CN108676814A - 一种天坛株痘苗病毒的荧光标记穿梭载体及其制备方法 - Google Patents
一种天坛株痘苗病毒的荧光标记穿梭载体及其制备方法 Download PDFInfo
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Abstract
本发明公开的一种天坛株痘苗病毒的荧光标记穿梭载体，穿梭载体含有mCherry红色荧光蛋白报告基因，编码mCherry的DNA序列位于天坛株痘苗病毒胸苷激酶基因TK的同源重组臂TKL和TKR之间，由P7.5启动子启动mCherry红色荧光报告基因表达，穿梭载体含有氨苄青霉素筛选标记和大肠杆菌复制起始位点pUC ori。本发明以mCherry红色荧光蛋白基因为报告基因可用于筛选重组痘苗病毒，重组病毒感染肿瘤动物模型后可作为特异性分子探针成为追踪靶目标并实现小动物活体成像；同时可提高痘苗病毒的肿瘤细胞选择性。
Description
技术领域
本发明属于生物基因工程技术领域，具体涉及一种天坛株痘苗病毒的荧光标记穿梭载体，本发明还涉及一种天坛株痘苗病毒的荧光标记穿梭载体的制备方法。
背景技术
肿瘤生物治疗技术是近年来兴起的一种全新的肿瘤治疗手段，具有副作用小、抗肿瘤活性高、适应性广等特点。将生物技术用于肿瘤治疗，既可靶向性地杀伤肿瘤细胞同时降低对正常细胞和组织的损害，是改变和提高患者自身机体免疫功能的治本方法，为最终攻克肿瘤带来希望，其应用前景广阔。
痘苗病毒作为第一个成功应用于人类的疫苗，引起了长时间普遍的关注和研究。痘苗病毒具有许多理想的生物治疗载体属性，使其有可能成为疫苗和溶瘤病毒的优良载体。痘苗病毒能够选择性地感染肿瘤细胞，并在其中复制，最终使肿瘤细胞裂解。胸苷激酶TK为痘苗病毒DNA复制所必需，正常细胞中TK浓度较低，不足以供TK去除的痘苗病毒复制；而肿瘤细胞DNA代谢旺盛，有较高浓度的TK存在，所以TK去除的痘苗病毒更倾向于在肿瘤细胞中复制，这已在包括结肠癌、黑素瘤、肉瘤等在内的多种肿瘤细胞中得到证实。
活体生物荧光成像技术是近年来发展起来的一项分子、基因表达的分析检测技术。研究人员能够直接监控活体生物体内肿瘤的生长及转移、特定基因的表达等生物学过程。因其不需要宰杀动物、操作简单、所得结果直观、灵敏度高等特点，已广泛应用于生命科学、医学研究及药物开发等领域。在所有的红色荧光蛋白中mCherry应用广泛，mCherry是一种来自于蘑菇珊瑚(mushroom coral)的红色荧光蛋白，常用于标记细胞或者分子，进行示踪实验。相对于其他荧光，mCherry的好处在于它的颜色和应用最多的绿色荧光蛋白(GFP)能进行共同标记，并且mCherry相对于其他单体荧光蛋白来说也具有卓越的光稳定型，mCherry的最大吸收/发射峰分别位于587nm和610nm，对光致漂白耐受，荧光非常稳定。
发明内容
本发明的目的是提供一种天坛株痘苗病毒的荧光标记穿梭载体，用于制备重组痘苗病毒，利用活体生物荧光成像技术动态监测重组病毒在肿瘤动物模型中的全身分布和在靶组织中的复制情况。
本发明的目的是提供一种天坛株痘苗病毒的荧光标记穿梭载体的制备方法。
本发明所采用的第一个技术方案是，一种天坛株痘苗病毒的荧光标记穿梭载体，穿梭载体含有mCherry红色荧光蛋白报告基因。
本发明的特征在于，
编码mCherry的DNA序列位于天坛株痘苗病毒胸苷激酶基因TK的同源重组臂TKL和TKR之间，由P7.5启动子启动mCherry红色荧光报告基因表达，穿梭载体含有氨苄青霉素筛选标记和大肠杆菌复制起始位点pUC ori。
穿梭载体的核苷酸序列如序列1所示。
穿梭载体的出发载体为pMV-VTTTK，pMV-VTTTK载体包含痘苗病毒天坛株的TK基因的同源重组臂TKL和TKR。
pMV-VTTTK载体序列如序列2所示。
本发明所采用的第二个技术方案是，一种天坛株痘苗病毒的荧光标记穿梭载体的制备方法，包括以下步骤：
步骤1，根据GenBank:AY678264.1mCherry红色荧光蛋白报告基因DNA序列，设计扩增引物，以pbI111L-mCherry质粒为模板，PCR扩增获得红色荧光蛋白报告基因完整编码序列mCherry CDS；
PCR扩增参数为：
PCR反应体系为：5×PrimerSTAR Buffer(Mg2+plus)10μl，dNTP Mixture4μl，mcherryF(10μM)1μl，mcherryR(10μM)1μl，模板DNA 1μl，PrimerSTAR HS DNA Polymerase(2.5U/μl)0.5μl，加灭菌蒸馏水至50μl；
PCR反应条件为：94℃预变性2min，98℃变性10s，55℃退火15s，72℃延伸1min，循环35次；72℃延伸2min，扩增产物保存于4℃；
PCR结果：PCR产物上样电泳，mCherryCDS片段为771bp，切胶回收目的条带；
步骤2，将步骤1得到的mCherryCDS通过酶切连接方法连接至pMV-VTTTK，获得穿梭载体pMV-VTTTK-mCherry。
本发明的特征还在于，
步骤1中扩增引物为：
mcherryF:5’-CATGAATTCATGGTGAGCAAGGGCGAGGAG-3’
mcherryR:5’-CATGGATCCTTACTTGTACAGCTCGTCCAT-3’。
步骤1中mCherry CDS基因完整编码序列如序列3所示。
步骤2中酶切连接方法具体为：
用限制性内切酶EcoRI和BamHI对mCherryCDS片段进行双酶切，与经过同样双酶切的pMV-VTTTK质粒通过T4连接酶进行连接，得到质粒pMV-VTTTK-mCherry。
本发明的第三个技术方案：一种天坛株痘苗病毒的荧光标记穿梭载体在制备去除天坛株痘苗病毒TK基因的重组痘苗病毒作为作为活病毒载体疫苗及亚单位疫苗制备及生物制药中的应用。
本发明的有益效果是：
(1)本发明天坛株痘苗病毒的荧光标记穿梭载体，用于制备重组痘苗病毒，以mCherry红色荧光蛋白基因为报告基因可用于筛选重组痘苗病毒，重组病毒感染肿瘤动物模型后可作为特异性分子探针成为追踪靶目标并实现小动物活体成像；
(2)本发明天坛株痘苗病毒的荧光标记穿梭载体可用于制备重组痘苗病毒，以胸苷激酶基因TK为打靶目标，去除TK基因既可作为筛选标记，同时可提高痘苗病毒的肿瘤细胞选择性。
附图说明
图1是本发明实施例中pMV-VTTTK质粒结构示意图；
图2是本发明实施例中带有相应酶切位点的mCherryCDS片段的PCR扩增产物图；
图3是本发明实施例中EcoRI和BamHI双酶切pMV-VTTTK-mCherry和空载体质粒鉴定结果图；
图4是本发明实施例中pMV-VTTTK-mCherry荧光穿梭质粒结构示意图。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
一.主要试剂和材料：
PrimerSTAR高保真DNA聚合酶、限制性内切酶、T4连接酶均购自Takara公司，质粒小提试剂盒、胶回收试剂盒购自天根生化科技公司；
本实施例中天坛株痘苗病毒(Vaccinia virus Tian Tan strain)的全基因组DNA序列为GenBank:AF095689.1(VRL 14-FEB-2000)；
天坛株痘苗病毒和pMV-VTTTK质粒、pbI111L-mCherry质粒均保藏在西安医学院分子病毒与病毒免疫学实验室；
细胞系CV-1和143TK-购自中国典型培养物保藏中心。
二.引物设计与合成：
根据GenBank序列号为AY678264.1的mCherry红色荧光蛋白基因的完整编码序列设计克隆引物，将mCherry红色荧光蛋白基因的完整编码序列克隆至pMV-VTTTK质粒，得到pMV-VTTTK-mCherry穿梭载体。
引物序列合成由Invitrogen公司完成，引物序列信息如下：
mcherryF：5’-CATGAATTCATGGTGAGCAAGGGCGAGGAG-3’(下划线之前部分为保护碱基，下划线部分为EcoRI酶切位点识别序列，其后序列为GenBank:AY678264.1的第1-21位，即本发明序列3的第1-21位)。
mcherryR：5’-CATGGATCCTTACTTGTACAGCTCGTCCAT-3’(下划线之前部分为保护碱基，下划线部分为BamHI酶切位点识别序列，其后序列为GenBank:AY678264.1的第691-711位的反向互补序列，即本发明序列3的第691-711位的反向互补序列)。
三.pMV-VTTTK-mCherry的构建和鉴定；
1.如图1所示，以pbI111L-mCherry质粒为模板，以引物对mCherry完整编码区进行PCR扩增，获得带有相应酶切位点的mCherryCDS，扩增结果如图2所示：
PCR扩增反应体系为：5×PrimerSTAR Buffer(Mg2+plus)10μl，dNTP Mixture 4μl，mcherryF(10μM)1μl，mcherryR(10μM)1μl，模板DNA 1μl，PrimerSTAR HS DNA Polymerase(2.5U/μl)0.5μl，加灭菌蒸馏水至50μl；
PCR扩增反应条件为：94℃预变性2min，98℃变性10s，55℃退火15s，72℃延伸1min，循环35次；72℃延伸2min，扩增产物保存于4℃；
PCR扩增结果：PCR产物上样电泳，mCherryCDS片段为771bp，切胶回收目的片段。
2.用限制性内切酶EcoRI和BamHI分别对得到的mCherryCDS片段和pMV-VTTTK质粒进行双酶切，经过同样双酶切的mCherryCDS片段和pMV-VTTTK质粒通过T4连接酶进行连接，获得重组质粒，酶切鉴定和测序，经鉴定和测序后得到质粒pMV-VTTTK-mCherry。
如图3所示，为EcoRI和BamHI双酶切pMV-VTTTK-mCherry和空载体质粒鉴定结果图；
本发明序列4为得到质粒pMV-VTTTK-mCherry，其中第86-91位、803-808位分别为EcoRI和BamHI酶切位点识别序列，第86-808位为连接入的mCherry CDS序列，其余为载体上序列，经鉴定质粒pMV-VTTTK-mCherry序列正确。如图4所示，为pMV-VTTTK-mCherry荧光穿梭质粒结构示意图。
序列表
<110> 西安医学院
<120> 一种天坛株痘苗病毒的荧光标记穿梭载体及其制备方法
<130> 无
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4910
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 1
aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 60
gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 120
tgtaaaacga cggccagtcg aaccacgcaa tgcgtctcga tccgcagtgt cttgcgtctc 180
tttactgcag acagatattc cgacaaagga ttgattacta taaatggaga atgttcctaa 240
tgtatacttt aatcctgtgt ttatagagcc cacgtttaaa cattctttat taagtgttta 300
taaacacaga ttaatagttt tatttgaagt attcgttgta ttcattctaa tatatgtatt 360
ttttagatct gaattaaata tgttcttcat gcctaaacga aaaatacccg atcctattga 420
tagattacga cgtgctaatc tagcgtgtga agacgataaa ttaatgatct atggattacc 480
atggatgaca actcaaacat ctgcgttatc aataaatagt aaaccgatag tgtataaaga 540
ttgtgcaaag cttttgcgat caataaatgg atcacaacca gtatctctta acgatgttct 600
tcgcagatga tgattcattt tttaagtatt tggctagtca agatgatgaa tcttcattat 660
ctgatatatt gcaaatcact caatatctag actttctgtt attattattg atccaatcaa 720
aaaataaatt agaagccgtg ggtcattgtt atcaatctct ttcagaggaa tacagacaat 780
tgacaaaatt cacagactct caagatttta aaaaactgtt taacaaggtc cctattgtta 840
cagatggaag ggtcaaactt aataaaggat atttgttcga ctttgtgatt agtttgatgc 900
gattcaaaaa agaatcctct ctagctacca ccgcaataga tcctattaga tacatagatc 960
ctcgtcgcga tatcgcattt tctaacgtga tggatatatt aaagtcgaat aaagtgaaca 1020
ataattaatt ctttattgtc atcatgaacg gcggacatat tcagttgatg tcgacaagct 1080
tcaactgtcc atgggcccgc ggccgctcga gcatctggta atttatagca tagaaaaaaa 1140
caaaatgaaa ttctactata tttttacata catatattct aaatatgaaa gtggtgattg 1200
tgactagcgt agcatcgctc atctatatac tatatagtaa taccaataga gctcaagact 1260
acgactagta aactgataca atctcttata tgtgggtaat gttctcgatg tcgatagcca 1320
tatgcccggt agttgcgata tacataaact gatcactaat tccaaaccca cccgcttttt 1380
atagtaagtt tttcacccat aaataataaa tacaatggaa ttcatggtga gcaagggcga 1440
ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc 1500
cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac 1560
ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct 1620
gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga 1680
ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga 1740
cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa 1800
ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat 1860
gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat 1920
caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta 1980
caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat 2040
cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca 2100
ctccaccggc ggcatggacg agctgtacaa gtaaggatcc ggcttccttt tctaaacgat 2160
tgggtgagga aaccgagata gaaataatag gaggtaatga tatgtatcaa tcggtgtgta 2220
gaaagtgtta catcgactca taatattata ttttttatct aaaaaactaa aaataaacat 2280
tgattaaatt ttaatataat acttaaaaat ggatgttgtg tcgttagata aaccgtttat 2340
gtattttgag gaaattgata atgagttaga ttacgaacca gaaagtgcaa atgaggtcgc 2400
aaaaaaactg ccgtatcaag gacagttaaa actattacta ggagaattat tttttcttag 2460
taagttacag cgacacggta tattagatgg tgccaccgta gtgtatatag gatctgctcc 2520
cggtacacat atacgttatt tgagagatca tttctataat ttaggagtga tcatcaaatg 2580
gatgctaatt gacggccgcc atcatgatcc tattttaaat ggattgcgtg atgtgactct 2640
agtgactcgg ttcgttgatg aggaatatct acgatccatc aaaaaacaac tgcatccttc 2700
taagattatt ttaatttctg atgtgagatc caaacgagga ggaaatgaac ctagtacggc 2760
ggatttacta agtaattacg ctctacaaaa tgtcatgatt agtattttaa accccgtggc 2820
atctagtctt aaatggagat gcccgtttcc agatcaatgg atcaaggact tttatatccc 2880
acacggtaat aaaatgttac aaccttttgc tccttcatat tcagctgaaa tgagattatt 2940
aagtatttat accggtgaga acatgagact gactcggtac ctaaagagac ggagtcactg 3000
ccaaccgaga cggtcatagc tgtttcctgt gtgccgcttc ctcgctcact gactcgctgc 3060
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttac 3120
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 3180
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 3240
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 3300
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 3360
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 3420
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 3480
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 3540
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 3600
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 3660
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 3720
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 3780
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 3840
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 3900
agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 3960
ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 4020
gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 4080
catctggccc cagtgctgca ataataccgc gggacccacg ctcaccggct ccagatttat 4140
cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 4200
cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 4260
gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt atcacgctcg tcgtttggta 4320
tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgc 4380
gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgccg 4440
tgttatcact catggttatg gcagcactac ataattctct tactgtcatg ccatccgtaa 4500
gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 4560
gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 4620
taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 4680
tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 4740
ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 4800
taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 4860
tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 4910
<210> 2
<211> 4229
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 2
aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 60
gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 120
tgtaaaacga cggccagtcg aaccacgcaa tgcgtctcga tccgcagtgt cttgcgtctc 180
tttactgcag acagatattc cgacaaagga ttgattacta taaatggaga atgttcctaa 240
tgtatacttt aatcctgtgt ttatagagcc cacgtttaaa cattctttat taagtgttta 300
taaacacaga ttaatagttt tatttgaagt attcgttgta ttcattctaa tatatgtatt 360
ttttagatct gaattaaata tgttcttcat gcctaaacga aaaatacccg atcctattga 420
tagattacga cgtgctaatc tagcgtgtga agacgataaa ttaatgatct atggattacc 480
atggatgaca actcaaacat ctgcgttatc aataaatagt aaaccgatag tgtataaaga 540
ttgtgcaaag cttttgcgat caataaatgg atcacaacca gtatctctta acgatgttct 600
tcgcagatga tgattcattt tttaagtatt tggctagtca agatgatgaa tcttcattat 660
ctgatatatt gcaaatcact caatatctag actttctgtt attattattg atccaatcaa 720
aaaataaatt agaagccgtg ggtcattgtt atcaatctct ttcagaggaa tacagacaat 780
tgacaaaatt cacagactct caagatttta aaaaactgtt taacaaggtc cctattgtta 840
cagatggaag ggtcaaactt aataaaggat atttgttcga ctttgtgatt agtttgatgc 900
gattcaaaaa agaatcctct ctagctacca ccgcaataga tcctattaga tacatagatc 960
ctcgtcgcga tatcgcattt tctaacgtga tggatatatt aaagtcgaat aaagtgaaca 1020
ataattaatt ctttattgtc atcatgaacg gcggacatat tcagttgatg tcgacaagct 1080
tcaactgtcc atgggcccgc ggccgctcga gcatctggta atttatagca tagaaaaaaa 1140
caaaatgaaa ttctactata tttttacata catatattct aaatatgaaa gtggtgattg 1200
tgactagcgt agcatcgctc atctatatac tatatagtaa taccaataga gctcaagact 1260
acgactagta aactgataca atctcttata tgtgggtaat gttctcgatg tcgatagcca 1320
tatgcccggt agttgcgata tacataaact gatcactaat tccaaaccca cccgcttttt 1380
atagtaagtt tttcacccat aaataataaa tacaatggaa ttcgcgcgcg atatcggcgc 1440
ctattcgtct agaggatccg gcttcctttt ctaaacgatt gggtgaggaa accgagatag 1500
aaataatagg aggtaatgat atgtatcaat cggtgtgtag aaagtgttac atcgactcat 1560
aatattatat tttttatcta aaaaactaaa aataaacatt gattaaattt taatataata 1620
cttaaaaatg gatgttgtgt cgttagataa accgtttatg tattttgagg aaattgataa 1680
tgagttagat tacgaaccag aaagtgcaaa tgaggtcgca aaaaaactgc cgtatcaagg 1740
acagttaaaa ctattactag gagaattatt ttttcttagt aagttacagc gacacggtat 1800
attagatggt gccaccgtag tgtatatagg atctgctccc ggtacacata tacgttattt 1860
gagagatcat ttctataatt taggagtgat catcaaatgg atgctaattg acggccgcca 1920
tcatgatcct attttaaatg gattgcgtga tgtgactcta gtgactcggt tcgttgatga 1980
ggaatatcta cgatccatca aaaaacaact gcatccttct aagattattt taatttctga 2040
tgtgagatcc aaacgaggag gaaatgaacc tagtacggcg gatttactaa gtaattacgc 2100
tctacaaaat gtcatgatta gtattttaaa ccccgtggca tctagtctta aatggagatg 2160
cccgtttcca gatcaatgga tcaaggactt ttatatccca cacggtaata aaatgttaca 2220
accttttgct ccttcatatt cagctgaaat gagattatta agtatttata ccggtgagaa 2280
catgagactg actcggtacc taaagagacg gagtcactgc caaccgagac ggtcatagct 2340
gtttcctgtg tgccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 2400
gagcggtatc agctcactca aaggcggtaa tacggttacc cacagaatca ggggataacg 2460
caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 2520
tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 2580
gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 2640
ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 2700
cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 2760
tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 2820
tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 2880
cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 2940
agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 3000
agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 3060
gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 3120
aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 3180
ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 3240
gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 3300
taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 3360
tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 3420
taataccgcg ggacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 3480
gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 3540
gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 3600
tcgctacagg catcgtggta tcacgctcgt cgtttggtat ggcttcattc agctccggtt 3660
cccaacgatc aaggcgagtt acatgatccc ccatgttgcg caaaaaagcg gttagctcct 3720
tcggtcctcc gatcgttgtc agaagtaagt tggccgccgt gttatcactc atggttatgg 3780
cagcactaca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 3840
agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 3900
cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 3960
aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 4020
aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 4080
gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 4140
gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 4200
tgagcggata catatttgaa tgtatttag 4229
<210> 3
<211> 711
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60
gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120
cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180
ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240
cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300
gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360
ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420
atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480
gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540
gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600
aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660
cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta a 711
<210> 4
<211> 904
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 4
gtatcgatca tcataactga tcactaattc caaacccacc cgctttttat agtaagtttt 60
tcacccataa ataataaata caatggaatt catggtgagc aagggcgagg aggataacat 120
ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca 180
cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa 240
gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt 300
catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct 360
gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt 420
gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg 480
cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc 540
ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct 600
gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa 660
gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa 720
cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg 780
catggacgag ctgtacaagt aaggatccgg cttccttttc taaacgattg ggtgaggaaa 840
ccgagataga aataatagga ggtaatgata tgtatcaatc ggtgtgtaga aagtgttaca 900
tcga 904
Claims (10)
1.一种天坛株痘苗病毒的荧光标记穿梭载体，其特征在于，所述穿梭载体含有mCherry红色荧光蛋白报告基因。
2.根据权利要求1所述的一种天坛株痘苗病毒的荧光标记穿梭载体，其特征在于，编码所述mCherry的DNA序列位于天坛株痘苗病毒胸苷激酶基因TK的同源重组臂TKL和TKR之间，由P7.5启动子启动mCherry红色荧光报告基因表达，所述穿梭载体含有氨苄青霉素筛选标记和大肠杆菌复制起始位点pUC ori。
3.根据权利要求1所述的一种天坛株痘苗病毒的荧光标记穿梭载体，其特征在于，所述穿梭载体的核苷酸序列如序列1所示。
4.根据权利要求1所述的一种天坛株痘苗病毒的荧光标记穿梭载体，其特征在于，所述穿梭载体的出发载体为pMV-VTTTK，pMV-VTTTK载体包含痘苗病毒天坛株的TK基因的同源重组臂TKL和TKR。
5.根据权利要求4所述的一种天坛株痘苗病毒的荧光标记穿梭载体，其特征在于，所述pMV-VTTTK载体序列如序列2所示。
6.如权利要求1-5任一所述的一种天坛株痘苗病毒的荧光标记穿梭载体的制备方法，其特征在于，包括以下步骤：
步骤1，根据GenBank:AY678264.1mCherry红色荧光蛋白报告基因DNA序列，设计扩增引物，以pbI111L-mCherry质粒为模板，PCR扩增获得红色荧光蛋白报告基因完整编码序列mCherry CDS；
PCR扩增参数为：
PCR反应体系为：5×PrimerSTAR Buffer(Mg2+plus)10μl，dNTP Mixture 4μl，mcherryF(10μM)1μl，mcherryR(10μM)1μl，模板DNA 1μl，PrimerSTAR HS DNA Polymerase(2.5U/μl)0.5μl，加灭菌蒸馏水至50μl；
PCR反应条件为：94℃预变性2min，98℃变性10s，55℃退火15s，72℃延伸1min，循环35次；72℃延伸2min，扩增产物保存于4℃；
PCR结果：PCR产物上样电泳，mCherryCDS片段为771bp，切胶回收目的条带；
步骤2，将步骤1得到的mCherryCDS通过酶切连接方法连接至pMV-VTTTK，获得穿梭载体pMV-VTTTK-mCherry。
7.根据权利要求6所述的一种天坛株痘苗病毒的荧光标记穿梭载体的制备方法，其特征在于，所述步骤1中扩增引物为：
mcherryF:5’-CATGAATTCATGGTGAGCAAGGGCGAGGAG-3’
mcherryR:5’-CATGGATCCTTACTTGTACAGCTCGTCCAT-3’。
8.根据权利要求6所述的一种天坛株痘苗病毒的荧光标记穿梭载体的制备方法，其特征在于，所述步骤1中mCherry CDS基因完整编码序列如序列3所示。
9.根据权利要求6所述的一种天坛株痘苗病毒的荧光标记穿梭载体的制备方法，其特征在于，所述步骤2中酶切连接方法具体为：
用限制性内切酶EcoRI和BamHI对mCherryCDS片段进行双酶切，与经过同样双酶切的pMV-VTTTK质粒通过T4连接酶进行连接，得到质粒pMV-VTTTK-mCherry。
10.一种天坛株痘苗病毒的荧光标记穿梭载体在制备去除天坛株痘苗病毒TK基因的重组痘苗病毒作为作为活病毒载体疫苗及亚单位疫苗制备及生物制药中的应用。
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