CN109750056A - Remove the target practice plasmid and preparation method thereof of the Temple of Heaven strain VGF gene - Google Patents
Remove the target practice plasmid and preparation method thereof of the Temple of Heaven strain VGF gene Download PDFInfo
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- CN109750056A CN109750056A CN201910012357.4A CN201910012357A CN109750056A CN 109750056 A CN109750056 A CN 109750056A CN 201910012357 A CN201910012357 A CN 201910012357A CN 109750056 A CN109750056 A CN 109750056A
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- vtt
- segment
- vgfr
- target practice
- egfp
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Abstract
The invention discloses a kind of target practice plasmids for removing the Temple of Heaven strain VGF gene, upstream homologous recombination arm VTT-VGFL and downstream homologous recombination arm VTT-VGFR including vaccinia virus Tiantan strain VGF gene, fluorescent marker gene EGFP is plugged between upstream homologous recombination arm VTT-VGFL and downstream homologous recombination arm VTT-VGFR, fluorescent marker gene EGFP is started by promoter P7.5 to express, and the nucleotide sequence of the fluorescent marker gene EGFP is as shown in sequence 4.The invention also discloses the preparation methods of the target practice plasmid of removal the Temple of Heaven strain VGF gene.The target practice plasmid of removal the Temple of Heaven strain VGF gene of the invention, can remove the VGF gene in Tiantan strain vaccinia virus, to prepare the attenuation Tiantan strain vaccinia virus of removal VGF gene.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, are related to a kind of target practice plasmid for removing the Temple of Heaven strain VGF gene, tool
Body is a kind of fluorescent marker target practice plasmid for removing the Temple of Heaven strain VGF gene, and the invention further relates to the removal the Temple of Heaven strain VGF genes
The preparation method of target practice plasmid.
Background technique
Tumor biotherapy technology is the completely new oncotherapy means of one kind of rising in recent years, has Small side effects, resists
The features such as tumor promotion height, wide adaptability.
Vaccinia virus (vaccinia virus, VACV) has natural taxis to tumour as oncolytic virus, optional
Infected tumor's cell of selecting property, and replicated in it, it will not be removed quickly by immune system.Vaccinia virus Tiantan strain (Tian
Tan strain, VTT) it is that the distinctive strain vaccinia virus in China need to be by phase in order to study and improve the oncolytic effect of VTT
VTT is transformed in the target practice plasmid answered.Viral growth factors (virus growth factor, VGF) are a kind of and epidermal growth factors
The homologous secreted protein of son can promote cell Proliferation in conjunction with the EGF-R ELISA of cell surface, this is to disease
Duplication is of great significance poison in the normal tissue, but not essential in tumour cell, and therefore, the VGF for removing VTT can increase
Strong tumor-selective improves Viral safety.For the VGF gene removal for realizing VTT, it would be desirable to for the target practice of VGF gene
Plasmid, but have no the target practice plasmid for VGF gene in VTT on the market at present.
Summary of the invention
The object of the present invention is to provide a kind of target practice plasmid pMV-VTTVGFE with removal the Temple of Heaven strain VGF gene, being capable of target
VGF gene into removal Tiantan strain vaccinia virus, to prepare the Tiantan strain vaccinia virus of removal VGF gene.
It is a further object to provide a kind of preparation methods of target practice plasmid for removing the Temple of Heaven strain VGF gene.
The first technical solution of the present invention is to remove the target practice plasmid of the Temple of Heaven strain VGF gene, including bovine vaccine is sick
The upstream homologous recombination arm VTT-VGFL and downstream homologous recombination arm VTT-VGFR of malicious the Temple of Heaven strain VGF gene, upstream homologous recombination
Be plugged with fluorescent marker gene EGFP between arm VTT-VGFL and downstream homologous recombination arm VTT-VGFR, fluorescent marker gene EGFP by
Promoter P7.5 starting expression.
The characteristics of the first technical solution of the invention, also resides in:
Also 1 multiple cloning sites between upstream homologous recombination arm VTT-VGFL and downstream homologous recombination arm VTT-VGFR, by
Promoter P11 starting expression.
The nucleotide sequence of fluorescent marker target practice plasmid is as shown in sequence 1.
The carrier that sets out of target practice plasmid is pMV-Pro, and the carrier that sets out contains Escherichia coli replication origin
PUCori and amicillin resistance selection markers.
The another technical solution that the present invention uses is:
Remove the preparation method of the target practice plasmid of the Temple of Heaven strain VGF gene, which is characterized in that specifically real according to the following steps
It applies:
Step 1, synthetic primer is separately designed, VTT-VGFR segment, VTT-VGFL segment and EGFP piece are obtained by PCR
Section, wherein the nucleotide sequence of VTT-VGFR is as shown in sequence table 2, the nucleotide sequence of VTT-VGFL segment such as 3 institute of sequence table
Show, the nucleotide sequence of fluorescent marker gene EGFP is as shown in sequence 4；
Step 2, VTT-VGFR segment, VTT-VGFR segment and EGFP segment are successively passed through to the conversion of digestion, connection
Method is connected on carrier pMV-Pro, obtains target practice plasmid.
The characteristics of another technical solution of the present invention, also resides in:
Using VTT virus as template in step 1, synthetic primer is designed, using VACV-VTT gene DNA as template, PCR is taken
VTT-VGFR segment with BamHI/KpnI restriction enzyme site and the VTT-VGFL segment for carrying PstI/HindIII restriction enzyme site；
Using p-EGFP-Luc plasmid as template, synthetic primer is designed, PCR, which is obtained, carries EcoRI/BamHI restriction enzyme site
EGFP segment；
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 archaeal dna polymerase of (10 μM) 2 μ l, 5 × PrimeSTAR buffer, 0.5 μ l, adds DEPC water to totality
50 μ l of product.
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 recycles: PCR product is through 1.5% agarose gel electrophoresis, VTT-VGFR 876bp, VTT-VGFL
857bp, EGFP 742bp, gel extraction purpose band obtain VTT-VGFR segment, VTT-VGFL segment and EGFP segment.
The amplimer of VTT-VGFR are as follows:
VTT-VGFR-F:5 '-CGGGATCCCCGGGTCTACAAGATATGGTTGTGCC-3’
VTT-VGFR-R:5 '-GGGGTACCGGAAACACCGATATGTGGAG-3'；
The amplimer of VTT-VGFL are as follows:
VTT-VGFL-F:5 '-AACTGCAGCCATTTGTCATTGTCTTAACCC-3’
VTT-VGFL-R:5 '-CCCAAGCTTCAGCGAACAACAACATCAGA-3'；
The amplimer of fluorescent marker gene EGFP are as follows:
EGFP-F:5 '-GGAATTCATGGTGAGCAAGGGCGA-3’
EGFP-R:5 '-CGCGGATCCAGATCTTTACTTGTACAGCTCGTCCATG-3’。
Step 2 is specific to be carried out as steps described below:
Step 2.1, by MV-PRO plasmid and VTT-VGFR segment after BamHI/KpnI double digestion purification and recovery, will return
The segment of receipts is attached, converts, interstitial granules pMV-VTTVGFR in acquisition；
Step 2.2, middle interstitial granules pMV-VTTVGFR and segment VTT-VGFL is pure after PstI/HindIII double digestion
Change recycling, the segment of recycling is attached, is converted, initial target practice plasmid pMV-VTTVGF is obtained；
Step 2.3, target practice plasmid pMV-VTTVGF and segment EGFP are purified back after EcoRI/BamHI double digestion
It receives, the segment of recycling is attached, is converted, obtain the target practice plasmid pMV-VTTVGFE for having fluorescent marker function.
The beneficial effects of the invention are as follows
The target practice plasmid of removal the Temple of Heaven strain VGF gene of the invention, can target the VGF in removal Tiantan strain vaccinia virus
Gene, to prepare the recombination Tiantan strain vaccinia virus of removal VGF gene；
The target practice plasmid of removal the Temple of Heaven strain VGF gene of the invention can be the Temple of Heaven strain containing fluorescent marker gene EGFP
Vaccinia virus increases tracking function, can analyze the distribution situation of oncolytic vaccinia virus in vivo, the distribution especially in tumour,
Live vaccine is researched and developed as carrier for vaccinia virus and tumor biotherapy provides good tool.
Detailed description of the invention
Fig. 1 be present invention removal the Temple of Heaven strain VGF gene target practice plasmid in set out the plasmid map of plasmid pMV-PRO；
Fig. 2 is the pcr amplified fragment of VTT-VGFR in the target practice plasmid of present invention removal the Temple of Heaven strain VGF gene；
Fig. 3 is the pcr amplified fragment of VTT-VGFL in the target practice plasmid of present invention removal the Temple of Heaven strain VGF gene；
Fig. 4 is the pcr amplified fragment of EGFP in the target practice plasmid of present invention removal the Temple of Heaven strain VGF gene；
Fig. 5 is the schematic diagram of the target practice plasmid of present invention removal the Temple of Heaven strain VGF gene
Fig. 6 is middle interstitial granules pMV-VTTVGFR in the preparation method of the target practice plasmid of present invention removal the Temple of Heaven strain VGF gene
Digestion qualification figure；
Fig. 7 present invention removes target practice plasmid pMV-VTTVGF in the preparation method of the target practice plasmid of the Temple of Heaven strain VGF gene
Digestion qualification figure；
Fig. 8 present invention, which removes, marks target practice plasmid pMV- in the preparation method of the target practice plasmid of the Temple of Heaven strain VGF gene
The digestion qualification figure of VTTVGFE.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
One, main agents and material:
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).
Wild-type vaccinia strain the Temple of Heaven strain in following embodiment is real in Xi'an Medical University's molecule virus and virological immunology
Test room (this laboratory, similarly hereinafter) preservation.PMV-PRO plasmid and p-EGFP-Luc plasmid are by this laboratory preservation.
Two, remove the synthesis of the target practice plasmid of the Temple of Heaven strain VGF gene:
The present invention is used to remove the target practice plasmid pMV-VTTVGFE building process of vaccinia virus Tiantan strain VGF gene: will be wild
7325-8179 (i.e. the positions 1-855 of sequence 2, be named as VTT-VGFR) of the DNA sequence dna of raw vaccinia virus Tiantan strain and the
Successively it is cloned into plasmid map pMV- as shown in Figure 1 for 6143-6960 (i.e. the position 1-840 of sequence 3, be named as VTT-VGFL)
PRO plasmid obtains the target practice plasmid pMV-VTTVGF comprising vaccinia virus Tiantan strain VGF gene left and right sides homologous recombination arm；?
It is inserted into EGFP gene sequence (i.e. the position 1-720 of sequence 4) between multiple cloning sites after P7.5 promoter, obtains target practice plasmid pMV-
VTTVGFE。
The synthesis for removing the target practice plasmid of the Temple of Heaven strain VGF gene, is specifically operated as steps described below:
Step 1, synthetic primer is separately designed, VTT-VGFR segment, VTT-VGFL segment and fluorescent marker are obtained by PCR
Gene EGFP segment:
Step 1.1, design of primers and synthesis
Expand the primer pair of VTT-VGFR are as follows:
VTT-VGFR-F:5 '-CGGGATCCCCGGGTCTACAAGATATGGTTGTGCC-3 ' (is partially before underscore
Base is protected, underscore part is BamHI endonuclease recognition sequence, and sequence is 1-21 of sequence 2 thereafter)
VTT-VGFR-R:5 '-GGGGTACC(part is protection alkali to GGAAACACCGATATGTGGAG-3 ' before underscore
Base, underscore part are KpnI endonuclease recognition sequence, and sequence is 836-855 reverse complementary sequences of sequence 2 thereafter)
Expand the primer pair of VTT-VGFL are as follows:
VTT-VGFL-F:5 '-AACTGCAG(part is protection to CCATTTGTCATTGTCTTAACCC-3 ' before underscore
Base, underscore part are PstI endonuclease recognition sequence, and sequence is 1-22 of sequence 3 thereafter)
VTT-VGFL-R:5 '-CCCAAGCTT(part is protection alkali to CAGCGAACAACAACATCAGA-3 ' before underscore
Base, underscore part are HindIII endonuclease recognition sequence, and sequence is 721-840 reverse complemental sequences of sequence 3 thereafter
Column)
Expand the primer pair of EGFP gene are as follows:
EGFP-F:5 '-GGAATTC(part is protection base, underscore to ATGGTGAGCAAGGGCGA-3 ' before underscore
Part is EcoRI endonuclease recognition sequence, and sequence is 1-17 of sequence 4 thereafter)
EGFP-R:5 '-CGCGGATCC(part is guarantor to AGATCTTTACTTGTACAGCTCGTCCATG-3 ' before underscore
Base is protected, underscore part is BamHI endonuclease recognition sequence, and sequence is 699-720 reverse complementals of sequence 4 thereafter
Sequence)
Step 1.2, the acquisition of VTT-VGFR segment, VTT-VGFL segment and EGFP segment:
A is extracted wild type VACV-VTT genomic DNA: being mentioned using the viral genome extracts kit of Biomiga company
Viral DNA is taken, is operated by the specification provided in kit；
B, using VACV-VTT genomic DNA as template, using VTT-VGFR-F/VTT-VGFR-R as primer, PCR is carried
The VTT-VGFR segment of BamHI/KpnI restriction enzyme site；Using VTT-VGFL-F/VTT-VGFL-R as primer, PCR is carried
The VTT-VGFL segment of PstI/HindIII restriction enzyme site；It is to draw with EGFP-F/EGFP-R using p-EGFP-Luc plasmid as template
Object, PCR obtain the EGFP segment for carrying EcoRI/BamHI restriction enzyme site；
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 archaeal dna polymerase of (10 μM) 2 μ l, 5 × PrimeSTAR buffer, 0.5 μ l, adds DEPC water to totality
50 μ l of product.
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；
Step 1.3, PCR fragment recycles: PCR product is through 1.5% agarose gel electrophoresis, as shown in Fig. 2, VTT-VGFR is
876bp；As shown in figure 3, VTT-VGFL is 857bp；As shown in figure 4, EGFP is 742bp；Gel extraction purpose band obtains core
Nucleotide sequence VTT-VGFR segment as shown in sequence table 2, nucleotide sequence VTT-VGFL segment and core as shown in sequence table 3
Nucleotide sequence EGFP segment as shown in sequence 4.
Step 2, the building of target practice plasmid pMV-VTTVGFE:
Step 2.1, by pMV-PRO plasmid and VTT-VGFR segment after BamHI/KpnI double digestion purification and recovery, will
The segment of recycling is attached, converts, interstitial granules pMV-VTTVGFR in acquisition；
Step 2.2, middle interstitial granules pMV-VTTVGFR and segment VTT-VGFL is pure after PstI/HindIII double digestion
Change recycling, the segment of recycling is attached, is converted, initial target practice plasmid pMV-VTTVGF is obtained；
Step 2.3, target plasmid pMV-VTTVGF and segment EGFP are purified back after EcoRI/BamHI double digestion
It receives, the segment of recycling is attached, is converted, the target practice plasmid pMV- with fluorescent marker function as shown in Figure 5 is obtained
VTTVGFE。
Three, the identification for the granulation that each step of step 2 constructs
The middle interstitial granules pMV-VTTVGFR that step 2.1 is obtained is identified through BamHI/KpnI double digestion, as shown in fig. 6, going out
Show two bar segment of 870bp and 2511bp, it is consistent with expection；
The initial target practice plasmid pMV-VTTVGF that step 2.2 is obtained is identified through PstI/HindIII double digestion, such as Fig. 7 institute
Show two bar segment of 842bp and 3367bp occur, it is consistent with expection；
The fluorescent marker target practice plasmid pMV-VTTVGFE that step 2.3 is obtained is through EcoRI/BamHI double digestion, such as Fig. 8 institute
Show two bar segment of 732bp and 4173bp occur, it is consistent with expection；
Therefore target practice plasmid pMV-VTTVGFE of the invention is constructed successfully.
<110>Xi'an Medical University
<120>the target practice plasmid and preparation method thereof of the Temple of Heaven strain VGF gene is removed
<130>nothing
<210>1
<211>4905
<212>DNA
<213>pMV-VTTVGFE
<400>1
AAAAATAAAC AAATAGGGGT TCCGCGCACA TTTCCCCGAA AAGTGCCACC TGACGTCTAA 60
GAAACCATTA TTATCATGAC ATTAACCTAT AAAAATAGGC GTATCACGAG GCCCTTTCGT 120
TGTAAAACGA CGGCCAGTCG AACCACGCAA TGCGTCTCGA TCCGCAGTGT CTTGCGTCTC 180
TTTACTGCAG CCATTTGTCA TTGTCTTAAC CCATTCGTTG ATTACTTCCT TTGTTTGGTT 240
AGCATTATTA AAGTTTACAG TTTGAAAATC GTCTTTTATT TTTTGTAGGA AGGAGGCGTG 300
GAACTCGATA CTATCGCTAC CGTATATTTT ATTTGCGGTA GCTAGTGTCG CACAATACGG 360
AATATCTACG TCCATGTCAT TATTGTCATC GGGTGTATTC TCATTCATAT TCTCTATATA 420
TTTTGATAGT TGTTCAGCTG TAGAACCAGC TGCTCCATGA TTTAGAATAG ATAAAGTAGA 480
TAAAATGGAA ACTGGAGAAA TCAAAACATT TTCATCAGGG TGTTTTACGA TTAGTTCTTT 540
AAAGATATCC ATGGTATAGA CCAAACAATA ACGATAACGA TATATATCAT AAATAAATAA 600
TGTTAAATTT CAGTTTATGT TTGTACCCCG TATTCATACT TAACAAATTG GTATTGCGTA 660
CACAATCAAT CATATTACAT ACCATTAATA ATGCAAGCAT AAAAAATCGT TAGTAGATGT 720
TTCTAAATAT AGGTTCCGTA AGCAAAGAAT ATAAGAATGA AGCGGTAATG ATAAAATCAA 780
TCGTTATCTA AAATGATCAT ACTCATTTAT TTTATTCTAT TATATTAACA CATACATTTT 840
TAACAGCAAC ACATTCAATA TTGTATTGTT ATTTTTATAT TATTTACACA ATTAACAATA 900
TATTATTAGT TTATATTACT GAATTAATAA TATAAAATTC CCAATCTTGT CATAAACACA 960
CACTGAGAAA CAGCATAAAC ACAAAATCCA TCAAAAATGT TGATAAATTA TCTGATGTTG 1020
TTGTTCGCTG AAGCTTCAAC TGTCCATGGG CCCGCGGCCG CTCGAGCATC TGGTAATTTA 1080
TAGCATAGAA AAAAACAAAA TGAAATTCTA CTATATTTTT ACATACATAT ATTCTAAATA 1140
TGAAAGTGGT GATTGTGACT AGCGTAGCAT CGCTCATCTA TATACTATAT AGTAATACCA 1200
ATAGAGCTCA AGACTACGAC TAGTAAACTG ATACAATCTC TTATATGTGG GTAATGTTCT 1260
CGATGTCGAT AGCCATATGC CCGGTAGTTG CGATATACAT AAACTGATCA CTAATTCCAA 1320
ACCCACCCGC TTTTTATAGT AAGTTTTTCA CCCATAAATA ATAAATACAA TGGAATTCAT 1380
GGTGAGCAAG GGCGAGGAGC TGTTCACCGG GGTGGTGCCC ATCCTGGTCG AGCTGGACGG 1440
CGACGTAAAC GGCCACAAGT TCAGCGTGTC CGGCGAGGGC GAGGGCGATG CCACCTACGG 1500
CAAGCTGACC CTGAAGTTCA TCTGCACCAC CGGCAAGCTG CCCGTGCCCT GGCCCACCCT 1560
CGTGACCACC CTGACCTACG GCGTGCAGTG CTTCAGCCGC TACCCCGACC ACATGAAGCA 1620
GCACGACTTC TTCAAGTCCG CCATGCCCGA AGGCTACGTC CAGGAGCGCA CCATCTTCTT 1680
CAAGGACGAC GGCAACTACA AGACCCGCGC CGAGGTGAAG TTCGAGGGCG ACACCCTGGT 1740
GAACCGCATC GAGCTGAAGG GCATCGACTT CAAGGAGGAC GGCAACATCC TGGGGCACAA 1800
GCTGGAGTAC AACTACAACA GCCACAACGT CTATATCATG GCCGACAAGC AGAAGAACGG 1860
CATCAAGGTG AACTTCAAGA TCCGCCACAA CATCGAGGAC GGCAGCGTGC AGCTCGCCGA 1920
CCACTACCAG CAGAACACCC CCATCGGCGA CGGCCCCGTG CTGCTGCCCG ACAACCACTA 1980
CCTGAGCACC CAGTCCGCCC TGAGCAAAGA CCCCAACGAG AAGCGCGATC ACATGGTCCT 2040
GCTGGAGTTC GTGACCGCCG CCGGGATCAC TCTCGGCATG GACGAGCTGT ACAAGTAAAG 2100
ATCTGGATCC CCGGGTCTAC AAGATATGGT TGTGCCATAA TTTTTATAAA TTTTTTTATG 2160
AGTATTTTTA CAAAAAAAAT GTATAAAGTG TATGTCTTAT GTATATTTAT AAAAATGCTA 2220
AGTATGCGAT GTATCTATGT TATTTGTATT TATCTAAACA ATACCTCTAC CTCTAGATAT 2280
TATACAAAAA TTTTTTATTT CAGCATATTA AAGTAAAATC TAGTTACCTT GAAAATGAAT 2340
ACAGTGGGTG GTTCCGTATC ACCAGTAAGA ACATAATAGT CGAATACAGT ATCCGATTGA 2400
GATTTTGCAT ACAATACTAG TCTAGAAAGA AATTTGTAAT CATCTTCTGT GACGGGAGTC 2460
CATATATCTG TATCATCGTC TAGTTTATCA GTGTCCCATG CTATATTCCT GTTATCATCA 2520
TTAGTTAATG AAAATAACTC TCGTGCTTCA GAAAAGTCAA ATATTGTATC CATACATACA 2580
TCTCCAAAAC TATCGCTTAT ACGTTTATCT TTAACGATAC CTATACCTAG ATGGTTATTT 2640
ACTAACAGAC ATTTTCCAGA TCTATTGACT ATAACTCCTA TAGTTTCCAC ATCAACCAAG 2700
TAATGATCAT CTATTGTTAT ATAACAATAA CATAACTCTT TTCCATTTTT ATCAGTATGT 2760
ATATCTATAT CAACGTCGTC GTTGTAGTGA ATAGTAGTCA TTGATCTATT ATATGAAACG 2820
GATATGTCTA GAACGGCAAT TGTCTTACGT CCAGTTAACA CTTTCGTTGA TTTAAAGTCT 2880
AGAGTCTTTG CAAACATAAT ATCCTTATCC GACTTTATAT TTCCTGTAGG GTGGTATAAT 2940
TTTATTTTGC CTCCACATAT CGGTGTTTCC GGTACCTAAA GAGACGGAGT CACTGCCAAC 3000
CGAGACGGTC ATAGCTGTTT CCTGTGTGCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG 3060
GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTACCCACA 3120
GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC 3180
CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC 3240
AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG 3300
TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC 3360
CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG CTGTAGGTAT 3420
CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG 3480
CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC 3540
TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT 3600
GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT 3660
ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC 3720
AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA 3780
AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC 3840
GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC 3900
CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT 3960
GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA 4020
TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT 4080
GGCCCCAGTG CTGCAATAAT ACCGCGGGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA 4140
ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC 4200
ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG 4260
CGCAACGTTG TTGCCATCGC TACAGGCATC GTGGTATCAC GCTCGTCGTT TGGTATGGCT 4320
TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGCGCAAA 4380
AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCCGTGTTA 4440
TCACTCATGG TTATGGCAGC ACTACATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC 4500
TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG 4560
AGTTGCTCTT GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA 4620
GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG 4680
AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC 4740
ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG 4800
GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT 4860
CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAG 4905
<210>2
<211>855
<212>DNA
<213>VTT-VGFR
<400>2
TCTACAAGAT ATGGTTGTGC CATAATTTTT ATAAATTTTT TTATGAGTAT TTTTACAAAA 60
AAAATGTATA AAGTGTATGT CTTATGTATA TTTATAAAAA TGCTAAGTAT GCGATGTATC 120
TATGTTATTT GTATTTATCT AAACAATACC TCTACCTCTA GATATTATAC AAAAATTTTT 180
TATTTCGGCA TATTAAAGTA AAATCTAGTT ACCTTGAAAA TGAATACAGT GGGTGGTTCC 240
GTATCACCAG TAAGAACATA ATAGTCGAAT ACAGTATCCG ATTGAGATTT TGCATACAAT 300
ACTAGTCTAG AAAGAAATTT GTAATCATTT TCTGTGACGG GAGTCCATAT ATCTGTATCA 360
TCGTCTAGTT TATCAGTGTC CCATGCTATA TTCCTGTTAT CATCATTAGT TAATGAAAAT 420
AACTCTCGTG CTTCAGAAAA GTCAAATATT GTATCCATAC ATACATCTCC AAAACTATCG 480
CTTATACGTT TATCTTTAAC GATACCTATA CCTAGATGGT TATTTACTAA CAGACATTTT 540
CCAGATCTAT TGACTATAAC TCCTATAGTT TCCACATCAA CCAAGTAATG ATCATCTATT 600
GTTATATAAC AATAACATAA CTCTTTTCCA TTTTTATCAG TATGTATATC TATATCAACG 660
TCGTCGTTGT AGTGAATAGT AGTCATTGAT CTATTATATG AAACGGATAT GTCTAGAACG 720
GCAATTGTCT TACGTCCAGT TAACACTTTC GTTGATTTAA AGTCTAGAGT CTTTGCAAAC 780
ATAATATCCT TATCCGACTT TATATTTCCT GTAGGGTGGT ATAATTTTAT TTTGCCTCCA 840
CATATCGGTG TTTCC 855
<210>3
<211>840
<212>DNA
<213>VTT-VGFL
<400>3
CCATTTGTCA TTGTCTTAAC CCATTCGTTG ATTACTTCCT TTGTTTGGTT AGCATTATTA 60
AAGTTTACAG TTTGAAAATC GTCTTTTATT TTTTGTAGGA AGGAGGCGTG GAACTCGATA 120
CTATCGCTAC CGTATATTTT ATTTGCGGTA GCTAGTGTCG CACAATACGG AATATCTACG 180
TCCATGTCAT TATTGTCATC GGGTGTATTC TCATTCATAT TCTCTATATA TTTTGATAGT 240
TGTTCAGCTG TAGAACCAGC TGCTCCATGA TTTAGAATAG ATAAAGTAGA TAAAATGGAA 300
ACTGGAGAAA TCAAAACATT TTCATCAGGG TGTTTTACGA TTAGTTCTTT AAAGATATCC 360
ATGGTATAGA CCAAACAATA ACGATAACGA TATATATCAT AAATAAATAA TGTTAAATTT 420
CAGTTTATGT TTGTACCCCG TATTCATACT TAACAAATTG GTATTGCGTA CACAATCAAT 480
CATATTACAT ACCATTAATA ATGCAAGCAT AAAAAATCGT TAGTAGATGT TTCTAAATAT 540
AGGTTCCGTA AGCAAAGAAT ATAAGAATGA AGCGGTAATG ATAAAATCAA TCGTTATCTA 600
AAATGATCAT ACTCATTTAT TTTATTCTAT TATATTAACA CATACATTTT TAACAGCAAC 660
ACATTCAATA TTGTATTGTT ATTTTTATAT TATTTACACA ATTAACAATA TATTATTAGT 720
TTATATTACT GAATTAATAA TATAAAATTC CCAATCTTGT CATAAACACA CACTGAGAAA 780
CAGCATAAAC ACAAAATCCA TCAAAAATGT TGATAAATTA TCTGATGTTG TTGTTCGCTG 840
<210>4
<211>720
<21 2>DNA
<213>EGFP
<400>4
ATGGTGAGCA AGGGCGAGGA GCTGTTCACC GGGGTGGTGC CCATCCTGGT CGAGCTGGAC 60
GGCGACGTAA ACGGCCACAA GTTCAGCGTG TCCGGCGAGG GCGAGGGCGA TGCCACCTAC 120
GGCAAGCTGA CCCTGAAGTT CATCTGCACC ACCGGCAAGC TGCCCGTGCC CTGGCCCACC 180
CTCGTGACCA CCCTGACCTA CGGCGTGCAG TGCTTCAGCC GCTACCCCGA CCACATGAAG 240
CAGCACGACT TCTTCAAGTC CGCCATGCCC GAAGGCTACG TCCAGGAGCG CACCATCTTC 300
TTCAAGGACG ACGGCAACTA CAAGACCCGC GCCGAGGTGA AGTTCGAGGG CGACACCCTG 360
GTGAACCGCA TCGAGCTGAA GGGCATCGAC TTCAAGGAGG ACGGCAACAT CCTGGGGCAC 420
AAGCTGGAGT ACAACTACAA CAGCCACAAC GTCTATATCA TGGCCGACAA GCAGAAGAAC 480
GGCATCAAGG TGAACTTCAA GATCCGCCAC AACATCGAGG ACGGCAGCGT GCAGCTCGCC 540
GACCACTACC AGCAGAACAC CCCCATCGGC GACGGCCCCG TGCTGCTGCC CGACAACCAC 600
TACCTGAGCA CCCAGTCCGC CCTGAGCAAA GACCCCAACG AGAAGCGCGA TCACATGGTC 660
CTGCTGGAGT TCGTGACCGC CGCCGGGATC ACTCTCGGCA TGGACGAGCT GTACAAGTAA 720
Claims (8)
1. removing the target practice plasmid of the Temple of Heaven strain VGF gene, which is characterized in that the upstream including vaccinia virus Tiantan strain VGF gene
Homologous recombination arm VTT-VGFL and downstream homologous recombination arm VTT-VGFR, the upstream homologous recombination arm VTT-VGFL and downstream are same
It is plugged with fluorescent marker gene EGFP between source recombination arm VTT-VGFR, the fluorescent marker gene EGFP is started by promoter P7.5
Expression.
2. the target practice plasmid of removal the Temple of Heaven strain VGF gene according to claim 1, which is characterized in that the upstream is homologous
There are also 1 multiple cloning sites between recombination arm VTT-VGFL and downstream homologous recombination arm VTT-VGFR, start table by promoter P11
It reaches.
3. the target practice plasmid of removal the Temple of Heaven strain VGF gene according to claim 2, which is characterized in that the fluorescent marker
The nucleotide sequence of target practice plasmid is as shown in sequence 1.
4. the target practice plasmid of removal the Temple of Heaven strain VGF gene according to claim 1, which is characterized in that the target practice plasmid
The carrier that sets out be pMV-Pro, it is anti-that the carrier that sets out contains Escherichia coli replication origin pUCori and ampicillin
Property selection markers.
5. removing the preparation method of the target practice plasmid of the Temple of Heaven strain VGF gene, which is characterized in that be specifically implemented according to the following steps:
Step 1, synthetic primer is separately designed, VTT-VGFR segment, VTT-VGFL segment and fluorescent marker gene are obtained by PCR
EGFP segment, wherein the nucleotide sequence of VTT-VGFR segment is as shown in sequence table 2, and the nucleotide sequence of VTT-VGFL segment is such as
Shown in sequence table 3, the nucleotide sequence of fluorescent marker gene EGFP segment is as shown in sequence 4；
Step 2, by VTT-VGFR segment, VTT-VGFR segment and fluorescent marker gene EGFP segment successively pass through digestion, connection,
The method of conversion is connected on carrier pMV-Pro, obtains target practice plasmid.
6. the preparation method of the target practice plasmid of removal the Temple of Heaven strain VGF gene according to claim 5, which is characterized in that institute
It states using VTT virus as template in step 1, designs synthetic primer, using VACV-VTT gene DNA as template, PCR is carried
The VTT-VGFR segment of BamHI/KpnI restriction enzyme site and the VTT-VGFL segment for carrying PstI/HindIII restriction enzyme site；
Using p-EGFP-Luc plasmid as template, synthetic primer is designed, PCR obtains the EGFP for carrying EcoRI/BamHI restriction enzyme site
Segment；
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 μM)
2 μ l, 5 × PrimeSTAR buffer, 10 μ l, PrimeSTAR HS archaeal dna polymerase, 0.5 μ l, adds DEPC water to 50 μ of total volume
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: PCR product is through 1.5% agarose gel electrophoresis, VTT-VGFR 876bp, VTT-VGFL 857bp,
EGFP is 742bp, and gel extraction purpose band obtains VTT-VGFR segment, VTT-VGFL segment and EGFP segment.
7. the preparation method of the target practice plasmid according to claim 6 except the Temple of Heaven strain VGF gene, which is characterized in that
Expand the primer of VTT-VGFR are as follows:
VTT-VGFR-F:5 '-CGGGATCCCCGGGTCTACAAGATATGGTTGTGCC-3’
VTT-VGFR-R:5 '-GGGGTACCGGAAACACCGATATGTGGAG-3'；
Expand the primer of VTT-VGFL are as follows:
VTT-VGFL-F:5 '-AACTGCAGCCATTTGTCATTGTCTTAACCC-3’
VTT-VGFL-R:5 '-CCCAAGCTTCAGCGAACAACAACATCAGA-3'；
The primer of amplification fluorescent marker gene EGFP are as follows:
EGFP-F:5 '-GGAATTCATGGTGAGCAAGGGCGA-3’
EGFP-R:5 '-CGCGGATCCAGATCTTTACTTGTACAGCTCGTCCATG-3’。
8. the preparation method of the target practice plasmid according to the removal the Temple of Heaven strain VGF gene described in claim 5, which is characterized in that described
Step 2 is specific to be carried out as steps described below:
Step 2.1, by MV-PRO segment and VTT-VGFR segment after BamHI/KpnI double digestion purification and recovery, by recycling
Segment is attached, converts, interstitial granules pMV-VTTVGFR in acquisition；
Step 2.2, the middle interstitial granules pMV-VTTVGFR and VTT-VGFL segment is pure after PstI/HindIII double digestion
Change recycling, the segment of recycling is attached, is converted, initial target practice plasmid pMV-VTTVGF is obtained；
Step 2.3, initial target practice plasmid pMV-VTTVGF and the EGFP segment is pure after EcoRI/BamHI double digestion
Change recycling, the segment of recycling is attached, is converted, obtains the target practice plasmid pMV-VTTVGFE for having fluorescent marker function.
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