CN113025631A - 一种表达双报告标签的重组副流感病毒5型 - Google Patents
一种表达双报告标签的重组副流感病毒5型 Download PDFInfo
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Abstract
本发明的目的是提供一种共表达增强型绿色荧光蛋白(eGFP)和NanoLuc荧光素酶(NLuc)的重组副流感病毒5型(PIV5)，其中用于拯救共表达eGFP和NLuc的重组PIV5核酸片段的结构为5′‑NP‑V/P‑eGFP‑T2A‑NLuc‑M‑F‑SH‑HN‑L‑3′；其中NP、V/P、M、F、SH、HN和L为PIV5的自身基因；eGFP‑T2A‑NLuc为融合序列；其中T2A为自剪切肽序列，负责“切断”eGFP和NLuc，将二者分离。本发明通过反向遗传技术拯救的重组PIV5，既不能导致MDBK细胞病变，也不能诱导空斑形成，但对于eGFP和NLuc的表达水平较高。重组PIV5的生长动力学与NLuc的表达动力学呈正相关。eGFP‑T2A‑NLuc融合序列在重组PIV5连续20次传代过程中基因较为稳定。
Description
技术领域
本发明属于重组病毒构建技术领域，具体涉及一种表达双报告标签的重组副流感病毒5型。
背景技术
副流感病毒5型(parainfluenza virus 5，PIV5)，旧称“猴病毒5型”，属于副粘病毒科(Paramyxoviridae)、正腮腺炎病毒属(Orthorubulavirus)成员。PIV5基因组为单股、负链、线性的RNA，全长15246nt，包含七个非重叠基因，顺序依次为3′-NP-V/P-M-F-SH-HN-L-5′，其中P基因包含V基因，对应编码V蛋白(非结构蛋白)。病毒经纯化后电镜下观察形状不定，但多呈球形或椭球形，外披脂质囊膜，囊膜外表面嵌有铆钉状纤突。囊膜内表面紧邻基质蛋白，其单体之间相互作用构成了整个病毒的外骨架，内部则含有鲱鱼骨状螺旋型核衣壳。
PIV5可感染多种哺乳动物，如牛、猪、犬等，也包括能感染人。该病毒除了引起狗的犬窝咳之外，通常不能导致其他易感动物表现出临床症状。另外，PIV5可感染多种细胞系，如Vero细胞、BHK细胞、MDBK细胞等，但通常不引起或只引起轻微的细胞病变，这便在多个方面限制了PIV5的应用，如病毒滴度测定。而表达荧光标签的重组PIV5，由于具有实时荧光指示作用，因此可以解决野生型病毒不诱导细胞病变所带来的限制。
发明内容
本发明目的是提供一种共表达增强型绿色荧光蛋白(enhancedgreenfluorescence protein，eGFP)和NanoLuc萤光素酶(NanoLuc luciferase,NLuc)的重组副流感病毒5型PIV5(rPIV5-GN)，从而弥补现有技术的不足。
本发明首先提供一种用于拯救共表达eGFP和NLuc的重组PIV5核酸片段(rPIV5-GNcDNA clone)，所述核酸片段的结构如下：5′-NP-V/P-eGFP-T2A-NLuc-M-F-SH-HN-L-3′；其中，NP、V/P、M、F、SH、HN、L为PIV5病毒的自身基因；eGFP-T2A-NLuc为插入的融合序列；eGFP-T2A-NLuc的上游包含M基因起始序列(M GS)和Pme I酶切位点，下游包含Not I酶切位点及V/P基因终止序列(V/P GE)；
作为实施例的一种具体记载，所述重组PIV5核酸片段的序列为SEQ IDNO:1；
本发明所提供的核酸片段用于在细胞内拯救能够共表达eGFP和NLuc的重组副流感病毒5型；
所述的细胞为BSR-T7/5细胞和Madin-Darby bovine kidney(MDBK)细胞，两者分别用于重组病毒的拯救和传代；
本发明再一个方面提供一种能够共表达eGFP和NLuc的重组副流感病毒5型，所述的病毒是使用共表达eGFP和NLuc的重组PIV5核酸片段的重组质粒在细胞内拯救获得的。
本发明还提供用于检测所述的重组副流感病毒5型的引物对，其上游引物的序列为5′-GCAATCATCCGCAGTGCAATTTGA-3′；
下游引物的序列为5′-TGCGGGAATGCTGATGGATGGCAT-3′。
本发明通过反向遗传技术拯救的rPIV5-GN在细胞内对于eGFP及NLuc的表达水平较高。eGFP的表达可直接通过荧光显微镜观察；Nluc的表达在发光底物作用下，黑暗环境中可发出肉眼可见的淡蓝色荧光。且本发明的eGFP-T2A-NLuc融合序列在病毒传代过程中稳定。
附图说明
图1：eGFP-T2A-NLuc融合蛋白序列模式图。
图2：rPIV5-GN cDNA clone模式图。
图3：三个辅助质粒模式图。
图4：四质粒共转染BSR-T7/5细胞42h图。
图5：共转染60h荧光素酶活性检测图。
图6：rPIV5-GN拯救后MDBK细胞连续传代图。
图7：第七代rPIV5-GN感染48h荧光素酶活性检测图。
图8：第七代rPIV5-GN的RT-PCR及PCR分析图。
图9：第八代rPIV5-GN蛋白质谱分析图。
图10：rPIV5-GN感染MDBK细胞噬斑分析图。
图11：第七代rPIV5-GN生长曲线图。
图12：第七代rPIV5-GN对NLuc表达的动力学曲线图。
图13：第16、18、20代rPIV5-GN对eGFP的表达图。
图14：第16、18、20代rPIV5-GN的荧光素酶活性检测图。
图15：第16、18、20代rPIV5-GN的RT-PCR检测图。
具体实施方式
本发明所涉及的eGFP是目前应用较为广泛的荧光蛋白标签，开放阅读框(openreading frame,ORF)长度为720bp。NLuc是一个经基因工程改造的新型小分子酶，分子量为19.1kD。NLuc可催化一种新型底物furimazine，从而产生高强度、辉光型发光现象。NLuc催化的生物发光反应不依赖ATP，自发光背景低，光信号较传统荧光素显著增强，同时抑制背景发光，可获得极高检测灵敏度。NLuc作为一种荧光示踪蛋白(Genbank登录号：MH037010)，因ORF较短(516bp)，因此极大方便了基因操作。
Thosea asigna virus 2A(T2A)为自剪切肽，完整序列为“EGRGSLLTCGDVEENPG↓P”，其通过核糖体跳跃机制进行自我“剪切”，“↓”所示为自剪切位点。因此T2A序列可作为Linker连接两个外源蛋白，当融合蛋白在宿主细胞内表达，由于核糖体跳跃机制，融合蛋白将被“剪切”，形成两个独立行使生物学功能的蛋白。
本发明构建了一种含eGFP-T2A-NLuc融合序列的rPIV5-GN cDNA clone，将其与分别表达PIV5 NP、P、L蛋白的真核表达质粒共转染BSR-T7/5细胞，成功拯救了rPIV5-GN，进而对重组病毒进行鉴定及特性分析。
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目的，本发明的保护范围并不仅限于说明书的具体实施例的记载。
实施例1、rPIV5-GN cDNA clone的构建
本实施例首先构建eGFP-T2A-NLuc融合蛋白序列，上游包含M基因起始序列(M GS)和Pme I酶切位点，下游包含Not I酶切位点及V/P基因终止序列(V/P GE)。eGFP-T2A-NLuc融合蛋白序列如图1所示，箭头所示为自剪切位点。在此基础上构建用于拯救共表达eGFP和NLuc的重组PIV5核酸片段rPIV5-GN cDNA clone。该片段呈“5′-NP-V/P-eGFP-T2A-NLuc-M-F-SH-HN-L-3′”分布(图2)。其中NP、V/P、M、F、SH、HN、L基因为PIV5毒株(GenBank:NC_006430.1)的相关基因；eGFP为增强型绿色荧光蛋白ORF；T2A为自剪切肽序列；NLuc为NanoLuc荧光素酶的ORF。
本实施例构建的rPIV5-GN cDNA clone的核苷酸序列为SEQ ID NO:1。
将核苷酸序列为SEQ ID NO:1的rPIV5-GN cDNA clone的全长序列进行核酸合成，亚克隆至pBR322质粒上。这样在pBR322质粒上，rPIV5-GN cDNA clone上游受T7启动子序列调控，下游受丁肝病毒核酶及T7终止子序列调控。
实施例2、rPIV5-GN的拯救
复苏BSR-T7/5细胞，在DMEM培养基中传代2次，DMEM培养基包含penicillin(100U/mL)、streptomycin(100μg/mL)、amphotericin B(0.25μg/mL)、G418(500μg/mL)及10％FBS(fetal bovine serum)。转染前一天，在12孔板中接种BSR-T7/5细胞，次日待细胞达到70％汇合度，将rPIV5-GN cDNA clone质粒及三个辅助质粒(图3)共转染BSR-T7/5细胞，四者每孔的转染量分别为2μg、1μg、0.5μg、0.5μg。按每孔加75μL Opti-MEM和8μLLipofectamine2000的比例配制溶液I。再按每孔加75μL Opti-MEM和4μg质粒配制溶液II。将溶液I和II轻柔混匀，室温放置15min以形成脂质体-DNA复合物。向细胞孔中逐滴加入脂质体-DNA混合液。将12孔板置于CO2培养箱转染8h，吸弃转染液，加入DMEM，置于培养箱中培养。转染后42h(42hpt)置于荧光显微镜下观察，可看到少量细胞发出绿色荧光(图4)。转染后60h(60hpt)收获一个转染孔的细胞液，与发光底物混合后，置于发光成像仪内观察，可看到明显的荧光(图5)。
实施例3、rPIV5-GN的鉴定
将拯救的rPIV5-GN在MDBK细胞中进行连续盲传，传至第七代。随着病毒盲传，eGFP达到了理想的表达水平，但整个传代过程中未观察到细胞病变(图6)。同时，第七代rPIV5-GN感染细胞48h后，也可高效表达NLuc(图7)。第7代病毒液冻融一次，收集上清提取总RNA，用于病毒的RT-PCR检测。上游引物序列：5′-GCAATCATCCGCAGTGCAATTTGA-3′，下游引物序列：5′-TGCGGGAATGCTGATGGATGGCAT-3′，预扩增1578bp片段。RT-PCR反应采用HighFidelity One Step RT-PCR Kit(Takara)，反应条件设定如下，45℃10min,94℃2min,经历30个循环：98℃(10s),55℃(15s),68℃(16s)。为消除残留质粒污染，另设PCR对照。PCR反应采用2×PrimeSTAR Max Premix(Takara)，反应条件设定为30个循环：98℃(10s),55℃(10s),72℃(16s)。RT-PCR和PCR反应结束后，同时进行琼脂糖凝胶电泳，结果如图8所示，仅RT-PCR扩增出了特异性条带(1578bp)。第8代rPIV5-GN病毒液进行蛋白质谱分析，结果显示NP、P、M、F、HN、L蛋白皆可匹配特异性肽段图谱，其中L蛋白对应图谱如图9。
实施例4、rPIV5-GN空斑分析
将rPIV5-GN进行10倍连续稀释，并将其分别接种6孔板中的MDBK细胞，将培养板置于培养箱中孵育2h，吸弃病毒液，D-PBS润洗一遍，补加1％低熔点琼脂糖凝胶的DMEM(含10％FBS)，置于培养箱中培养72h，荧光显微镜下观察细胞。然后采用4％多聚甲醛固定细胞，0.2％结晶紫染色细胞，自来水轻柔冲洗后晾干6孔板，观察是否有空斑形成。结果表明，虽然eGFP可以高效表达，但病毒感染的细胞并未有空斑形成(图10)。
实施例5、rPIV5-GN生长动力学及NLuc表达动力学
将MDBK细胞接种五个12孔板(1.5×106细胞/孔，3孔/板)，置于培养箱静置2h后，将rPIV5-GN接种所有细胞孔(MOI＝0.001)，置于培养箱培养2h后，吸弃病毒液，补加DMEM，将所有12孔板置于培养箱培养。第0、24、48、72及96h，分别随机取出一个12孔板，冻融一次后，离心收集上清采用TCID50分析法进行病毒滴度测定，由于rPIV5-GN感染不引起MDBK细胞病变，因此TCID50测定过程中，可通过eGFP指示病毒感染的细胞孔。同时，收获的上清进行荧光素酶催化底物发光分析。对于每个样品，取100μL上清与0.5μL底物在白色不透明96孔板中彻底混合后，置于Tecan微型板分析仪中进行定量分析。采用GraphPad Prism软件，将病毒滴度及荧光分析的数据进行处理，每个时间节点包括三个重复，绘制病毒生长曲线(图11)及Nluc表达动力学曲线(图12)。结果表明，rPIV5-GN感染MDBK细胞72h内，病毒的生长和NLuc的表达呈正相关；72h后，病毒滴度仍然缓慢上升，但NLuc表达量开始下降。
实施例6、rPIV5-GN的传代稳定性
将rPIV5-GN在MDBK细胞中连续传代(72h/代)，共计20代，每一代都可观察到eGFP的高效表达。第16、18及20代eGFP的表达情况如图13所示。收获第16、18及20代的病毒液，冻融一次后，进行荧光素酶活性分析，如图14所示，三个病毒子代皆可有效表达Nluc。同时采用上游(5′-GCAATCATCCGCAGTGCAATTTGA-3′)及下游(5′-TGCGGGAATGCTGATGGATGGCAT-3′)引物，对第16、18及20代的病毒液进行RT-PCR分析，结果如图15所示，三个病毒子代都扩增出了特异性目的片段(1578bp)。
上述结果表明，本发明方法制备的rPIV5-GN，其外源eGFP-T2A-NLuc融合序列在重组病毒传代过程中稳定存在。
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，在不背离本发明说明书具体技术思路的情况下，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。
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<110> 青岛农业大学
<120> 一种表达双报告标签的重组副流感病毒5型
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 16662
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
accaagggga aaatgaagtg gtgactcaaa tcatcgaaga ccctcgagat tacataggtc 60
cggaacctat ggccttcgtg accgacctcg agtcagagta gttcaataag gacctatcaa 120
gtttgggcaa tttttcgtcc ccgacacaaa aatgtcatcc gtgcttaaag catatgagcg 180
attcacgctc actcaagaac tgcaagatca gagtgaggaa ggtacaatcc cacctacaac 240
actaaaaccg gtaatcaggg tatttatact aacctctaat aacccagagc taagatcccg 300
gcttcttcta ttctgcctac ggattgttct cagtaatggt gcaagggatt cccatcgctt 360
tggagcatta ctcacaatgt tttcgctacc atcagccaca atgctcaatc atgtcaaatt 420
agctgaccag tcaccagaag ctgatatcga aagggtagag atcgatggct ttgaggaggg 480
atcattccgc ttaatcccca atgcacgttc aggtatgagc cgtggagaga tcaatgccta 540
tgctgcactt gcagaagatc tacctgacac actaaaccat gcaacacctt tcgttgattc 600
cgaagtcgag ggaactgcat gggatgagat tgagactttc ttagatatgt gttacagtgt 660
cctaatgcag gcatggatag tgacttgcaa gtgcatgact gcgccagacc aacctgctgc 720
ttctattgag aaacgcctgc aaaaatatcg tcagcaaggc aggatcaacc cgagatatct 780
cctgcaaccg gaggctcgac gaataatcca gaatgtaatc cggaagggaa tggtggtcag 840
acatttcctc acctttgaac tgcagcttgc ccgagcacaa agccttgtat caaataggta 900
ttatgctatg gtaggggatg ttggaaagta tatagagaat tgtggaatgg gaggcttctt 960
tttgacacta aaatatgcat taggaactag atggcccaca cttgctttag ctgcattttc 1020
aggagagcta acaaagctaa agtccctcat ggcattatac cagacccttg gtgagcaggc 1080
ccgatatttg gccctattgg agtcaccaca tttgatggat tttgctgcag caaactaccc 1140
actgctatat agctatgcta tgggaatagg ctatgtgtta gatgtcaaca tgaggaacta 1200
cgctttctcc agatcataca tgaacaagac atatttccaa ttgggaatgg aaactgcaag 1260
aaaacaacag ggtgcagttg acatgaggat ggcagaagat ctcggtctaa ctcaagccga 1320
acgcaccgag atggcaaata cacttgccaa attgaccaca gcaaatcgag gggcagacac 1380
caggggagga gtcaacccgt tctcatctgt cactgggaca actcaggtgc ccgctgcagc 1440
aacaggtgac acactcgaga gttacatggc agcggatcga ctgaggcaga gatatgctga 1500
tgcaggcacc catgatgatg agatgccacc attggaagag gaggaagagg acgacacatc 1560
tgcaggtcca cgcactggac caactcttga acaagtggcc ttggacatcc agaacgcagc 1620
agttggagct cccatccata cagatgacct gaatgccgca ctgggtgatc ttgacatcta 1680
gacaattcag atcccaatct aaaattgaca tacctaattg attagttaga tggaactaca 1740
gtggattcca taaggttcct gcctaccatc ggctttaaag aaaaaaatag gcccggacgg 1800
gttagcaaca agcgactgcc ggtgccaaca gcgcaatcca caatctacaa tggatcccac 1860
tgatctgagc ttctccccag atgagatcaa taagctcata gagacaggcc tgaatactgt 1920
agagtatttt acttcccaac aagtcacagg aacatcctct cttggaaaga atacaatacc 1980
accaggggtc acaggactac taaccaatgc tgcagaggca aagatccaag agtcaactaa 2040
ccatcagaag ggctcagttg gtgggggtgc aaaaccaaag aaaccgcgac caaaaattgc 2100
cattgtgcca gcagatgaca aaacagtgcc cggaaagccg atcccaaacc ctctattagg 2160
tctggactcc accccgagca cccaaactgt gcttgatcta agtgggaaaa cattaccatc 2220
aggatcctat aagggggtta agcttgcgaa atttggaaaa gaaaatctga tgacacggtt 2280
catcgaggaa cccagagaga atcctatcgc aaccagttcc cccatcgatt ttaagagggg 2340
cagggatacc ggcgggttcc atagaaggga gtactcaatc ggatgggtgg gagatgaagt 2400
caaggtcact gagtggtgca atccatcctg ttctccaatc accgctgcag caaggcgatt 2460
tgaatgcact tgtcaccagt gtccagtcac ttgctctgaa tgtgaacgag atacttaata 2520
cagtgagaaa tttggactct cggatgaatc aactggagac aaaagtagat cgcattctct 2580
catctcagtc tctaatccag accatcaaga atgacatagt tggacttaaa gcagggatgg 2640
ctactttaga aggaatgatt acaactgtga aaatcatgga cccgggagtt cccagtaatg 2700
ttactgtgga agatgtacgc aagacactaa gtaaccatgc tgttgttgtg ccagaatcat 2760
tcaatgatag tttcttgact caatctgaag atgtaatttc acttgatgag ttggctcgac 2820
caactgcaac aagtgttaag aagattgtca ggaaggttcc tcctcagaag gatctgactg 2880
gattgaagat tacactagag caattggcaa aggattgcat cagcaaaccg aagatgaggg 2940
aagagtatct cctcaaaatc aaccaggctt ccagtgaggc tcagctaatt gacctcaaga 3000
aagcaatcat ccgcagtgca atttgatcaa gaaacaccca attacactac actggtatga 3060
cactgtacta accctgaggg ttttagaaaa aacgattaac gataaataag cccgaacact 3120
acacactacc tgaggcagcc gtttaaacat ggtgagcaag ggcgaggagc tgttcaccgg 3180
ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 3240
cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 3300
cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg 3360
cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 3420
aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 3480
cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 3540
caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 3600
ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 3660
catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 3720
cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga 3780
ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 3840
tctcggcatg gacgagctgt acaaggaggg cagaggaagt ctgctaacat gcggtgacgt 3900
cgaggagaat cctggcccaa tggtcttcac actcgaagat ttcgttgggg actggcgaca 3960
gacagccggc tacaacctgg accaagtcct tgaacaggga ggtgtgtcca gtttgtttca 4020
gaatctcggg gtgtccgtaa ctccgatcca aaggattgtc ctgagcggtg aaaatgggct 4080
gaagatcgac atccatgtca tcatcccgta tgaaggtctg agcggcgacc aaatgggcca 4140
gatcgaaaaa atttttaagg tggtgtaccc tgtggatgat catcacttta aggtgatcct 4200
gcactatggc acactggtaa tcgacggggt tacgccgaac atgatcgact atttcggacg 4260
gccgtatgaa ggcatcgccg tgttcgacgg caaaaagatc actgtaacag ggaccctgtg 4320
gaacggcaac aaaattatcg acgagcgcct gatcaacccc gacggctccc tgctgttccg 4380
agtaaccatc aacggagtga ccggctggcg gctgtgcgaa cgcattctgg cgtagcggcc 4440
gctcaagaaa cacccaatta cactacactg gtatgacact gtactaaccc tgagggtttt 4500
agaaaaaacg attaacgata aataagcccg aacactacac actacctgag gcagccatgc 4560
catccatcag cattcccgca gaccccacca atccacgtca atcaataaaa gcgttcccaa 4620
ttgtgatcaa cagtgatggg ggtgagaaag gccgcttggt taaacaacta cgcacaacct 4680
acttgaatga cctagatact catgagccac tggtgacatt cataaatacc tatggattca 4740
tctacgaaca ggatcggggg aataccattg tcggagagga tcaacttggg aagaaaagag 4800
aggctgtgac cgctgcaatg gttacccttg gatgtgggcc taatctacca tcattaggga 4860
atgtcctggg acaactgagg gaattccagg tcactgttag gaagacatcc agcaaagcgg 4920
aagagatggt ctttgaaatt gttaagtatc cgagaatatt tcggggtcat acattaatcc 4980
agaaaggact agtctgtgtc tccgcagaaa aatttgttaa gtcaccaggg aaaatacaat 5040
ctggaatgga ctatctcttc attccgacat ttctgtcagt gacttactgt ccagctgcaa 5100
tcaaatttca ggtacctggc cccatgttga aaatgagatc aagatacact cagagcttac 5160
aacttgaact aatgataaga atcctgtgta agcccgattc gccacttatg aaggtccata 5220
cccctgacaa ggagggaaga ggatgtcttg tatcagtatg gctgcatgta tgcaacatct 5280
tcaaatcagg aaacaagaat ggcagtgagt ggcaggaata ctggatgaga aagtgtgcta 5340
acatgcaact tgaagtgtcg attgcagata tgtggggacc aactatcata attcatgcca 5400
gaggtcacat tcccaaaagt gctaagttgt tttttggaaa gggtggatgg agctgccatc 5460
cacttcacga agttgttcca agtgtcacta aaacactatg gtccgtgggc tgtgagatta 5520
caaaggcgaa ggcaataata caagagagta gcatctctct tctcgtggag actactgaca 5580
tcataagtcc aaaagtcaaa atttcatcta agcatcgccg ctttgtgaaa tcaaattggg 5640
gtctgttcaa gaaaactaaa tcactgccta acctgacgga gctggaatga ctgacctcta 5700
atcgagacta caccgccgca aactataggt gggtggtacc tcagtgatta atcttgtaag 5760
cactgatcgt aggctacaac acactaatat tatccagatt agagagctta attagctctg 5820
tattaataat aacactacta ttccaataac tggaatcacc agcttgattt atctccaaaa 5880
tgattcaaag aaaacaaatc atattaagac tatcctaagc acgaacccat atcgtccttc 5940
aaatcatggg tactataatt caatttctgg tggtctcctg tctattggca ggagcaggca 6000
gccttgatcc agcagccctc atgcaaatcg gtgtcattcc aacaaatgtc cggcaactta 6060
tgtattatac tgaggcctca tcagcattca ttgttgtgaa gttaatgcct acaattgact 6120
cgccgattag tggatgtaat ataacatcaa tttcaagcta taatgcaaca gtgacaaaac 6180
tcctacagcc gatcggtgag aatttggaga cgattaggaa ccagttgatt ccaactcgga 6240
ggagacgccg gtttgcaggg gtggtgattg gattagctgc attaggagta gctactgccg 6300
cacaggtcac tgccgcagtg gcactagtaa aggcaaatga aaatgctgcg gctatactca 6360
atctcaaaaa tgcaatccaa aaaacaaatg cagcagttgc agatgtggtc caggccacac 6420
aatcactagg aacggcagtt caagcagttc aagatcacat aaacagtgtg gtaagtccag 6480
caattacagc agccaattgt aaggcccaag atgctatcat tggctcaatc ctcaatctct 6540
atttgaccga gttgacaacc atcttccaca atcaaattac aaaccctgca ttgagtccca 6600
ttacaattca agctttaagg atcctactgg ggagtacctt gccgactgtg gtcgaaaaat 6660
ctttcaatac ccagataagt gcagctgagc ttctctcatc agggttattg acaggccaga 6720
ttgtgggatt agatttgacc tatatgcaga tggtcataaa aattgagctg ccaactttaa 6780
ctgtacaacc tgcaacccag atcatagatc tggccaccat ttctgcattc attaacaatc 6840
aagaagtcat ggcccaatta ccaacacgtg ttatggtgac tggcagcttg atccaagcct 6900
atcccgcatc gcaatgcacc attacaccca acactgtgta ctgtaggtat aatgatgccc 6960
aagtactctc agatgatact atggcttgcc tccaaggtaa cttgacaaga tgcaccttct 7020
ctccagtggt tgggagcttt ctcactcgat tcgtgctgtt cgatggaata gtttatgcaa 7080
attgcaggtc gatgttgtgc aagtgcatgc aacctgctgc tgtgatccta cagccgagtt 7140
catcccctgt aactgtcatt gacatgtaca aatgtgtgag tctgcagctt gacaatctca 7200
gattcaccat cactcaattg gccaatgtaa cctacaatag caccatcaag cttgaatcat 7260
cccagatctt gtctattgat ccgttggata tatcccaaaa tctagctgcg gtgaataaga 7320
gtctaagtga tgcactacaa cacttagcac aaagtgacac atatctttct gcaatcacat 7380
cagctacgac tacaagtgta ttatccataa tagcaatctg tcttggatcg ttaggtttaa 7440
tattaataat cttgctcagt gtagttgtgt ggaagttatt gaccattgtc gttgctaatc 7500
gaaatagaat ggagaatttt gtttatcata aataagcatt ccaccactca cgatctgatc 7560
tcagtgagaa aaatcaacct gcaactcttg gaacaagata agacagtcat ccattagtaa 7620
tttttaagaa aaaaacgata ggaccgaacc tagtattgaa agaaccgtct cggtcaatct 7680
aggtaatcga gctgataccg tctcggaaag ctcaaatcat gctgcctgat ccggaagatc 7740
cggaaagcaa gaaagctaca aggagagcag gaaacctaat tatctgcttc ctattcatct 7800
tctttctgtt tgtaaccttc attgttccaa ctctaagaca cttgctgtcc taacacctgc 7860
tataggctat ccactgcatc atctctcctg ccatacttcc tactcacatc atatctattt 7920
taaagaaaaa ataggcccga acactaatcg tgccggcagt gccactgcac acacaacact 7980
acacatacaa tacactacaa tggttgcaga agatgcccct gttagggcca cttgccgagt 8040
attatttcga acaacaactt taatctttct atgcacacta ctagcattaa gcatctctat 8100
cctttatgag agtttaataa cccaaaagca aatcatgagc caagcaggct caactggatc 8160
taattctgga ttaggaagta tcactgatct tcttaataat attctctctg tcgcaaatca 8220
gattatatat aactctgcag tcgctctacc tctacaattg gacactcttg aatcaacact 8280
ccttacagcc attaagtctc ttcaaaccag tgacaagcta gaacagaact gctcgtggag 8340
tgctgcactg attaatgata atagatacat taatggcatc aatcagttct atttttcaat 8400
tgctgagggt cgcaatctga cacttggccc acttcttaat atgcctagtt tcattccaac 8460
tgccacgaca ccagagggct gcaccaggat cccatcattc tcgctcacta agacacactg 8520
gtgttataca cacaatgtta tcctgaatgg atgccaggat catgtatcct caaatcaatt 8580
tgtttccatg ggaatcattg aacccacttc tgccgggttt ccattctttc gaaccctaaa 8640
gactctatat ctcagcgatg gggtcaatcg taagagctgc tctatcagta cagttccggg 8700
gggttgtatg atgtactgtt ttgtttctac tcaaccagag agggatgact acttttctgc 8760
cgctcctcca gaacaacgaa ttattataat gtactataat gatacaatcg tggagcgcat 8820
aattaatcca cccggggtac tagatgtatg ggcaacattg aacccaggaa caggaagcgg 8880
ggtatattat ttaggttggg tgctctttcc aatatatggc ggcgtgatta aaggtacgag 8940
tttatggaat aatcaagcaa ataaatactt tatcccccag atggttgctg ctctctgctc 9000
acaaaaccag gcaactcaag tccaaaatgc taagtcatca tactatagca gctggtttgg 9060
caatcgaatg attcagtctg ggatcctggc atgtcctctt cgacaggatc taaccaatga 9120
gtgtttagtt ctgccctttt ctaatgatca ggtgcttatg ggtgctgaag ggagattata 9180
catgtatggt gactcggtgt attactatca aagaagcaat agttggtggc ctatgaccat 9240
gctgtataag gtaaccataa cattcactaa tggtcagcca tctgctatat cagctcagaa 9300
tgtgcccaca cagcaggtcc ctagacctgg gacaggagac tgctctgcaa ccaatagatg 9360
tcccggtttt tgcttgacag gagtgtatgc cgatgcctgg ttactgacca acccttcgtc 9420
taccagtaca tttggatcag aagcaacctt cactggttct tatctcaaca cagcaactca 9480
gcgtatcaat ccgacgatgt atatcgcgaa caacacacag atcataagct cacagcaatt 9540
tggatcaagc ggtcaagaag cagcatatgg ccacacaacc tgttttaggg acacaggctc 9600
tgttatggta tactgtatct atattattga attgtcctca tctctcttag gacaatttca 9660
gattgtccca tttatccgtc aggtgacact atcctaaagg cagaagcctt caggtctgac 9720
ccagccaatc aaagcattat accagaccat ggaatgcata ccaaacatta ttgacactaa 9780
tgacacacaa aattggtttt aagaaaaacc aagagaacaa taggccagaa tggctgggtc 9840
tcgggagata ttactccctg aagtccatct caattcacca attgtaaagc ataagctata 9900
ctattacatt ctacttggaa acctcccaaa tgagatcgac cttgacgatt taggtccatt 9960
acataatcaa aattggaatc agatagcaca tgaagagtct aacttagctc aacgcttggt 10020
aaatgtaaga aattttctaa ttacccacat ccctgatctt agaaagggcc attggcaaga 10080
gtatgtcaat gtaatactgt ggccgcgaat tcttcccttg atcccggatt ttaaaatcaa 10140
tgaccaattg cctctgctca aaaattggga caagttagtt aaagaatcat gttcagtaat 10200
caatgcaggt acttcccagt gcattcagaa tctcagctat ggactgacag gtcgtgggaa 10260
cctctttaca cgatcacgtg aactctctgg tgaccgcagg gatattgatc ttaagacagt 10320
tgtggcagca tggcatgact cagactggaa aagaataagt gatttttgga ttatgatcaa 10380
attccagatg agacaattaa ttgttaggca aacagatcat aatgattctg atttaatcac 10440
gtatatcgaa aatagagaag gcataatcat cataacccct gaactggtag cattatttaa 10500
cactgagaat catacactaa catacatgac ctttgaaatt gtactgatgg tttcagatat 10560
gtacgaaggt cgtcacaaca ttttatcact atgcacagtt agcacttacc tgaatcctct 10620
gaagaaaaga ataacatatt tattgagcct tgtagataac ttagcttttc agataggtga 10680
tgctgtatat aacataattg ctttgctaga atcctttgta tatgcacagt tgcaaatgtc 10740
agatcccatc ccagaactca gaggacaatt ccatgcattc gtatgttctg agattcttga 10800
tgcactaaga ggaactaata gtttcaccca ggatgaatta agaactgtga caactaattt 10860
gatatcccca ttccaagatc tgaccccaga tcttacggct gaattgctct gtataatgag 10920
gctttgggga caccccatgc tcactgccag tcaagctgca ggaaaggtac gcgagtctat 10980
gtgtgctgga aaagtattag actttcccac cattatgaaa acactagcct ttttccatac 11040
tattctgatc aatggataca ggaggaagca tcatggagta tggccaccct taaacttacc 11100
gggtaatgct tcaaagggtc tcacggaact tatgaatgac aatactgaaa taagctatga 11160
attcacactt aagcattgga aggaagtctc tcttataaaa ttcaagaaat gttttgatgc 11220
agacgcaggt gaggaactca gtatatttat gaaagataag gcaattagtg ccccaaaaca 11280
agactggatg agtgtgttta gaagaagcct aatcaaacag cgccatcagc atcatcaggt 11340
ccccctacca aatccattca atcgacggct gttgctaaac tttctcggag atgacaaatt 11400
cgacccgaat gtggagctac agtatgtaac atcaggtgag tatctacatg atgacacgtt 11460
ttgtgcatca tattcactaa aagagaagga aattaaacct gatggtcgaa tttttgcaaa 11520
gttgactaag agaatgagat catgtcaagt tatagcagaa tctcttttag cgaaccatgc 11580
tgggaagtta atgaaagaga atggtgttgt gatgaatcag ctatcattaa caaaatcact 11640
attaacaatg agtcagattg gaataatatc cgagaaagct agaaagtcaa ctcgagataa 11700
cataaatcaa cctggtttcc agaatatcca gagaaataaa tcacatcact ccaagcaagt 11760
caatcagcga gatccaagtg atgactttga attggcagca tcttttttaa ctactgatct 11820
caaaaaatat tgtttacaat ggaggtacca gacaattatc ccatttgctc aatcattaaa 11880
cagaatgtat ggttatcctc atctctttga gtggattcac ttacggctaa tgcgtagtac 11940
actttacgtg ggggatccct tcaacccacc agcagatacc agtcaatttg atctagataa 12000
agtaattaat ggagatatct tcattgtatc acccagaggt ggaattgaag ggctgtgtca 12060
aaaggcttgg acaatgatat ctatcgctgt gataattcta tctgccacag agtctggcac 12120
acgagtaatg agtatggtgc agggagataa tcaagcaatt gctgtcacca cacgagtacc 12180
aaggagcctg ccgactcttg agaaaaagac tattgctttt agatcttgta atctattctt 12240
tgagaggtta aaatgtaata attttggatt aggtcaccat ttgaaagaac aagagactat 12300
cattagttct cacttctttg tttatagcaa gagaatattc tatcagggga ggattctaac 12360
gcaagcctta aaaaatgcta gtaagctctg cttgacagct gatgtcctag gagaatgcac 12420
ccaatcatca tgttctaatc ttgcaactac tgtcatgagg ttaactgaga atggtgttga 12480
aaaagatatc tgtttctact tgaatatcta tatgaccatc aaacagctct cctatgatat 12540
catcttccct caagtgtcaa ttcctggaga tcagatcaca ttagaataca taaataatcc 12600
acacctggta tcacgattgg ctcttttgcc atcccagtta ggaggtctaa actacctgtc 12660
atgcagtagg ctgttcaatc gaaacatagg cgacccggtg gtttccgcag ttgcagatct 12720
taagagatta attaaatcag gatgtatgga ttactggatc ctttataact tattagggag 12780
aaaaccggga aacggctcat gggctacttt agcagctgac ccgtactcaa tcaatataga 12840
gtatcaatac cctccaacta cagctcttaa gaggcacacc caacaagctc tgatggaact 12900
cagtacgaat ccaatgttac gtggcatatt ctctgacaat gcacaggcag aagaaaataa 12960
ccttgctagg tttctcctgg atagggaggt gatctttccg cgtgtagctc acatcatcat 13020
tgagcaaacc agtgtcggga ggagaaaaca gattcaagga tatttggatt caactagatc 13080
gataatgagg aaatcactag aaattaagcc cttatccaat aggaagctta atgaaatact 13140
ggattacaac atcaattacc tagcttacaa tttggcatta ctcaagaatg ctattgaacc 13200
tccgacttat ttgaaggcaa tgacacttga aacatgtagc atcgacattg caaggaacct 13260
ccggaagctc tcctgggccc cactcttggg tgggagaaat cttgaaggat tagagacgcc 13320
agatcccatt gaaattactg caggagcatt aattgttgga tcgggctact gtgaacagtg 13380
tgctgcagga gacaatcgat tcacatggtt tttcttgcca tctggtatcg agataggagg 13440
ggatccccgt gataatcctc ctatccgtgt accgtacatt ggctccagga ctgatgagag 13500
gagggtagcc tcaatggcat acatcagggg tgcctcgagt agcttaaaag cagttcttag 13560
actggcggga gtgtacatct gggcattcgg agatactctg gagaattgga tagatgcact 13620
ggatttgtct cacactagag ttaacatcac acttgaacag ctgcaatccc tcaccccact 13680
tccaacctct gccaatctaa cccatcggtt ggatgatggc acaactaccc taaagtttac 13740
tcctgcgagc tcttacacct tttcaagttt cactcatata tcaaatgatg agcaatacct 13800
gacaattaat gacaaaactg cagattcaaa tataatctac caacagttaa tgatcactgg 13860
actcggaatc ttagaaacat ggaataatcc cccaatcaat agaacattcg aagaatctac 13920
cctacatttg cacactggtg catcatgttg tgtccgacct gtggactcct gcattctctc 13980
agaagcatta acagtcaagc cacatattac agtaccgtac agcaataaat ttgtatttga 14040
tgaggacccg ctatctgaat atgaaactgc aaaactggaa tcgttatcat tccaagccca 14100
attaggcaac attgatgctg tagatatgac aggtaaatta acattattgt cccaattcac 14160
tgcaaggcag attatcaatg caatcactgg actcgatgag tctgtctctc ttactaatga 14220
tgccattgtt gcatcagact atgtctccaa ttggattagt gaatgcatgt ataccaaatt 14280
agatgaatta tttatgtatt gtgggtggga actactattg gaactatcct atcaaatgta 14340
ttatctgagg gtagttgggt ggagtaatat agtggattat tcttacatga tcttgagaag 14400
aatcccgggt gcagcattaa acaatctggc atctacatta agtcatccaa aacttttccg 14460
acgagctatc aacctagata tagttgcccc cttaaatgct cctcattttg catctctgga 14520
ctacatcaag atgagtgtgg atgcaatact ctggggctgt aaaagagtca tcaatgtgct 14580
ctccaatgga ggggacttag aattagttgt gacatctgaa gatagcctta ttctcagtga 14640
ccgatccatg aatctcattg caaggaaatt aactttatta tcactgattc accataatgg 14700
tttggaacta ccaaagatta aggggttctc tcctgatgag aagtgtttcg ctttgacaga 14760
atttttgagg aaagtggtga actcagggtt gagttcaata gagaacctat caaattttat 14820
gtacaatgtg gagaacccac ggcttgcagc attcgccagc aacaattact acctgaccag 14880
aaaattattg aattcaatac gagatactga gtcgggtcaa gtagcagtca cctcatatta 14940
tgaatcatta gaatatattg atagtcttaa gctaacccca catgtgcctg gcacctcatg 15000
cattgaggat gatagtctat gtacaaatga ttacataatc tggatcatag agtctaatgc 15060
aaacttggag aagtatccaa ttccaaatag ccctgaggat gattccaatt tccataactt 15120
taagttgaat gctccatcgc accatacctt acgcccatta gggttgtcat caactgcttg 15180
gtataagggt ataagctgct gcaggtacct tgagcgatta aagctaccac aaggtgatca 15240
tttatatatt gcagaaggta gtggtgccag tatgacaatc atagaatacc tattcccagg 15300
aagaaagata tattacaatt ctttatttag tagtggtgac aatcccccac aaagaaatta 15360
tgcaccaatg cctactcagt tcattgagag tgtcccatac aagctctggc aagcacacac 15420
agatcaatat cccgagattt ttgaggactt catccctcta tggaacggaa acgccgccat 15480
gactgacata ggaatgacag cttgtgtaga attcatcatc aatcgagtcg gcccaaggac 15540
ttgcagttta gtacatgtag atttggaatc aagtgcaagc ttaaatcaac aatgcctgtc 15600
aaagccgata attaatgcta tcatcactgc tacaactgtt ttgtgccctc atggggtgct 15660
tattctgaaa tatagttggt tgccatttac tagatttagt actttgatca ctttcttatg 15720
gtgctacttt gagagaatca ctgttcttag gagcacatat tctgatccag ctaatcatga 15780
ggtttattta atttgtatcc ttgccaacaa ctttgcattc cagactgtct cgcaggcaac 15840
aggaatggcg atgactttaa ctgatcaagg gtttactttg atatcacctg aaagaataaa 15900
tcagtattgg gatggtcact tgaagcaaga acgtatcgta gcagaagcaa ttgataaggt 15960
ggttctagga gaaaatgctc tatttaattc gagtgataat gaattaattc tcaaatgtgg 16020
agggacacca aatgcacgga atctcatcga tatcgagcca gtcgcaactt tcatagaatt 16080
tgaacaattg atctgcacaa tgttgacaac ccacttgaag gaaataattg atataacaag 16140
gtctggaacc caggattatg aaagtttatt actcactcct tacaatttag gtcttcttgg 16200
taaaatcagt acgatagtga gattattaac agaaaggatt ctaaatcata ctatcaggaa 16260
ttggttgatc ctcccacctt cgctccggat gatcgtgaag caggacttgg aattcggcat 16320
attcaggatt acttccatcc tcaattctga tcggttcctg aagctttctc caaataggaa 16380
atacttgatt gcacaattaa ctgcaggcta cattaggaaa ttgattgagg gggattgcaa 16440
tatcgatcta accagaccta tccaaaagca aatctggaaa gcattaggtt gtgtagtcta 16500
ttgtcacgat ccaatggatc aaagggagtc aacagagttt attgatataa atattaatga 16560
agaaatagac cgcgggatcg atggcgagga aatctaaaca tatcaagaat cagaattagt 16620
ttaagaaaaa agaagaggat taatcttggt tttccccttg gt 16662
Claims (6)
1.一种用于拯救表达eGFP-T2A-NLuc融合序列的重组副流感病毒5型的核酸片段，其特征在于，所述核酸片段的结构为3′-NP-V/P-M-F-SH-HN-L-5′，其中NP、V/P、M、F、SH、HN和L为副流感病毒5型的自身基因；eGFP-T2A-NLuc为融合序列，其上游包含M基因起始序列和PmeI酶切位点，下游包含Not I酶切位点及V/P基因终止序列；eGFP为增强型绿色荧光蛋白序列；T2A为Thosea asigna virus 2A自剪切肽序列；Nluc为NanoLuc荧光素酶序列。
2.如权利要求1所述的核酸片段，其特征在于，所述核酸片段的序列为SEQ ID NO:1。
3.权利要求1或2所述的核酸片段在细胞内拯救重组副流感病毒5型的应用。
4.如权利要求3所述的应用，其特征在于，所述的细胞为BSR-T7/5细胞和MDBK细胞。
5.一种能共表达eGFP和NLuc的重组副流感病毒5型，其特征在于，所述的重组副流感病毒5型是使用权利要求1或2所述的核酸片段在细胞内拯救获得的。
6.一种用于检测权利要求5所述的重组副流感病毒5型的引物对，其特征在于，所述的引物对中，其上游引物序列为5′-GCAATCATCCGCAGTGCAATTTGA-3′，下游引物序列为5′-TGCGGGAATGCTGATGGATGGCAT-3′。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040208895A1 (en) * | 2000-03-21 | 2004-10-21 | Medimmune Vaccines, Inc. | Recombinant parainfluenza virus expression systems and vaccines |
| CN104911152A (zh) * | 2015-06-08 | 2015-09-16 | 东北农业大学 | 一株重组传染性造血器官坏死病毒rIHNV HLJ-09株及其构建方法和应用 |
| CN111733170A (zh) * | 2020-07-01 | 2020-10-02 | 青岛农业大学 | 一种表达荧光素酶的重组犬麻疹病毒 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040208895A1 (en) * | 2000-03-21 | 2004-10-21 | Medimmune Vaccines, Inc. | Recombinant parainfluenza virus expression systems and vaccines |
| CN104911152A (zh) * | 2015-06-08 | 2015-09-16 | 东北农业大学 | 一株重组传染性造血器官坏死病毒rIHNV HLJ-09株及其构建方法和应用 |
| CN111733170A (zh) * | 2020-07-01 | 2020-10-02 | 青岛农业大学 | 一种表达荧光素酶的重组犬麻疹病毒 |
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