CN112322658A - 一种共表达小反刍兽疫病毒h和f蛋白的重组山羊痘病毒 - Google Patents
一种共表达小反刍兽疫病毒h和f蛋白的重组山羊痘病毒 Download PDFInfo
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Abstract
本发明提供一种用于重组山羊痘病毒构建的重组转移载体。所述的转移载体主要包含：TK基因的左臂和右臂、两个逆向的p7.5K启动子及其分别调控的小反刍兽疫病毒H基因和F基因、p11K启动子、增强型绿色荧光蛋白(eGFP)基因、两个顺向Loxp序列。所述的转移载体与山羊痘病毒在羊睾丸细胞内发生同源重组，获得表达eGFP的重组山羊痘病毒，在Cre酶作用下，eGFP基因被特异性敲除，获得潜在共表达小反刍兽疫病毒H和F蛋白的重组山羊痘病毒。
Description
技术领域
本发明属于重组病毒构建技术领域，具体涉及一种共表达小反刍兽疫病毒H和F蛋白的重组羊痘病毒。
背景技术
小反刍兽疫(peste des petits ruminants,PPR)，是一种由小反刍兽疫病毒(peste des petits ruminants virus,PPRV)感染所引起的急性或亚急性传染病，现为国际动物卫生组织(OIE)必须通报疫病。该病通过直接或间接接触传播，以呼吸道和消化道为主要感染途径，病原可通过眼鼻口分泌物及飞沫传播。该病最易感染绵羊和山羊，而山羊比绵羊、新生羊比成年羊更易发病，发病率较高，急性感染的死亡率可达100％。
PPRV属副粘病毒科(Paramyxoviridae)、麻疹病毒属(Morbillivirus)成员。PPRV基因组为不分节段的单股负链RNA，全长15948nt。PPRV H基因开放阅读框(ORF)全长1830nt，编码病毒的H蛋白。该病毒全长609aa，分子质量约为68kD，属于II型糖蛋白。
成熟病毒粒子的H蛋白是由二硫键连接的两个同源二聚体，从而形成了一个铆钉样结构的四聚体，镶嵌在病毒囊膜表面。PPRV F基因较为保守，由2411nt构成。F蛋白由546个aa构成，分子质量约59kD。
成熟病毒粒子的F蛋白前体(F0)的104～108aa为蛋白水解区域，F0在该区域将被水解为由二硫键连接的F1和F2，其氨基酸切割位点及细胞蛋白酶的切割能力决定了病毒的毒力。PPRV的H和F蛋白是病毒的免疫保护性蛋白，在体内可诱导理想的免疫保护作用。
山羊痘病毒(goatpox virus,GTPV)属于痘病毒科(Poxviridae)、羊痘病毒属(Capripoxvirus)成员。GTPV基因组为两端共价闭合、线性双股DNA，对外源基因容纳性较高，若经适当修饰，可成为理想的活疫苗载体。胸苷激酶(thymidine kinase,TK)基因是痘病毒复制的非必需基因，因此作为同源重组基因广泛用于重组GTPV构建。外源抗原基因插入至TK基因会破坏其表达，但不显著影响重组GTPV的复制。若外源蛋白能在体内表达且诱导理想的免疫保护作用，重组病毒将是潜在的活疫苗候选株。
发明内容
本发明提供一种共表达小反刍兽疫病毒H和F蛋白的重组羊痘病毒GTPV，从而弥补现有技术的不足。
本发明首先提供一种用于重组山羊痘病毒构建的重组转移载体，所述的重组转移载体携带氨苄青霉素抗性基因，还包含有如下的作用元件：TK基因的左臂(TKL)和右臂(TKR)；两个转录方向相反的p7.5K启动子；逆向的PPRV H和F基因；p11K启动子；增强型绿色荧光蛋白(eGFP)筛选marker；两个顺向Loxp序列。
所述的重组转移载体H和F基因上游皆为p7.5K启动子，下游皆为“ATTTTTAT”早期转录终止信号。
筛选标记marker为eGFP基因，受p11K启动子调控，使后期筛选更为便捷。eGFP基因和p11K启动子两端为两个顺向Loxp序列。
所述的重组转移载体，其一种具体的核苷酸序列为SEQ ID NO:1；
本发明另一个方面还提供种共表达小反刍兽疫病毒H和F蛋白的重组羊痘病毒GTPV，是将上述的重组转移载体转移到永生化羊睾丸(GT)细胞内，通过TK基因左右同源臂与亲本GTPV发生同源重组，借助于eGFP筛选marker的指示作用，通过连续多次噬斑纯化，获得含H、F及eGFP基因的重组山羊痘病毒(GTPV-HF-eGFP)。GTPV-HF-eGFP在细胞内Cre重组酶作用下，特异性敲除筛选marker，通过几轮噬斑纯化，筛选到潜在共表达H和F蛋白的重组GTPV(GTPV-HF)。
附图说明
图1：重组转移载体pLSEG-HF模式图。
图2：GTPV-HF-eGFP噬斑纯化。a：绿色荧光噬斑；b：亮场对照。
图3：F3/R3上下游引物退火位点。
图4：第8代GTPV-HF-eGFP噬斑纯化后PCR鉴定。M：DNA Marker；G：GTPV-HF-eGFP。
图5：筛选marker敲除的测序鉴定。黄色阴影序列：单一Loxp序列。
图6：重组山羊痘病毒(GTPV-HF)和母源山羊痘病毒(GTPV)生长曲线的比较。
具体实施方式
本发明利用分子克隆及同源重组技术，构建了一种重组山羊痘病毒转移载体(pLSEG-HF)，利用该重组载体与母源GTPV的同源重组，获得带有筛选marker的重组病毒。利用Cre-Loxp重组技术，对筛选marker进行特异性敲除，最终获得一株潜在共表达PPRV H和F蛋白的重组GTPV。
作为实施例的一种具体记载，其中H和F蛋白为PPRV(GenBank:KX421388)中的H和F蛋白。
以下通过实施例来进一步阐明本发明。但是应理解，所述实施例只是举例说明的目的，并不意欲限制本发明的范围和精神。
实施例1.重组山羊痘病毒转移载体pLSEG-HF的构建
所构建的pLSEG-HF模式图如图1所示。TK基因的左臂(TKL)和右臂(TKR)分别位于外源序列的上游和下游，二者为GTPV的同源重组位点；PPRV H和F基因的方向相反，上游分别为p7.5K启动子，下游分别为“ATTTTTAT”早期转录终止信号；eGFP筛选marker受p11K启动子转录调控。重组载体全长9 533bp，氨苄青霉素抗性。
实施例2.GTPV-HF-eGFP的构建
在六孔细胞培养板中接种GT细胞，培养至80－90％致密单层，接种GTPV AV41疫苗株，37℃作用2h。按照每孔加入150μL无血清Opti-MEM和6μL Lipofectamine 2000的比例，配制溶液I；按照每孔加入150μL无血清Opti-MEM和3μg pLSEG-HF的比例，配制溶液II。将溶液II逐滴加入溶液I中，轻柔混匀，室温静置20min。在此期间，吸弃六孔板内病毒液，每孔加入1mL Opti-MEM，再逐滴加入孵育后的脂质体与pLSEG-HF混合液。37℃5％CO2培养箱培养6h，吸弃转染液，加入细胞维持液培养72–96h。通常转染后24h，细胞单层便可明显观察到绿色荧光，而只感染病毒和只转染质粒的对照孔无绿色荧光。
实施例3.GTPV-HF-eGFP的纯化
采用连续噬斑纯化的方法将GTPV-HF-eGFP从亲本毒种群中分离出来。将重组后病毒液冻融2次，按10–1至10–6连续稀释度接种六孔板中的GT细胞，37℃5％CO2培养箱培养2h。吸弃病毒液，每孔加入2mL含1％低熔点琼脂糖的维持培养基，继续培养5–7天。培养过程中，荧光显微镜下可观察到细胞单层出现绿色荧光噬斑(图2a)。抠取荧光噬斑，加至500μL培养基中，冻融2次，进行下一轮噬斑纯化，重复进行8次。
实施例4.GTPV-HF-eGFP纯化后的PCR鉴定
GTPV-HF-eGFP经历连续8次噬斑纯化后，采用病毒核酸提取试剂盒提取病毒液总核酸，进行PCR检测。首先根据TKL和TKR序列，设计一对特异性引物F3/R3。上游引物F3序列为：5’-AAACATTTTATCAATATTGCACGAA-3’；下游引物R3序列为：5’-TACACAGAATTACCATATCGGATAT-3’。二者退火位点如图3所示，扩增目的基因5 862bp。PCR反应体系采用2×PrimeSTAR Max Premix配制，经历30个反应循环，反应条件为：98℃变性10s，55℃退火10s，72℃延伸30s。PCR检测结果如图4，在5 862bp处扩增出了目的条带，且未检测出亲本毒的特异性条带(209bp)，说明重组病毒已纯化成功。
实施例5.GTPV-HF-eGFP筛选marker的敲除
将表达Cre重组酶的质粒转染至GT细胞，通过G418连续筛选3代，获得表达Cre重组酶的GT细胞(GT-Cre细胞)。将GTPV-HF-eGFP接种GT-Cre细胞，置于37℃5％CO2培养箱培养内培养。在此期间，重组病毒两个Loxp之间序列在Cre重组酶作用下被敲除。3d后冻融病毒感染的细胞2次，收集上清，其中含有无筛选marker的重组病毒(GTPV-HF)。
实施例6.GTPV-HF的纯化及鉴定
采用连续噬斑纯化法将GTPV-HF从亲本毒种群(GTPV-HF-eGFP)中分离出来。将收获的GTPV-HF病毒液冻融2次，按10–1至10–6连续稀释度接种六孔板中的GT细胞，37℃5％CO2培养箱培养2h。吸弃病毒液，每孔加入2mL含1％低熔点琼脂糖的维持培养基，继续培养7-10天。在荧光显微镜下，排除绿色荧光斑干扰，抠取白斑，将其在培养基中冻融2次，收获病毒液，再重复进行2-3轮噬斑纯化。采用F3/R3上下游引物，对纯化的GTPV-HF进行PCR扩增。扩增产物进行Sanger测序，如图5所示，黄色阴影所示为单一Loxp序列，说明筛选marker已敲除，且成功获得纯化的GTPV-HF。
实施例7.GTPV-HF生长曲线测定
将GT细胞同时接种多个35mm细胞培养皿，次日将GTPV-HF及亲本毒(GTPV AV41疫苗株)分别平行感染多个GT细胞(MOI＝0.1)。感染0,24,48,72和96h后，分别将对应培养皿连续冻融2次并收获病毒液，测定不同培养时间点的病毒滴度，绘制重组毒和亲本毒的生长曲线(图6)。结果表明，外源片段的插入，并未显著影响病毒在细胞内的生长动力学。
应该理解，尽管参考其示例性实施方案，对本发明进行具体地显示和描述，但是本领域的普通技术人员应该理解，在不背离由后附的权利要求所定义的本发明的精神和范围的条件下，可以在其中进行各种形式和细节的变化，可以进行各种实施方案的任意组合。
序列表
<110> 中国动物卫生与流行病学中心
<120> 一种共表达小反刍兽疫病毒H和F蛋白的重组山羊痘病毒
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9533
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aagcttttta ttagtgggtt acctaatact atgtactcaa aggaagcact atcattaaat 60
agacaaccga taacatataa atattgtaat gatcttttac aatcaataaa tggatcacag 120
caagtattta ttaacgatat tcttagaaaa tgatgactcc ttttttaaat acttatcaga 180
acaagatgat gaaacagcta tgtctgatat cgaaactatt gtaacatatt taaatttttt 240
attgtcattg ttaattagat caaaggataa attagagtcg ataggttatt attatgaacc 300
actgtctgaa gaatgtaaaa cattagttga tttttccaat atgaaaaatt ttaggatatt 360
atttaataag attcctataa atatactaaa taaacaaata actgtaaata aagggtactt 420
atcagatttt gttacgacat tgatgagatt aaaaaaagaa ctttttttag aatcaccaga 480
gccgataaca tatatagacc ctagaaaaga tccaacattt ttaaacattt tatcaatatt 540
gcacgaaaat aattgaacaa atatttttta aaaaaaaatg gactatggat atatacattt 600
aattatagga cctatgtttt ctggcaaaag tactgaattg ataagaatag ttaaaagtat 660
gcccggtagt tgcgatatac ataaactgat cactaattcc aaacccaccc actttttata 720
gtaagttttt cacccataaa taataaatac aataattaat ttctcgtaaa agtagaaaat 780
atattctaat ttattgcacg gtaaggaagt agaatcataa agaacagtca gatctggatc 840
tgcagggatc caccatgtcc gcacaaaggg aaaggatcaa tgccttctac aaagacaatc 900
ctcacaataa gaaccatagg gtgatcctgg atagagaacg cttggtcatt gaaagaccct 960
acatcttgct tggagtcctg ctggtaatgt tcctgagtct aatcggactg ctggccattg 1020
cagggatcag gcttcaccgg gccaccgttg gaacttcaga gatccagagt cggctgaata 1080
ccaatattaa gttgaccgaa tctattgatc accagactaa ggatgtctta actccccttt 1140
ttaaaatcat tggcgatgaa gtcggcatca gaattccaca gaaattcagt gatcttgtca 1200
agttcatctc cgataagatt aaattcctca accctgatag agagtatgat ttcagggatc 1260
tccggtggtg catgaatccc cccgagagag tcaaaattaa ttttgatcag ttttgtgagt 1320
acaaagctgc ggttaagtca attgaacata tatttgagtc accactcaac aagtcaaaaa 1380
agctgcaatc attgactctc gggcccggaa caggctgtct aggcaggaca gtaacaaaag 1440
ctcatttctc agaacttaca ctgaccttaa tggacctgga tctagagatg aagcacaacg 1500
tgtcctcagt gtttaccgta gttgaagagg ggttattcgg aagaacatat accgtctgga 1560
gatccgatgc cagggatccg agcaccgatc taggtatcgg ccatttttta agagtcttcg 1620
agattggact gataagagat ctcgggctgg gtccccctgt ttttcatatg accaactatc 1680
tcacagtgaa catgagtgat gactatcgga gatgtctttt agcggtaggg gagttgaagt 1740
tgacagccct atgcacctca tctgagactg tgacactgag tgagagagga gctccaaaga 1800
gggagcctct tgtggttgtg atacttaatc tggctggacc cactctaggg ggcgaactat 1860
acagtgtctt gcctacctcc gatctcatgg tggagaaact ctatttatct tcacatagag 1920
ggatcatcaa ggatgacgag gccaattggg tagtgccgtc taccgatgtt cgtgatcttc 1980
agaacaaagg tgaatgtctg gtggaagcat gcaagactcg acctccttca ttttgcaatg 2040
gcacaggatc aggcccgtgg tcagagggga gaatccctgc ttacggggtg atcagggtca 2100
gtcttgactt agctagtgac ccggatgtag ttatcacttc agtgtttggc ccactgattc 2160
ctcacctatc cggcatggat ctttacaaca acccgttttc aagagctata tggttggctg 2220
taccacctta tgagcagtca tttctaggaa tgataaatac aattggattc cctaacagag 2280
cagaggttat gccgcacatt ttgaccacag agatcagagg ccctcggggt cgttgccatg 2340
ttcccataga attgtcccgc agggttgatg acgatatcaa gatcgggtcc aacatggtca 2400
tattgccgac gatggacctg aggtatatta cagccactta tgatgtttcc aggagcgagc 2460
atgcaatcgt gtactatatc tatgacacag gtcgctcatc atcttacttc tacccagttc 2520
gactgaattt caaaggcaat cctctctctc tgaggataga gtgtttccct tggcgtcata 2580
aggtgtggtg ctaccatgat tgtcttatat acaacaccat aacagatgaa gaggtccata 2640
cgagagggct gaccggtata gaggtaacat gcaatccagt ttgaattttt atgcggccgc 2700
ggcctatatg gcccggtccg gttaactacg tagacgtcga ggatttcgcg tgggtcaatg 2760
ccgcgccaga tccacatcag acggttaatc atgcgatacc agtgagggat ggttttacca 2820
tcaagggccg actgcacagg cggttgtgcg ccgtgattaa agcggcggac tagcgtcgag 2880
gtttcaggat gtttaaagcg gggtttgaac agggtttcgc tcaggtttgc ctgtgtcatg 2940
gatgcagcct ccagaatact tactggaaac tattgtaacc cgcctgaagt taaaaagaac 3000
aacgcccggc agtgccaggc gttataaaaa ttacagtgat ctcacgtacg actttgaggt 3060
cccggtcaga tcgggtttga gccctgggtt gattttggag atagggattt ccttgttctt 3120
acacctcccc ttacagcagc atattaatgt gacaagccct agggatactc caatgcaagc 3180
tgccagtgct atgtacatgt tcccgccgaa aggtgcccct ttgacagtct tcagtatttg 3240
gtctgatgca tctaggagct cctttgcatt ctccagtctt gtgactgcat tgcctaaatt 3300
ggtgcctaca tccagttttt ctagggagat ggctggacct aagtctattt tgtgtaagta 3360
tacagaatcc ggatactctc gactgccgac ctgtattgtc actccatcca cctcaactac 3420
aggacacttg tcggatgcta caacagttag tagtttgtca ggatcttggc tgatgactgt 3480
ctccgttgtg taacatttgc acaaaactga cgcacaattt gcaatcaggt tcccttttga 3540
taggataaat ctattactta tggtccctga aactagggtt ctggcgcacg attttgttga 3600
tcccctgaaa cattcctgaa gcaatgggct cattgggtac aacgcattct ggctgcaaac 3660
tgtcccctct ggagtgaata cgcatgacgt ctcatcaaag ttcgatatca gatacccctg 3720
agtggctaca tatttaggga tagtagtata ccactcctgt gccccaatgt tgtatgttat 3780
agcttctatc ttgtgaacta tcactccctt gatctcagat aaggttgggt aggcgatgct 3840
aaggattata aagtaatctc ttgtgtccac atatgtaacc cgggccttta ttcccttgct 3900
ctctaggatg gcaaggaaat ccccaccgct atacccaagc ttgtccagga ttttattaat 3960
gtctccgcct aatgcataac tgagtgcctg gatcgatatt tcggcagcta tcgggtctcg 4020
aaggctgggc ccgaatatgg acaggatctc ggtgtagtac ctaaggagct tgaggccgag 4080
cttgtggcct accagctcgc atgacattct atgaacagaa gggacaagct cattgttgat 4140
ataatcctgg acgccctgta ctgccagtat ggtctcctta tttgcaagtc tgatttcttc 4200
tattgcctga ttcgacttct caagactggt ttttaaactc tcaattgctt gagagttcat 4260
caatgattga tgaagagcga ctcctgcagt tatctgagcg gctgtcgcaa ctccaagtgc 4320
tactccggcc agaacagctc cagcaaaacg acgggttcta cgcccaggtg tcagggtttg 4380
aattggtctt acattcttgg ttatcaccga cagagcatcc tctacaggtt ttaacactgt 4440
gatcagcaat ctcttgtact ctgcaatctc tgactttgta caattgttga tggctgttat 4500
atttggcatt aactttataa ccagagtttg gtggcttggc ctagtcatca ccttgtagct 4560
ggcactccct gttcctacaa tcccgatctt ggatagattg ccccagtgaa tctggcacgc 4620
aacaacattt gggagaagaa acagaaatgt caagattgcg acccgtgtca tggtggcatc 4680
tcgcaagcct gtcttgtgtc caagcttggg ggatcgatcc gtcactgttc tttatgattc 4740
tacttcctta ccgtgcaata aattagaata tattttctac ttttacgaga aattaattat 4800
tgtatttatt atttatgggt gaaaaactta ctataaaaag cgggtgggtt tggaattagt 4860
gatcagttta tgtatatcgc aactaccggg caataacttc gtatagcata cattatacga 4920
agttattatg ataaaaagtc gacttaatta attaggcctc tcgagcctag taagaaatat 4980
aaagaagaat atcccattgt taacataaac accataaagc gttattacga aataaaagat 5040
tcaaaaatga catgtataaa ttttgaatca cctataagtg attatgatca agtaaattat 5100
ttaaaagatt acataaatat aagtgatgat tattatctgt atgacggatc gatccagatc 5160
ggggaattcc gatcctagaa gcgatgctac gctagtcaca atcaccactt tcatatttag 5220
aatatatgta tgtaaaaata tagtagaatt tcattttgtt tttttctatg ctataaatga 5280
attccgaagc catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg 5340
tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg 5400
atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc 5460
cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg 5520
accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc 5580
gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg 5640
gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 5700
tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca 5760
agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg 5820
tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc 5880
ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg 5940
atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc 6000
tgtacaagtc cggactcaga tccaccggat ctagataact gatcataatc agccatacca 6060
catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac 6120
ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat 6180
aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 6240
gtttgtccaa actcatcaat gtatcttaac gcgactaata acttcgtata gcatacatta 6300
tacgaagtta tgaattcaaa tagcgcagta taaatgctgt gtagtaaaat acttaaaaga 6360
tatccgatat ggtaattctg tgtatacaca tgataataac catgtatctg ccatatcaac 6420
aactttatta tatgacgtcg ttgataaaat tatgaatttc gacattatag gtatagatga 6480
agggcaattt tttaaagata ttgtatcttt ttctgaaaat atggcaaata tgggaaagat 6540
aattataata gctgcactag atagcacgtt tcaacgaaaa gaatttaatg atatattgaa 6600
attgataccg ttatctgaaa aagtaacaaa attaaacgct gtatgtatgg aatgttataa 6660
agacgccgca ttttctaaga ggatcactaa agaaaaggaa atagaactca tcgggggtaa 6720
agaaaaatat aaatctgttt gtaggaaatg ttatttttta gaataataaa tattaatgaa 6780
aaaaaatcaa aaaaaagtga tctatttact tattaaacta tatagttaat aagtaaaatg 6840
ggtatcagac acgagttaga tattttgctt gtttctgaaa atctcgcact gaagaatgtt 6900
gaacttctta aaggtgatag ttatggatgt actattaata taaaagttaa tcaacaaaaa 6960
aaattggatt ttattattat attacggccc gattggacag aggtaaggaa tgttaaaaaa 7020
ataaatatgg tatgtaacgg tgttgttatt gatacaacac taattaaaaa atctttttac 7080
gaagaagtat attcatcatc tgtaacagtt tttcaaaata ctaccgttga attttttagt 7140
gatactagta agaaatataa agaagaatat cccattgtta acataaacac cataaagcgt 7200
tattacgaaa taaaagattc aaaaatgaca tgtataaatt ttgaatcacc tataagtgat 7260
tatgatcaag taaattattt aaaagattac ataaatataa gtgatgatta ttatctgtat 7320
gacgaattaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 7380
tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 7440
cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 7500
cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 7560
tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 7620
tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 7680
atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 7740
gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc 7800
aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 7860
aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 7920
gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 7980
cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 8040
atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 8100
tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 8160
ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 8220
ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 8280
ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 8340
tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 8400
tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 8460
aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 8520
tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 8580
tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 8640
gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 8700
agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 8760
tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 8820
ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 8880
cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 8940
tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 9000
acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 9060
gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 9120
ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 9180
tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 9240
attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 9300
cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc 9360
tccttacgca tctgtgcggt atttcacacc gcatacttgg tgtttggctt cagcaacaag 9420
tacaactctc cattcaccat aatcatcttt gacctctaca attccaccat caggtttgat 9480
gctggaattt gaaacaaata aagtaccgcc aagatcaggg tcaatttttt tta 9533
Claims (4)
1.一种用于重组山羊痘病毒构建的转移载体(pLSEG-HF)，其特征在于，所述的转移载体包含TK基因的左臂(TKL)和右臂(TKR)，两个p7.5K启动子和一个p11K启动子，PPRV H基因和F基因，eGFP基因，两个Loxp序列。pLSEG-HF全长9 533bp，氨苄青霉素抗性。
2.如权利要求1所述的转移载体，其特征在于，所述的转移载体的核酸序列为SEQ IDNO:1。
3.如权利要求1所述的重组转移载体，在制备潜在共表达PPRV H和F蛋白的重组山羊痘病毒中的应用。
4.一种制备潜在共表达PPRV H和F蛋白的重组山羊痘病毒的方法，其特征在于，所述的方法是权利要求1所述的转移载体与山羊痘病毒在羊睾丸细胞内发生同源重组后，获得含有筛选marker的重组山羊痘病毒，在Cre酶作用下，重组山羊痘病毒的筛选marker特异性被敲除，获得不含筛选marker的潜在共表达PPRV H和F蛋白的重组山羊痘病毒。
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