CN110157721A - A kind of tracer target practice plasmid of vaccinia virus Tiantan strain and preparation method thereof - Google Patents
A kind of tracer target practice plasmid of vaccinia virus Tiantan strain and preparation method thereof Download PDFInfo
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- CN110157721A CN110157721A CN201910012569.2A CN201910012569A CN110157721A CN 110157721 A CN110157721 A CN 110157721A CN 201910012569 A CN201910012569 A CN 201910012569A CN 110157721 A CN110157721 A CN 110157721A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
Abstract
The invention discloses a kind of tracer target practice plasmids of vaccinia virus Tiantan strain, upstream homologous recombination arm VTT-TKL and downstream homologous recombination arm VTT-TKR including vaccinia virus Tiantan strain TK gene, it is inserted into Luciferase reporter gene and EGFP Green Fluorescent Protein gene, Luciferase reporter gene and EGFP Green Fluorescent Protein gene between upstream homologous recombination arm VTT-TKL and downstream homologous recombination arm VTT-TKR, expression is started by promoter P7.5.The invention also discloses the preparation methods of the tracer target practice plasmid of the vaccinia virus Tiantan strain.A kind of tracer target practice plasmid of vaccinia virus Tiantan strain of the present invention can target the TK gene in removal vaccinia virus Tiantan strain, prepare the recombination the Temple of Heaven strain virus of removal TK gene.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, are related to a kind of tracer target practice plasmid of vaccinia virus Tiantan strain,
The invention further relates to the preparation methods of the tracer target practice plasmid of the vaccinia virus Tiantan strain.
Background technique
Vaccinia virus Tiantan strain (Vaccinia virus Tian Tan, VTT) is the distinctive vaccine strain in China, in order to mention
The oncolytic effect of high vaccinia virus Tiantan strain VTT, need to be by corresponding target practice plasmid.Thymidine kinase TK can be viral dna replication
High-caliber nucleotide pond is provided, TK level is lower in normal cell, and removal virus TK gene is more likely to virus in tumour
It is replicated in cell, viral tumor-selective cannot can be improved early in the TK gene for replicating in normal cell, therefore removing VTT.
At present be mainly used for infection prevention disease for the target practice plasmid of vaccinia virus Tiantan strain, for antitumor application thereof compared with
It is few.These target practice plasmids or lack tracer reporter gene or lack fluorescent screening label, so as to form recombination VTT disease
Poison otherwise can not tracer or traceability is weaker or more difficult screening, therefore be unable to satisfy current attenuation oncolytic bovine vaccine disease
The needs of poison research.
Summary of the invention
The object of the present invention is to provide a kind of tracer target practice plasmids of vaccinia virus Tiantan strain, can target removal bovine vaccine
Viral the Temple of Heaven strain TK gene, and it is made to carry trace labelling gene, prepare the recombination with traceability of removal TK gene
The Temple of Heaven strain virus.
Another object of the present invention is to provide a kind of preparation method of the tracer target practice plasmid of vaccinia virus Tiantan strain.
The technical scheme adopted by the invention is that a kind of tracer target practice plasmid of vaccinia virus Tiantan strain, including bovine vaccine disease
The upstream homologous recombination arm VTT-TKL and downstream homologous recombination arm VTT-TKR of malicious the Temple of Heaven strain TK gene, upstream homologous recombination arm
EGFP Green Fluorescent Protein gene, EGFP green fluorescence egg are inserted between VTT-TKL and downstream homologous recombination arm VTT-TKR
White marker gene is started by promoter P7.5 expresses.
The characteristics of the first technical solution of the invention, also resides in:
Target practice plasmid further includes Luciferase reporter gene, and it is homologous heavy that Luciferase reporter gene also is located at upstream
Between group arm VTT-TKL and downstream homologous recombination arm VTT-TKR, Luciferase reporter gene starts table by promoter P7.5
It reaches, the nucleotide sequence of target practice plasmid is as shown in sequence table 1.
The carrier pMV-VTTTK-Luc that sets out of target practice plasmid.
The another technical solution that the present invention uses is:
A kind of preparation method of the tracer target practice plasmid of vaccinia virus Tiantan strain specifically carries out as steps described below:
Step 1, fluorescent marker tracer genetic fragment E-Luc segment, the nucleotide sequence of E-Luc segment such as sequence are constructed
Shown in table 2；
Step 2, the E-Luc segment is connected in pMV-VTTTK-Luc plasmid with the method that digestion connects, is obtained
The tracer target practice plasmid of vaccinia virus Tiantan strain.
The characteristics of another technical solution of the present invention, also resides in:
Step 1 is specific to be carried out by the following method:
Step 1.1, using pEGFP-Luc plasmid as template, according to Luciferase reporter gene and EGFP green fluorescence egg
The fusion sequence of white marker gene, design primer；
Step 1.2, restriction enzyme site EcoRI is added in one end of primer, in the other end restriction enzyme site of the primer
XmaI obtains synthetic primer；
Step 1.3, using pEGFP-Luc plasmid as template, the sequence obtained using step 2 after PCR amplification, is obtained as primer
To signal tracer genetic fragment E-Luc segment.
Synthetic primer in step 1.2 are as follows:
ELuc-F:5 '-GGAATTCGCCACCATGGAAGACGCCA-3 '
ELuc-R:5 '-TCCCCCCGGGTTACTTGTACAGCTCGTCCATG-3.
In step 1.3, PCR reaction system are as follows: DNA 1 μ l, dNTP mix (2.5mM) 1 μ l, forward primer (10 μM) 2 μ
10 μ l, PrimeSTAR HS archaeal dna polymerase of l, 2 μ l, 5 × PrimeSTAR buffer of reverse primer (10 μM), 0.5 μ l, adds
DEPC water is to 50 μ l.
PCR reaction condition are as follows: 98 DEG C of initial denaturation 30s；98 DEG C of denaturation 10s, 55-60 DEG C of annealing 15s, 72 DEG C of extension 1min/
Kb is recycled 30 times；72 DEG C extend 5 minutes, are finally placed in 4 DEG C of preservations；
PCR fragment recycling: PCR product is through agarose gel electrophoresis, gel extraction E-Luc segment.
Step 2 is specific to be carried out as steps described below:
Step 2.1, by pMV-VTTTK-Luc plasmid through EcoRI/XmaI double digestion, large fragment conduct is recycled in gel electrophoresis
Carrier；
By E-Luc segment into EcoRI/XmaI double digestion is crossed, then purification and recovery obtains junction fragment；
Step 2.2, carrier and junction fragment are attached, converted, obtain the tracer target practice matter of vaccinia virus Tiantan strain
Grain.
Carrier and junction fragment are attached according to molecule clone technology in step 2.2, converted.
The beneficial effects of the present invention are:
A kind of tracer target practice plasmid of vaccinia virus Tiantan strain of the present invention can target in removal vaccinia virus Tiantan strain
TK gene, prepare removal TK gene the recombination the Temple of Heaven strain virus with traceability；
A kind of tracer target practice plasmid of vaccinia virus Tiantan strain of the present invention, EGFP Green Fluorescent Protein can be used for sieving
Select recombinant virus；Luciferase reporter gene can generate bioluminescence by produces chemiluminescence, play tracking function, be used for
The distribution situation of oncolytic VTT virus in vivo is analyzed, the distribution especially in tumour is vaccinia virus as carrier and researches and develops work
Vaccine and tumor biotherapy provide good tool.
Detailed description of the invention
Fig. 1 is a kind of plasmid map of the tracer target practice plasmid of vaccinia virus Tiantan strain of the present invention；
Fig. 2 is E-Luc segment pcr amplification product gel electrophoresis result；
Fig. 3 is the structural schematic diagram of carrier pMV-VTTTK-Luc plasmid；
Fig. 4 is a kind of digestion qualification result of the tracer target practice plasmid of vaccinia virus Tiantan strain of the present invention；
Figure 5-8 is a kind of sequencing qualification result of the tracer target practice plasmid of vaccinia virus Tiantan strain of the present invention.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
Material used in the present invention, reagent commercially obtain unless otherwise specified.
In the present invention genomic dna sequence of wild vaccinia virus Tiantan strain be No. GenBank be AF095689.1 sequence
It arranges (VRL 14-FEB-2000).Vaccinia virus Tiantan strain is in Xi'an Medical University's molecule virus and virological immunology laboratory (this
Laboratory, similarly hereinafter) preservation.Plasmid pEGFP-Luc and pMV-VTTTK-Luc is by this laboratory preservation.
One, a kind of tracer target practice plasmid of vaccinia virus Tiantan strain, as shown in Figure 1, including pMV-VTTTK-ELuc matter
Grain, pMV-VTTTK-Luc plasmid is inserted with the upstream homologous recombination arm VTT-TKL of vaccinia virus Tiantan strain TK gene and downstream
EGFP green is inserted between homologous recombination arm VTT-TKR, upstream homologous recombination arm VTT-TKL and downstream homologous recombination arm VTT-TKR
Fluorescent protein marker gene and Luciferase reporter gene, EGFP Green Fluorescent Protein gene and Luciferase report
It accuses gene and expression is started by Vaccinia promoters P7.5.
A kind of tracer target practice plasmid of vaccinia virus Tiantan strain of the present invention, homology arm VTT-TKL and VTT-TKR and VTT's
Homologous recombination occurs for the sequence of TK gene two sides, the recombination VTT disease for being formed and knocking out TK gene, express Luciferase and EGFP
Poison.EGFP fluorescent marker can be used for recombinant celo virus；Luciferase generates bioluminescence by produces chemiluminescence, plays
Tracking function is to determine the distribution situation of virus in vivo.
Two, a kind of preparation method of the tracer target practice plasmid of vaccinia virus Tiantan strain carries out as steps described below:
Step 1, fluorescent marker tracer genetic fragment E-Luc segment is constructed；
Step 1.1, using pEGFP-Luc plasmid as template, according to the fusion sequence of Luciferase and EGFP fusion
Column, design primer；
Step 1.2, restriction enzyme site EcoRI is added in one end of institute's primer, in the other end restriction enzyme site XmaI of primer,
Synthetic primer is obtained, the sequence information of synthetic primer is as follows:
ELuc-F:5 '-GGAATTCGCCACCATGGAAGACGCCA-3 ' (part is protection base before underscore, under
Dashed part is EcoRI endonuclease recognition sequence, and sequence is 1-19 of sequence 2 thereafter)
ELuc-R:5 '-TCCCCCCGGG(part is protection alkali to TTACTTGTACAGCTCGTCCATG-3 ' before underscore
Base, underscore part are XmaI endonuclease recognition sequence, and sequence is 2376-2397 reverse complemental sequences of sequence 2 thereafter
Column)；
Step 1.3, using pEGFP-Luc plasmid as template, after PCR amplification, signal tracer genetic fragment E- is obtained
Luc segment, the gene order of E-Luc segment are shown in sequence table 2.
PCR reaction system are as follows: DNA 1 μ l, dNTP mix (2.5mM) 1 μ l, 2 μ l of forward primer (10 μM), reverse primer
10 μ l, PrimeSTAR HS DNA polymerase of (10 μM) 2 μ l, 5 × PrimeSTAR buffer, 0.5 μ l, adds DEPC water to 50 μ
l。
PCR reaction condition are as follows: 98 DEG C of initial denaturation 30s；98 DEG C of denaturation 10s, 60 DEG C of annealing 15s, 72 DEG C of extension 1min/kb,
Circulation 30 times；72 DEG C extend 5 minutes, are finally placed in 4 DEG C of preservations；
PCR fragment recycling: for PCR product through agarose gel electrophoresis, the E-Luc with restriction enzyme site is 2414bp, electrophoresis knot
Fruit sees Fig. 2, gel extraction target fragment.
Step 2, the method that E-Luc segment digestion connects is connected in pMV-VTTTK-Luc plasmid, obtains bovine vaccine disease
The tracer target practice plasmid of malicious the Temple of Heaven strain:
Step 2.1, by pMV-VTTTK-Luc plasmid as shown in Figure 3 through EcoRI/XmaI double digestion, gel electrophoresis is returned
It receives 4197bp segment and obtains carrier；
E-Luc segment is obtained into junction fragment into EcoRI/XmaI double digestion, purification and recovery 2403bp segment is crossed；
Step 2.2, carrier and junction fragment are attached through molecule clone technology, converted, obtain vaccinia virus Tiantan
The tracer target practice plasmid of strain.
The tracer target practice plasmid for the vaccinia virus Tiantan strain that the present embodiment is obtained carries out digestion identification, and qualification result is such as
Shown in Fig. 4, the tracer target practice plasmid of vaccinia virus Tiantan strain through EcoRI/XmaI double digestion generate 4197bp segment and
2403bp, it is consistent with expected clip size；
The tracer target practice plasmid of the vaccinia virus Tiantan strain that the present embodiment is obtained carries out gene sequencing, as Fig. 5,
Shown in Fig. 6, Fig. 7 and Fig. 8, it was found from Fig. 5, Fig. 6, Fig. 7 and Fig. 8: there is no variation, insertion positions for display gene order
Correctly；Therefore, the tracer target practice plasmid construction success of vaccinia virus Tiantan strain.
SEQUENCE LISTING
<110>Xi'an Medical University
<120>the tracer target practice plasmid and preparation method thereof of a kind of vaccinia virus Tiantan strain
<160> 2
<210> 1
<211> 6600
<212> DNA
<213>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 attcattgta 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 atgaatctct 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 ttcgccacca tggaagacgc 1440
caaaaacata aagaaaggcc cggcgccatt ctatccgctg gaagatggaa ccgctggaga 1500
gcaactgcat aaggctatga agagatacgc cctggttcct ggaacaattg cttttacaga 1560
tgcacatatc gaggtggaca tcacttacgc tgagtacttc gaaatgtccg ttcggttggc 1620
agaagctatg aaacgatatg ggctgaatac aaatcacaga atcgtcgtat gcagtgaaaa 1680
ctctcttcaa ttctttatgc cggtgttggg cgcgttattt atcggagttg cagttgcgcc 1740
cgcgaacgac atttataatg aacgtgaatt gctcaacagt atgggcattt cgcagcctac 1800
cgtggtgttc gtttccaaaa aggggttgca aaaaattttg aacgtgcaaa aaaagctccc 1860
aatcatccaa aaaattatta tcatggattc taaaacggat taccagggat ttcagtcgat 1920
gtacacgttc gtcacatctc atctacctcc cggttttaat gaatacgatt ttgtgccaga 1980
gtccttcgat agggacaaga caattgcact gatcatgaac tcctctggat ctactggtct 2040
gcctaaaggt gtcgctctgc ctcatagaac tgcctgcgtg agattctcgc atgccagaga 2100
tcctattttt ggcaatcaaa tcattccgga tactgcgatt ttaagtgttg ttccattcca 2160
tcacggtttt ggaatgttta ctacactcgg atatttgata tgtggatttc gagtcgtctt 2220
aatgtataga tttgaagaag agctgtttct gaggagcctt caggattaca agattcaaag 2280
tgcgctgctg gtgccaaccc tattctcctt cttcgccaaa agcactctga ttgacaaata 2340
cgatttatct aatttacacg aaattgcttc tggtggcgct cccctctcta aggaagtcgg 2400
ggaagcggtt gccaagaggt tccatctgcc aggtatcagg caaggatatg ggctcactga 2460
gactacatca gctattctga ttacacccga gggggatgat aaaccgggcg cggtcggtaa 2520
agttgttcca ttttttgaag cgaaggttgt ggatctggat accgggaaaa cgctgggcgt 2580
taatcaaaga ggcgaactgt gtgtgagagg tcctatgatt atgtccggtt atgtaaacaa 2640
tccggaagcg accaacgcct tgattgacaa ggatggatgg ctacattctg gagacatagc 2700
ttactgggac gaagacgaac acttcttcat cgttgaccgc ctgaagtctc tgattaagta 2760
caaaggctat caggtggctc ccgctgaatt ggaatccatc ttgctccaac accccaacat 2820
cttcgacgca ggtgtcgcag gtcttcccga cgatgacgcc ggtgaacttc ccgccgccgt 2880
tgttgttttg gagcacggaa agacgatgac ggaaaaagag atcgtggatt acgtcgccag 2940
tcaagtaaca accgcgaaaa agttgcgcgg aggagttgtg tttgtggacg aagtaccgaa 3000
aggtcttacc ggaaaactcg acgcaagaaa aatcagagag atcctcataa aggccaagaa 3060
gggcggaaag atcgccgtgc gggatccacc ggtcgccacc atggtgagca agggcgagga 3120
gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 3180
gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt 3240
catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 3300
cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc 3360
cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta 3420
caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 3480
gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa 3540
cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa 3600
gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 3660
ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc 3720
cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 3780
cgccgggatc actctcggca tggacgagct gtacaagtaa cccgggatcc ggcttccttt 3840
tctaaacgat tgggtgagga aaccgagata gaaataatag gaggtaatga tatgtatcaa 3900
tcggtgtgta gaaagtgtta catcgactca taatattata ttttttatct aaaaaactaa 3960
aaataaacat tgattaaatt ttaatataat acttaaaaat ggatgttgtg tcgttagata 4020
aaccgtttat gtattttgag gaaattgata atgagttaga ttacgaacca gaaagtgcaa 4080
atgaggtcgc aaaaaaactg ccgtatcaag gacagttaaa actattacta ggagaattat 4140
tttttcttag taagttacag cgacacggta tattagatgg tgccaccgta gtgtatatag 4200
gatctgctcc cggtacacat atacgttatt tgagagatca tttctataat ttaggagtga 4260
tcatcaaatg gatgctaatt gacggccgcc atcatgatcc tattttaaat ggattgcgtg 4320
atgtgactct agtgactcgg ttcgttgatg aggaatatct acgatccatc aaaaaacaac 4380
tgcatccttc taagattatt ttaatttctg atgtgagatc caaacgagga ggaaatgaac 4440
ctagtacggc ggatttacta agtaattacg ctctacaaaa tgtcatgatt agtattttaa 4500
accccgtggc atctagtctt aaatggagat gcccgtttcc agatcaatgg atcaaggact 4560
tttatatccc acacggtaat aaaatgttac aaccttttgc tccttcatat tcagctgaaa 4620
tgagattatt aagtatttat accggtgaga acatgagact gactcggtac ctaaagagac 4680
ggagtcactg ccaaccgaga cggtcatagc tgtttcctgt gtgccgcttc ctcgctcact 4740
gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 4800
atacggttac ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 4860
caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 4920
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 4980
taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 5040
ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 5100
tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 5160
gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 5220
ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 5280
aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 5340
aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 5400
agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 5460
cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 5520
gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 5580
atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 5640
gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 5700
tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 5760
gagggcttac catctggccc cagtgctgca ataataccgc gggacccacg ctcaccggct 5820
ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 5880
actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 5940
ccagttaata gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt atcacgctcg 6000
tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 6060
cccatgttgc gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 6120
ttggccgccg tgttatcact catggttatg gcagcactac ataattctct tactgtcatg 6180
ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 6240
tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 6300
agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 6360
atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 6420
gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 6480
aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 6540
tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 6600
<210> 2
<211> 2397
<212> DNA
<213>artificial sequence
<400> 2
gccaccatgg aagacgccaa aaacataaag aaaggcccgg cgccattcta tccgctggaa 60
gatggaaccg ctggagagca actgcataag gctatgaaga gatacgccct ggttcctgga 120
acaattgctt ttacagatgc acatatcgag gtggacatca cttacgctga gtacttcgaa 180
atgtccgttc ggttggcaga agctatgaaa cgatatgggc tgaatacaaa tcacagaatc 240
gtcgtatgca gtgaaaactc tcttcaattc tttatgccgg tgttgggcgc gttatttatc 300
ggagttgcag ttgcgcccgc gaacgacatt tataatgaac gtgaattgct caacagtatg 360
ggcatttcgc agcctaccgt ggtgttcgtt tccaaaaagg ggttgcaaaa aattttgaac 420
gtgcaaaaaa agctcccaat catccaaaaa attattatca tggattctaa aacggattac 480
cagggatttc agtcgatgta cacgttcgtc acatctcatc tacctcccgg ttttaatgaa 540
tacgattttg tgccagagtc cttcgatagg gacaagacaa ttgcactgat catgaactcc 600
tctggatcta ctggtctgcc taaaggtgtc gctctgcctc atagaactgc ctgcgtgaga 660
ttctcgcatg ccagagatcc tatttttggc aatcaaatca ttccggatac tgcgatttta 720
agtgttgttc cattccatca cggttttgga atgtttacta cactcggata tttgatatgt 780
ggatttcgag tcgtcttaat gtatagattt gaagaagagc tgtttctgag gagccttcag 840
gattacaaga ttcaaagtgc gctgctggtg ccaaccctat tctccttctt cgccaaaagc 900
actctgattg acaaatacga tttatctaat ttacacgaaa ttgcttctgg tggcgctccc 960
ctctctaagg aagtcgggga agcggttgcc aagaggttcc atctgccagg tatcaggcaa 1020
ggatatgggc tcactgagac tacatcagct attctgatta cacccgaggg ggatgataaa 1080
ccgggcgcgg tcggtaaagt tgttccattt tttgaagcga aggttgtgga tctggatacc 1140
gggaaaacgc tgggcgttaa tcaaagaggc gaactgtgtg tgagaggtcc tatgattatg 1200
tccggttatg taaacaatcc ggaagcgacc aacgccttga ttgacaagga tggatggcta 1260
cattctggag acatagctta ctgggacgaa gacgaacact tcttcatcgt tgaccgcctg 1320
aagtctctga ttaagtacaa aggctatcag gtggctcccg ctgaattgga atccatcttg 1380
ctccaacacc ccaacatctt cgacgcaggt gtcgcaggtc ttcccgacga tgacgccggt 1440
gaacttcccg ccgccgttgt tgttttggag cacggaaaga cgatgacgga aaaagagatc 1500
gtggattacg tcgccagtca agtaacaacc gcgaaaaagt tgcgcggagg agttgtgttt 1560
gtggacgaag taccgaaagg tcttaccgga aaactcgacg caagaaaaat cagagagatc 1620
ctcataaagg ccaagaaggg cggaaagatc gccgtgcggg atccaccggt cgccaccatg 1680
gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 1740
gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 1800
aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 1860
gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 1920
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 1980
aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 2040
aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 2100
ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 2160
atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 2220
cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 2280
ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 2340
ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaa 2397
Claims (9)
1. a kind of tracer target practice plasmid of vaccinia virus Tiantan strain, which is characterized in that including vaccinia virus Tiantan strain TK gene
Upstream homologous recombination arm VTT-TKL and downstream homologous recombination arm VTT-TKR, the upstream homologous recombination arm VTT-TKL and downstream
EGFP Green Fluorescent Protein gene, the EGFP Green Fluorescent Protein gene are inserted between homologous recombination arm VTT-TKR
Started by promoter P7.5 and is expressed.
2. a kind of tracer target practice plasmid of vaccinia virus Tiantan strain according to claim 1, which is characterized in that the target practice
Plasmid further includes Luciferase reporter gene, and the Luciferase reporter gene also is located at upstream homologous recombination arm VTT-
Between TKL and downstream homologous recombination arm VTT-TKR, the Luciferase reporter gene is started by promoter P7.5 is expressed, institute
The nucleotide sequence of target practice plasmid is stated as shown in sequence table 1.
3. a kind of tracer target practice plasmid of vaccinia virus Tiantan strain according to claim 1, which is characterized in that the target practice
The carrier pMV-VTTTK-Luc that sets out of plasmid.
4. a kind of preparation method of the tracer target practice plasmid of vaccinia virus Tiantan strain, which is characterized in that specifically as steps described below
It carries out:
Step 1, fluorescent marker tracer genetic fragment E-Luc segment, the nucleotide sequence such as sequence table of the E-Luc segment are constructed
Shown in 2；
Step 2, the E-Luc segment is connected in pMV-VTTTK-Luc plasmid with the method that digestion connects, obtains bovine vaccine disease
The tracer target practice plasmid of malicious the Temple of Heaven strain.
5. a kind of preparation method of the tracer target practice plasmid of vaccinia virus Tiantan strain according to claim 4, feature exist
In the step 1 is specific to be carried out by the following method:
Step 1.1, using pEGFP-Luc plasmid as template, according to Luciferase reporter gene and EGFP green fluorescent protein mark
Remember the fusion sequence of gene, design primer；
Step 1.2, restriction enzyme site EcoRI is added in one end of the primer, in the other end restriction enzyme site XmaI of the primer,
Composition sequence is obtained as primer；
Step 1.3, using pEGFP-Luc plasmid as template, using the composition sequence as primer, after PCR amplification, signal is obtained
Tracer genetic fragment E-Luc segment E-Luc segment.
6. the preparation method of the tracer target practice plasmid of according to claim 5 kind of vaccinia virus Tiantan strain, which is characterized in that
Synthetic primer in the step 1.2 are as follows:
ELuc-F:5 '-GGAATTCGCCACCATGGAAGACGCCA-3 '
ELuc-R:5 '-TCCCCCCGGGTTACTTGTACAGCTCGTCCATG-3.
7. the preparation method of the tracer target practice plasmid of according to claim 5 kind of vaccinia virus Tiantan strain, which is characterized in that
In the step 1.3, PCR reaction system are as follows: DNA 1 μ l, dNTP mix (2.5mM) 1 μ l, 2 μ l of forward primer (10 μM), reversely
2 μ l, 5 × PrimeSTAR buffer of primer (10 μM), 10 μ l, PrimeSTAR HS archaeal dna polymerase, 0.5 μ l, adds DEPC water extremely
50μl。
PCR reaction condition are as follows: 98 DEG C of initial denaturation 30s；98 DEG C of denaturation 10s, 55-60 DEG C of annealing 15s, 72 DEG C of extension 1min/kb are followed
Ring 30 times；72 DEG C extend 5 minutes, are finally placed in 4 DEG C of preservations；
PCR fragment recycling: PCR product is through agarose gel electrophoresis, gel extraction E-Luc segment.
8. a kind of preparation method of the tracer target practice plasmid of vaccinia virus Tiantan strain according to claim 4, feature exist
In step 2 is specific to be carried out as steps described below:
Step 2.1, by pMV-VTTTK-Luc plasmid through EcoRI/XmaI double digestion, gel electrophoresis recycles large fragment as carrier；
By the E-Luc segment into EcoRI/XmaI double digestion is crossed, then purification and recovery obtains junction fragment；
Step 2.2, the carrier and junction fragment are attached, converted, obtain the tracer target practice matter of vaccinia virus Tiantan strain
Grain.
9. a kind of preparation method of the tracer target practice plasmid of vaccinia virus Tiantan strain according to claim 8, feature exist
In the carrier and junction fragment are attached according to molecule clone technology in the step 2.2, converted.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109750056A (en) * | 2019-01-07 | 2019-05-14 | 西安医学院 | Remove the target practice plasmid and preparation method thereof of the Temple of Heaven strain VGF gene |
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