCTGCAGGTTGGGACCCCGATCTGCCTCTAATTCGACTCGAGGCGGATGACGGGTCAATCACAGCACCCGAGCAAGGAACAATGGTTGGCGGCGTCATCGCTGAACCCAGCGCCCAGATGTCAACAGCTGCTGATATGGCCACCGGGAAAAGCGTTGATTCTGAGTGGGAGGCATTCTTCTCCTTTCACACCAGCGTCAATTGGAGTACATCTGAAACCCAAGGAAAGATTCTCTTCAAACAATCCTTAGGCCCTTTGCTCAACCCATATCTAGAACACCTTGCTAAGCTATATGTTGCGTGGTCTGGGTCGATTGAGGTTAGGTTCTCTATCTCTGGCTCTGGTGTCTTTGGTGGGAAGCTCGCAGCTATTGTTGTACCTCCTGGGGTTGATCCAGTGCAGAGTACTTCGATGCTACAATACCCCCATGTTTTGTTTGATGCTCGTCAGGTGGAACCAGTTATCTTCTGTCTTCCTGATCTAAGAAGCACCCTGTACCACCTTATGTCTGACACTGACACTACATCCTTGGTCATTATGGTGTACAATGATCTCATCAATCCCTATGCCAATGATGCCAACTCTTCTGGGTGTATTGTCACTGTCGAGACAAAACCTGGCCCTGACTTCAAGTTTCACCTCCTTAAGCCACCCGGATCTATGCTAACCCATGGCTCTATCCCTTCTGATTTAATTCCCAAAACATCTTCGCTCTGGATCGGTAACCGCTACTGGTCAGACATAACTGATTTTGTGATTCGGCCGTTTGTCTTCCAAGCAAATCGTCATTTTGACTTTAATCAAGAGACCGCAGGGTGGAGCACACCACGGTTTCGGCCTATATCTGTTACCATTACTGAACAGAACGGAGCAAAATTGGGCATTGGGGTGGCAACAGATTACATAGTGCCTGGAATCCCTGATGGCTGGCCTGACACCACAATTCCTGGGGAGTTGATACCAGCTGGTGATTACGCAATCACCAATGGTACTGGCAATGACATCACCACGGCTACAGGATATGACACTGCTGATATAATTAAGAACAATACCAACTTTAGGGGCATGTACATATGTGGTTCGCTCCAGCGTGCCTGGGGTGATAAGAAAATTTCCAACACTGCCTTTATCACCACTGCCACCCTAGATGGTGACAACAACAACAAGATCAATCCCTGTAATACCATAGACCAGTCAAAGATCGTCGTGTTTCAAGACAACCATGTTGGAAAGAAAGCGCAAACCTCAGACGATACATTGGCCCTGCTTGGTTACACTGGCATTGGTGAGCAGGCCATCGGGTCTGATAGGGACCGGGTTGTGCGCATCAGCACTCTCCCTGAAACTGGTGCTCGAGGCGGTAACCACCCAATTTTCTACAAGAACTCCATTAAATTGGGATATGTAATTAGGTCTATTGATGTCTTTAATTCACAAATCTTGCACACTTCCAGACAGTTATCGCTAAATCATTACCTACTCCCACCTGATTCTTTTGCCGTCTATAGAATAATTGACTCAAATGGCTCGTGGTTTGATATTGGAATTGATAGTGATGGGTTCTCTTTTGTTGGTGTTTCTGGCTTTGGTAAATTAGAATTTCCCCTTTCTGCCTCCTACATGGGAATACAATTGGCAAAGATCCGGCTTGCCTCTAACATTAGGAGTCCCATGACTAAGTTATGACCCGTCGACGAGTTCTAGCACCACGGGAATTGGAAGCTGCTCGTAAACTCCGTGAGATTTTCAACGCAGAGTACGTCGCACCTACGTTCACACTAGTCGATCCGGGGGATACGTCAAACGCGTATATTGTATGTAGGACCCCGGTGACCGAAGTAGTCTCTTCCATATCAAGAGGTATCGACAATAGAAAATCGGTAGATTCTTCATTTATTCGAATCGTCAGTAAATTAATCATTAGGAATGCTATTCACATGGGACTATCCGTCCTATGTGCATTTATATCCTATAATAAACCATGATAAATTTTATGTGGATATTTTATTAATCCTCCAAACCGTATGGGGGAGCACTTTTATAGAAATCTACCATAAAGAGTATATCCGTTAAATACCCGGGTTTGATTATATGTTTGTCAGTTGGTAGTTGAACTTCCATCGCCATCTAAGATGGACCATCAAACATCACTTATTAATGCCACAGATGATAATTGCTTAGACACGGATTCTAGTATAAACTTGCCAAGCATAGATAAATGCGAAATTGATGACAATTCAATTGCGGATGAAACTCTATCCGACAAGGGCTCCCCGGTCGCTATACCGCTATGCGCCACCATCGAGATCCCGCGTGGGAATGCGGACCGGCAGTCCCCAAGCCACGACGTACGAGGGGCCAATAGGACAAATTACGACTCCGATACCGGCTGTTATTATAGCGAGAGTGACAACGAGACGGCGACGCTGTTCATAAATAGAATAGGCAAACGCGAGACGGCCAAGAGACGGCGACGGAGGCGGTGTCTGGTGGCACTGGCCGTCTCAGGGGTGGCGACACTATGCGTGCTATCGGGATTATTAGGTGCGCTGCTGTGGCGGCTGATGGATGCCCCCGGGACGCGCCGGTGACGGGCTCGTTCAATAAACATAGCATACGTTATGACATGGTCTACCGCGTCTTATATGGGGACGATTGTTTTAGATTGGGTTTTCAGCGAGGCGCGTACAATATTGTACAGGGGAGTCTCCACGAACCCTAGGTTTTGGGTCGTAGATCACCACGGGGAGGGGATAGGGTACGAGTACATAAATCTTGTTGCTGGGATCGATCGTGTGATAAAGAATTTTGAGACCTTAGATGGCTTATGAAGGCGAG。
在FHV-1基因组的UL区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。根据上述TKhm1-VP2-TKhm2和gIhm1-VP1-gEhm2扩增片段可知,猫杯状病毒VP1基因的插入位置为猫疱疹病毒的US区域的gI/gE位置,猫细小病毒VP2基因的插入位置为猫疱疹病毒的UL区域的TK位置。申请人经大量筛选验证实验发现,将VP1基因和VP2基因分别插入猫疱疹病毒的US区域和UL区域,VP1基因和VP2基因之间基本不存在相互干扰,保证后续重组病毒能够发挥良好的免疫效果。
3、CRFK稳转细胞系的构建
在CMV、polyA基因(CMV和polyA基因均扩增自载体pcDNA3.1_+,GenBankaccessionno.MN996867.1)上分别选择5’-GN(20)GG或者5’-N(21)GG的序列位点,通过guidRNA在线设计工具(http://crispr.mit.edu/)和BLAST工具比对确定,所选的sgRNA靶标序列在基因中唯一位点,尽量避免脱靶的可能性。将设计合成的sgRNA放入沸水中自然降至室温成对变性、退火,退火之后形成具有粘性末端的DNA双链,可连入经BsmBI酶切线性化的LentiCRISPR真核表达载体中。其中,BsmBI酶切LentiCRISPR载体的体系为BsmBI酶5μL,10×3.1buffer 5μL,LentiCRISPR载体5μg,ddH2O up to 50μL,于55℃水浴锅酶切1h,电泳鉴定后使用omega胶回收试剂盒回收纯化;将sgRNA与线性化LentiCRISPR载体连接产物转化大肠杆菌感受态细胞(DH5α),并涂布氨苄抗性平板,挑取单克隆菌落。利用U6启动子通用引物(gactatcatatgcttaccgt)测序鉴定阳性克隆;37℃摇床培养阳性克隆12-16h,提取质粒,得到LentiCRISPR-CMV/polyA-sgRNA的表达载体。将上述表达载体与辅助质粒pSPAX2、pMD2.0G共转染293T细胞,转染48-72h后收取慢病毒,感染CRFK,使用嘌呤霉素加压筛选后即获得CMV/polyA-CRFK稳转细胞系。
表1 sgRNA引物信息
引物名称 | 引物序列 |
CMV-sgRNA-F | CACCgttattgacgtcaatgggcgg(SEQ ID NO:3) |
CMV-sgRNA-R | AAACccgcccattgacgtcaataac(SEQ ID NO:4) |
polyA-sgRNA-F | CACCggggaggggcaaacaacaga(SEQ ID NO:5) |
polyA-sgRNA-R | AAACtctgttgtttgcccctcccc(SEQ ID NO:6) |
4、重组病毒构建
将CMV/polyA-CRFK稳转细胞置于6孔板中进行培养(含胎牛血清10%、青霉素100U/ml和链霉素100U/ml),待细胞汇合度到70-80%后进行转染。将同源重组片段TKhm1-VP2-TKhm2、gIhm1-VP1-gEhm2混合转染,转染量比例为1:1,800μL转染液37℃孵育4h。转染细胞孵育完成后,感染rWH/2020-△TK/gI/gE-EGFP+-mCherry+毒株(MOI=0.01,该毒株记载在专利文献“CN114958783B一种三基因缺失的猫疱疹病毒I型重组病毒、猫传染性鼻气管炎活疫苗以及制备方法”中),1mL病毒液37℃孵育2h后换液;培养36-48h后,在显微镜下观察,出现不带红色和绿色两种荧光的细胞病变斑之后将其挑取出来进行多轮空斑纯化,直至所有病毒均不带红绿色双荧光,将完全纯化的病毒接种CRFK细胞,收获病毒液,按常规方法提取DNA后分别使用引物TK-F(cacaatcaggtcggcg)、TK-R(gtaaaccagtcaacatcctcg)及引物gI/gE-F(agtcagtagtgatacccgc)、gI/gE-R(gagactacttcggtcaccg)进行PCR扩增,对扩增出的片段进行序列测定,将序列测定后纯化的重组病毒命名为rWH2020-△TK/gI/gE-VP1-VP2。重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2的构建示意图见图1。图2中A-C分别为绿色荧光、红色荧光、明场视野下重组病毒rWH2020-△TK/gI/gE-VP1-VP2感染CRFK细胞后细胞病变结果图。
5、重组病毒的保存
将鉴定后的rWH2020-△TK/gI/gE-VP1-VP2病毒按照常规方法接种CRFK细胞,培养至90%以上细胞出现CPE(细胞病变效应)后收获病毒上清,分装后-80℃保存。
实施例2重组病毒rWH2020-△TK/gI/gE-VP1-VP2的遗传稳定性、纯净性检验
1、重组病毒rWH2020-△TK/gI/gE-VP1-VP2的遗传稳定性检验
将rWH2020-△TK/gI/gE-VP1-VP2在CRFK细胞上增殖连续传代至十代,利用常规方法提取各代病毒基因组DNA,分别使用引物TK-F(cacaatcaggtcggcg)、TK-R(gtaaaccagtcaacatcctcg)、gI/gE-F(agtcagtagtgatacccgc)、gI/gE-R(gagactacttcggtcaccg)、gB-F(ctccagacatggtaacggat)和gB-R(tgtaacggcataggaactcc)进行PCR扩增TK和gI/gE重组位点及gB基因验证重组病毒rWH2020-△TK/gI/gE-VP1-VP2的遗传稳定性。扩增体系为:2×phanta MIX 25μL,上游引物2μL,下游引物2μL,模板2μL,ddH2O 19μL。将上述试剂充分混匀,按下列条件进行扩增:95℃变性5min后进入循环,循环参数:95℃15s,55℃15s,72℃1min45s/15s,35个循环后72℃延伸5min,16℃2min。反应结束后,将PCR反应液进行1%琼脂糖凝胶电泳检测。图3为重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2的PCR鉴定结果;图中A是对TK基因进行扩增,M:DL5000 DNAMarker,泳道1是重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2扩增条带,泳道2是亲本毒株WH2020扩增条带,泳道3是阴性对照;图中B是对gI/gE基因进行扩增,M:DL5000 DNAMarker,泳道1是重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2扩增条带,泳道2是亲本毒株WH2020扩增条带,泳道3是阴性对照。电泳结果显示,重组毒rWH2020-△TK/gI/gE-VP1-VP2可稳定遗传(见图4中A-C,A:TK基因扩增,B:gI/gE基因扩增,C:gB基因扩增)。图5为重组病毒rWH2020-ΔTK/gI/gE-VP1-VP2表达VP1蛋白和VP2蛋白的荧光图。
2、重组病毒rWH2020-△TK/gI/gE-VP1-VP2的纯净性检验
将rWH2020-△TK/gI/gE-VP1-VP2在CRFK细胞上增殖连续传代至十代,按照《中国兽药典》附录记载的方法对不同代次病毒进行无菌、支原体、外源病毒检验,结果如下:
表2 rWH2020-△TK/gI/gE-VP1-VP2纯净性检验
实施例3重组病毒rWH2020-△TK/gI/gE-VP1-VP2的致病性试验
取约2月龄的FHV-1抗体阴性幼猫15只,随机分为3组,分别标记为A、B、C组,按照下表进行攻毒试验。攻毒后连续观察14日。
表3 rWH2020-△TK/gI/gE-VP1-VP2和WH2020对幼猫的致病性试验
组别 | 攻毒毒株 | 攻毒剂量 | 接种方式 | 备注 |
A | rWH2020-△TK/gI/gE-VP1-VP2 | 108TCID50 | 滴鼻 | |
B | DMEM培养液 | 1.0mL | 滴鼻 | 空白对照 |
C | WH2020 | 108TCID50 | 滴鼻 |
攻毒后每日观察并测定幼猫体温,观察是否出现猫传染性鼻气管炎临床症状和死亡。结果如表4所示:rWH2020-△TK/gI/gE-VP1-VP2滴鼻后未出现FHV-1相关的临床症状,而滴鼻接种WH2020的幼猫在攻毒后第三至四天有幼猫出现体温升高、打喷嚏、眼鼻分泌物和呼吸啰音等症状。空白对照组未出现任何临床症状,整个试验过程中均正常。
表4 rWH2020-△TK/gI/gE-VP1-VP2和WH2020对幼猫的致病性试验结果统计
组别 | 发热 | 眼鼻分泌物增多 | 打喷嚏 | 呼吸啰音 |
A | 0/5 | 0/5 | 0/5 | 0/5 |
B | 0/5 | 0/5 | 0/5 | 0/5 |
C | 5/5 | 5/5 | 5/5 | 5/5 |
实施例4猫传染性鼻气管炎活疫苗rWH2020-△TK/gI/gE-VP1-VP2的制备和检验
将rWH2020-△TK/gI/gE-VP1-VP2接种CRFK细胞,待90%以上细胞病变时收获病毒液。将收获病毒液稀释后与保护剂(谷氨酸钠8g/L、海藻糖70g/L、尿素5g/L、精氨酸1.6g/L)按体积比7:1混合后,按1mL/瓶分装进行冷冻真空干燥。按照《中国兽药典》附录检验无细菌与支原体污染,无外源病毒污染。
实施例5猫传染性鼻气管炎活疫苗rWH2020-△TK/gI/gE-VP1-VP2的免疫原性
选取8-12周龄猫疱疹病毒I型,猫细小病毒和猫杯状病毒抗原抗体均为阴性的幼猫10只,随机均分为2组,第1组皮下注射rWH2020-△TK/gI/gE-VP1-VP2(107.0TCID50),第2组注射1mLDMEM。第一次免疫21日后以相同剂量和免疫方式进行第二次免疫。二免21日后,所有试验猫采血,进行血清特异性抗体检测。在二次免疫21d后rWH2020-△TK/gI/gE-VP1-VP2疫苗免疫组产生了较高水平的FHV-1、FCV和FPV特异性抗体(见图6)。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.一种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的构建方法,其特征在于,包括以下步骤:
分别获取FCV病毒的VP1基因、FPV病毒的VP2基因以及FHV-1病毒的TK左右侧同源臂TKhm1、TKhm2和gI、gE同源臂gIhm1、gEhm2,利用同源重组和PCR扩增方法,获得TKhm1-VP2-TKhm2和gIhm1-VP1-gEhm2片段;
依据CMV、polyA基因的sgRNA引物,利用CRISPR基因编辑方法构建CMV、polyA基因的sgRNA表达载体,转染、收取慢病毒、感染CRFK细胞,获得CRFK稳转细胞系;
将TKhm1-VP2-TKhm2和gIhm1-VP1-gEhm2片段转染至CRFK稳转细胞系,得到转染细胞,将转染细胞感染rWH/2020-△TK/gI/gE-EGFP+-mCherry+毒株,得到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;
所述TKhm1-VP2-TKhm2片段的核苷酸序列如SEQ ID NO:1所示,所述gIhm1-VP1-gEhm2片段的核苷酸序列如SEQ ID NO:2所示;
所述sgRNA引物的核苷酸序列如SEQ ID NO:3-6所示。
2.一种如权利要求1所述构建方法获得的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。
3.一种如权利要求2所述的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在制备防治猫杯状病毒病的疫苗中的应用。
4.一种如权利要求2所述的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在制备防治猫细小病毒病的疫苗中的应用。
5.一种如权利要求2所述的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在制备防治猫传染性鼻气管炎的疫苗中的应用。
6.一种同时防治猫杯状病毒、猫细小病毒和猫疱疹病毒的活载体疫苗,其特征在于,所述活载体疫苗包含权利要求2所述的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。
7.根据权利要求6所述活载体疫苗,其特征在于,所述活载体疫苗还包含保护剂。
8.根据权利要求7所述活载体疫苗,其特征在于,将所述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接种细胞,收取病毒液,将病毒液与保护剂混合后,冷冻干燥,得到所述活载体疫苗。
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