CN110157721A - A tracer targeting plasmid of vaccinia virus Tiantan strain and preparation method thereof - Google Patents

Abstract

The invention discloses a tracer targeting plasmid of the Tiantan strain of vaccinia virus, comprising an upstream homologous recombination arm VTT‑TKL, a downstream homologous recombination arm VTT‑TKR, and an upstream homologous recombination arm VTT‑TKL of the TK gene of the vaccinia virus Tiantan strain. The Luciferase reporter gene and EGFP green fluorescent protein marker gene are inserted between the downstream homologous recombination arm VTT-TKR, and the Luciferase reporter gene and EGFP green fluorescent protein marker gene are expressed by the promoter P7.5. The invention also discloses a preparation method of the tracer targeting plasmid of the vaccinia virus Tiantan strain. The invention discloses a tracer targeting plasmid of the Tiantan strain of vaccinia virus, capable of targeting and removing the TK gene in the Tiantan strain of the vaccinia virus, and preparing a recombinant Tiantan strain virus with the TK gene removed.

Description

A tracer targeting plasmid of vaccinia virus Tiantan strain and preparation method thereof

technical field

The invention belongs to the technical field of biological genetic engineering, and relates to a tracer targeting plasmid of the Tiantan strain of vaccinia virus, and also relates to a preparation method of the tracer targeting plasmid of the Tiantan strain of vaccinia virus.

Background technique

Vaccinia virus Tian Tan (VTT) strain is a unique vaccine strain in my country. In order to improve the oncolytic efficacy of vaccinia virus Tian Tan strain VTT, the corresponding targeting plasmid is needed. Thymidine kinase TK can provide a high level of nucleotide pool for viral DNA replication, and the level of TK in normal cells is low. Removing the viral TK gene makes the virus more inclined to replicate in tumor cells and cannot replicate in normal cells earlier, so Removing the TK gene of VTT improves the tumor selectivity of the virus. At present, targeting plasmids targeting the Tiantan strain of vaccinia virus are mainly used for the prevention of infectious diseases, and are rarely used for anti-tumor applications. These targeting plasmids either lack a tracer reporter gene or a fluorescent selection marker, and the resulting recombinant VTT virus is either unable to be traced, or has a weak traceability, or is difficult to screen, so it cannot meet the requirements of the current attenuated oncolytic vaccinia virus. research needs.

Contents of the invention

The purpose of the present invention is to provide a tracer targeting plasmid of vaccinia virus Tiantan strain, which can target and remove the TK gene of vaccinia virus Tiantan strain, and make it carry a tracer marker gene, and prepare a recombination with traceability that removes the TK gene Tiantan strain virus.

Another object of the present invention is to provide a method for preparing a tracer targeting plasmid of vaccinia virus Tiantan strain.

The technical solution adopted in the present invention is a tracer targeting plasmid of the Tiantan strain of vaccinia virus, including the upstream homologous recombination arm VTT-TKL and the downstream homologous recombination arm VTT-TKR of the TK gene of the vaccinia virus Tiantan strain. The EGFP green fluorescent protein marker gene was inserted between the recombination arm VTT-TKL and the downstream homologous recombination arm VTT-TKR, and the expression of the EGFP green fluorescent protein marker gene was driven by the promoter P7.5.

The feature of the first technical solution of the present invention is also:

The targeting plasmid also includes a Luciferase reporter gene. The Luciferase reporter gene is also located between the upstream homologous recombination arm VTT-TKL and the downstream homologous recombination arm VTT-TKR. The Luciferase reporter gene is expressed by the promoter P7.5, and the nucleoside of the targeting plasmid The acid sequences are shown in Sequence Table 1.

The departure vector of the targeting plasmid is pMV-VTTTK-Luc.

Another kind of technical scheme that the present invention adopts is:

A method for preparing a tracer targeting plasmid of vaccinia virus Tiantan strain, specifically carried out according to the following steps:

Step 1, construct the fluorescent marker tracer gene fragment E-Luc fragment, the nucleotide sequence of the E-Luc fragment is as shown in Sequence Table 2;

In step 2, the E-Luc fragment was ligated into the pMV-VTTTK-Luc plasmid by restriction endonuclease ligation to obtain a tracer targeting plasmid of the Tiantan strain of vaccinia virus.

The feature of another kind of technical scheme of the present invention is also in:

Step 1 is specifically carried out as follows:

Step 1.1, using the pEGFP-Luc plasmid as a template, design primers according to the fusion sequence of the Luciferase reporter gene and the EGFP green fluorescent protein marker gene;

Step 1.2, adding a restriction site EcoRI at one end of the primer, and a restriction site XmaI at the other end of the primer to obtain a synthetic primer;

In step 1.3, the pEGFP-Luc plasmid is used as a template, and the sequence obtained in step 2 is used as a primer. After PCR amplification, the light-labeled tracer gene fragment E-Luc fragment is obtained.

The synthetic primers in step 1.2 are:

ELuc-F: 5'-GGAATTCGCCACCATGGAAGACGCCA-3'

ELuc-R: 5'-TCCCCCCGGGTTACTTGTACAGCTCGTCCATG-3.

In step 1.3, the PCR reaction system is: DNA 1 μl, dNTP mix (2.5mM) 1 μl, forward primer (10 μM) 2 μl, reverse primer (10 μM) 2 μl, 5×PrimeSTAR buffer 10 μl, PrimeSTAR HS DNA polymerase 0.5 μl, Add DEPC water to 50 μl.

The PCR reaction conditions are: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55-60°C for 15s, extension at 72°C for 1min/kb, cycled 30 times; extension at 72°C for 5 minutes, and finally stored at 4°C;

PCR fragment recovery: PCR products were subjected to agarose gel electrophoresis, and the E-Luc fragment was recovered by cutting the gel.

Step 2 is specifically carried out in accordance with the following steps:

Step 2.1, the pMV-VTTTK-Luc plasmid is digested with EcoRI/XmaI, and the large fragment is recovered by gel electrophoresis as a carrier;

The E-Luc fragment was subjected to EcoRI/XmaI double digestion, and then purified and recovered to obtain the ligated fragment;

In step 2.2, the vector and the ligated fragment are ligated and transformed to obtain the tracer targeting plasmid of the Tiantan strain of vaccinia virus.

In step 2.2, the vector and the connected fragments are connected and transformed according to the molecular cloning technique.

The beneficial effects of the present invention are:

The tracer targeting plasmid of a kind of vaccinia virus Tiantan strain of the present invention can be targeted to remove the TK gene in the vaccinia virus Tiantan strain, and prepare the recombinant Tiantan strain virus with traceability that removes the TK gene;

A tracer targeting plasmid of the Tiantan strain of vaccinia virus of the present invention, EGFP green fluorescent protein marker can be used to screen recombinant viruses; Luciferase reporter gene can generate bioluminescence by catalyzing fluorescein, and play a tracer function, which is used to analyze oncolytic VTT virus in The distribution in the body, especially in tumors, provides a good tool for vaccinia virus as a carrier to develop live vaccines and tumor biotherapy.

Description of drawings

Fig. 1 is the plasmid map of the tracer targeting plasmid of a kind of vaccinia virus Tiantan strain of the present invention;

Fig. 2 is the gel electrophoresis result of PCR amplification product of E-Luc fragment;

Fig. 3 is the structural representation of carrier pMV-VTTTK-Luc plasmid;

Fig. 4 is the enzyme digestion identification result of the tracer targeting plasmid of a kind of vaccinia virus Tiantan strain of the present invention;

Fig. 5-Fig. 8 is the sequence identification result of the tracer targeting plasmid of a kind of vaccinia virus Tiantan strain of the present invention.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

The materials and reagents used in the present invention can be obtained from commercial sources unless otherwise specified.

The genomic DNA sequence of the wild vaccinia virus Tiantan strain in the present invention is the sequence (VRL 14-FEB-2000) of GenBank number AF095689.1. The Tiantan strain of vaccinia virus was preserved in the Laboratory of Molecular Virology and Viral Immunology of Xi'an Medical College (this laboratory, the same below). Plasmids pEGFP-Luc and pMV-VTTTK-Luc are preserved by our laboratory.

One, a tracer targeting plasmid of the vaccinia virus Tiantan strain, as shown in Figure 1, includes the pMV-VTTTK-ELuc plasmid, and the pMV-VTTTK-Luc plasmid is inserted into the upstream homologous recombination arm VTT- Insert EGFP green fluorescent protein marker gene and Luciferase reporter gene between TKL and downstream homologous recombination arm VTT-TKR, upstream homologous recombination arm VTT-TKL and downstream homologous recombination arm VTT-TKR, EGFP green fluorescent protein marker gene and Luciferase reporter All the genes were expressed by vaccinia virus promoter P7.5.

A tracer targeting plasmid of the Tiantan strain of vaccinia virus of the present invention, the homologous arms VTT-TKL and VTT-TKR undergo homologous recombination with the sequences on both sides of the TK gene of VTT to form a recombination that knocks out the TK gene and expresses Luciferase and EGFP VTT virus. EGFP fluorescent labeling can be used to screen recombinant viruses; Luciferase catalyzes fluorescein to produce bioluminescence, and plays a tracing function to determine the distribution of viruses in vivo.

Two, a kind of preparation method of the tracer targeting plasmid of vaccinia virus Tiantan strain, carry out according to the following steps:

Step 1, constructing a fluorescently labeled tracer gene fragment E-Luc fragment;

Step 1.1, using the pEGFP-Luc plasmid as a template, according to the fusion sequence of Luciferase and EGFP fusion gene, design primers;

Step 1.2, add the enzyme cutting site EcoRI at one end of the primer, and the enzyme cutting site XmaI at the other end of the primer to obtain a synthetic primer. The sequence information of the synthetic primer is as follows:

ELuc-F: 5'-G GAATTC GCCACCATGGAAGACGCCA-3' (the part before the underline is the protected base, the underline part is the EcoRI endonuclease recognition sequence, and the sequence after that is the 1-19th position of sequence 2)

ELuc-R: 5'-TCCC CCCGGG TTACTTGTACAGCTCGTCCATG-3' (the part before the underline is the protective base, the underline part is the XmaI endonuclease recognition sequence, and the sequence thereafter is the reverse complementary sequence of the 2376-2397th position of sequence 2) ;

In step 1.3, the pEGFP-Luc plasmid is used as a template, and after PCR amplification, the light-labeled tracer gene fragment E-Luc fragment is obtained, and the gene sequence of the E-Luc fragment is shown in Sequence Table 2.

The PCR reaction system is: DNA 1μl, dNTP mix (2.5mM) 1μl, forward primer (10μM) 2μl, reverse primer (10μM) 2μl, 5×PrimeSTAR buffer 10μl, PrimeSTAR HS DNA polymerase 0.5μl, add DEPC water to 50 μl.

The PCR reaction conditions are: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 60°C for 15s, extension at 72°C for 1min/kb, and 30 cycles; extension at 72°C for 5 minutes, and finally stored at 4°C;

PCR fragment recovery: The PCR product was subjected to agarose gel electrophoresis, and the E-Luc with the enzyme cutting site was 2414bp. The electrophoresis result is shown in Figure 2, and the target fragment was recovered by cutting the gel.

Step 2. Ligate the E-Luc fragment into the pMV-VTTTK-Luc plasmid by enzyme-cut ligation to obtain the tracer targeting plasmid of the vaccinia virus Tiantan strain:

In step 2.1, the pMV-VTTTK-Luc plasmid shown in Figure 3 was digested with EcoRI/XmaI, and the 4197bp fragment was recovered by gel electrophoresis to obtain the vector;

The E-Luc fragment was subjected to EcoRI/XmaI double digestion, and the 2403bp fragment was purified and recovered to obtain the ligated fragment;

In step 2.2, the vector and the connecting fragment are ligated and transformed by molecular cloning techniques to obtain the tracer targeting plasmid of the Tiantan strain of vaccinia virus.

The tracer targeting plasmid of the vaccinia virus Tiantan strain obtained in this example was digested and identified, and the identification results are shown in Figure 4. The tracer targeting plasmid of the vaccinia virus Tiantan strain was digested with EcoRI/XmaI to produce a 4197bp fragment and a 2403bp fragment. Consistent with the expected fragment size;

The tracer targeting plasmid of the vaccinia virus Tiantan strain obtained in this embodiment is subjected to gene sequence determination, as shown in Fig. 5, Fig. 6, Fig. 7 and Fig. 8, as can be seen from Fig. 5, Fig. 6, Fig. 7 and Fig. 8: It showed that the gene sequence did not change, and the insertion position was correct; therefore, the tracer targeting plasmid of the vaccinia virus Tiantan strain was successfully constructed.

SEQUENCE LISTING

<110> Xi'an Medical College

<120> A tracer targeting plasmid of vaccinia virus Tiantan strain and its preparation method

<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 ttaatagttttatttgaagt 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 atggcccgc 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

cgattattct 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 gggtggtgc 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 tattatagatgg 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 ttatgtctc atgagcggat acatatttga atgtatttag 6600

<210> 2

<211> 2397

<212> DNA

<213> Artificial sequence

<400> 2

gccaccatgg aagacgccaa aaacataaag aaaggcccgg cgccaattcta 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

ctccaacaccc 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 agaacaccccc 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.