CN107604004A - Tracer target practice plasmid for vaccinia virus Tiantan strain TK genes and preparation method thereof - Google Patents

Abstract

Tracer target practice plasmid disclosed by the invention for vaccinia virus Tiantan strain TK genes, including the carrier pMV that sets out, the upstream homologous recombination arm VTT TKL and downstream homologous recombination arm VTT TKR of vaccinia virus Tiantan strain TK genes, there is vaccinia virus early late phase promoter P11 and late promoter P7.5 between VTT TKL and VTT TKR, catenation sequence between promoter P11 and promoter P7.5 has two restriction enzyme sites, promoter P11 downstream and promoter P7.5 downstream include 1 multiple cloning sites, tracer gene Luciferase is inserted at multiple cloning sites after promoter P7.5, the nucleotide sequence of the tracer target practice plasmid is as shown in sequence 1.The tracer target practice plasmid of the present invention, solve the problems, such as that existing vaccinia virus recombinant can not analyze the distribution situation of virus in vivo.Present invention also offers the preparation method of above-mentioned tracer target practice plasmid.

Description

Tracer target practice plasmid for vaccinia virus Tiantan strain TK genes and preparation method thereof

Technical field

The invention belongs to technical field of biological genetic engineering, and in particular to a kind of for vaccinia virus Tiantan strain TK genes Tracer target practice plasmid, the invention further relates to the preparation method of the tracer target practice plasmid for being used for vaccinia virus Tiantan strain TK genes.

Background technology

Vaccinia virus (vaccinia virus, VACV) belongs to Poxviridae, orthopoxvirus, is a kind of tunicary double Chain DNA virus, its genome are up to 200kbp.Carriers or oncolytic virus of the VACV as vaccine, compared to other viral vectors such as Retrovirus, human herpes simplex vicus and adenovirus etc., there are many unique advantages.And VACV exterminates smallpox as the mankind Vaccine, be up to century more than one in the application of human body, its security and validity are sufficiently verified in practice.Cause This, VACV is considered as comparatively ideal oncolytic virus carrier.

Vaccinia virus Tiantan strain (Vaccinia virus Tian Tan, VACV-VTT) is the distinctive Strain in China, by In region Slurry pump, the vaccinia virus for oncolytic virus transformation mainly includes：WR strains (Western reserve), Wei Family name's strain (Wyeth or NYCBOH), Liszt's strain (Lister) and Copenhagen strain (Copenhagen), for the molten of VACV-VTT The research of knurl effect is still few.In order to study and improve VACV-VTT oncolytic effect, need to be transformed by corresponding target practice plasmid VACV-VTT.At present, more than 20 kinds that China has been built are mainly used in infection prevention for the target practice plasmid of vaccinia virus Tiantan strain Property disease, and the recombinant virus built with this lacks tracking function, can not analyze the distribution situation of virus in vivo, especially exists Distribution in tumour, the needs of current attenuation oncolytic virus research can not be met.

The content of the invention

It is an object of the invention to provide a kind of tracer target practice plasmid for vaccinia virus Tiantan strain TK genes, solve Existing vaccinia virus recombinant can not analyze the distribution situation problem of virus in vivo.

Present invention also offers the preparation method of the above-mentioned tracer target practice plasmid for vaccinia virus Tiantan strain TK genes.

The first technical scheme of the present invention is：Tracer target practice matter for vaccinia virus Tiantan strain TK genes Grain, include the upstream homologous recombination arm VTT-TKL and downstream homologous recombination of the carrier pMV that sets out, vaccinia virus Tiantan strain TK genes Arm VTT-TKR, there is vaccinia virus early late phase promoter P11 and late promoter P7.5 between VTT-TKL and VTT-TKR, open Catenation sequence between mover P11 and promoter P7.5 has two restriction enzyme sites, under promoter P11 downstream and promoter P7.5 Swim and include 1 multiple cloning sites (Multiple cloning site, MCS), inserted at the multiple cloning sites after promoter P7.5 Enter tracer gene Luciferase, the nucleotide sequence of the tracer target practice plasmid is as shown in sequence 1.

The characteristics of the first technical scheme of the invention, also resides in,

The carrier pMV that sets out includes Escherichia coli replication origin pUC ori and amicillin resistance selection markers.

Promoter P11 and the order of promoter P7.5 promotor genes expression are on the contrary, promoter P11 be reverse starting, startup Sub- P7.5 starts to be positive.

Second of technical scheme of the present invention be：Tracer target practice plasmid for vaccinia virus Tiantan strain TK genes Preparation method, specifically implement according to following steps：

Step 1：Engineer and synthetic promoter P7.5 and promoter P11 sequences and two multiple cloning sites sequences, and It is cloned into pMV carriers, forms pMV-PRO plasmids；Synthetic primer is designed, PCR obtains upstream homologous recombination arm VTT-TKL with Swim homologous recombination arm VTT-TKR and tracer gene Luciferase fragments；

Step 2：By the pMV-PRO plasmids obtained in step 1 and VTT-TKR fragments respectively after BamHI/KpnI double digestions Purifying recovery, the fragment of recovery is attached, converted, interstitial granules pMV-VTTTKR in acquisition；

Step 3：The VTT-TKL fragments obtained in the pMV-VTTTKR plasmids and step 1 that are obtained in step 2 are passed through respectively Recovery is purified after PstI/SalI double digestions, the fragment of recovery is attached, converted, obtains target practice plasmid pMV-VTTTK；

Step 4：The Luciferase fragments obtained in the pMV-VTTTK plasmids and step 1 that are obtained in step 3 are passed through respectively Recovery is purified after EcoRI/BamHI double digestions, the fragment of recovery is attached, converted, obtains tracer target practice plasmid pMV- VTTTK-Luc。

The characteristics of second of technical scheme of the invention, also resides in,

The primer that synthesis is designed in step 1 specifically includes following three pairs of primers：

Expand VTT-TKR primer pair：

Forward primer VTT-TKR-F：5’-CGGGATCCGGCTTCCTTTTCTAAACGATTGG-3’；

Reverse primer VTT-TKR-R：5’-GGGGTACCGAGTCAGTCTCATGTTCTCACCG-3’；

Expand VTT-TKL primer pair：

Forward primer VTT-TKL-F：5’-AACTGCAGACAGATATTCCGACAAAGGATTG-3’；

Reverse primer VTT-TKL-R：5’-ACGCGTCGACATCAACTGAATATGTCCGCCG-3’；

Expand the primer pair of Luciferase genes：

Luc-F：5’-GGAATTCGCCACCATGGAAGACGCCA-3’；

Luc-R：5’-CGGGATCCTTACACGGCGATCTTTCCGC-3’。

Upstream homologous recombination arm VTT-TKL and downstream homologous recombination arm VTT-TKR and tracer gene are obtained in step 1 Luciferase's comprises the following steps that：

1) wild type VACV-VTT genomic DNAs are extracted；

2) using VACV-VTT genomic DNAs template, carried by primer PCR of VTT-TKR-F/VTT-TKR-R The VTT-TKR of BamHI/KpnI restriction enzyme sites, obtained using VTT-TKL-F/VTT-TKL-R as primer PCR and carry PstI/SalI enzymes The VTT-TKL of enzyme site；Using p-EGFP-Luc plasmids as template, obtained using Luc-F/Luc-R as primer PCR and carry EcoRI/ The Luciferase genetic fragments of BamHI restriction enzyme sites；

3) PCR primer obtains upstream homologous recombination arm VTT-TKL and downstream respectively through agarose gel electrophoresis and glue reclaim Homologous recombination arm VTT-TKR and tracer gene Luciferase fragments.

The invention has the advantages that

(1) tracer target practice plasmid of the present invention includes Luciferase genes, the TK bases of targeting knock out vaccinia virus Tiantan strain The homologous recombination arm of cause, the vaccinia virus recombinant the Temple of Heaven strain of the attenuation with traceability can be prepared, and TK gene delections can For recombinant celo virus；

(2) tracer target practice plasmid of the present invention includes the distinctive early late phase promoter and late promoter of bovine vaccine disease, not It is different with the expression quantity of the foreign gene under promoter, can meet the needs of different foreign gene requirements are different, two promoters Between restriction enzyme site can be inserted into new expression casette be used for express more foreign genes；

(3) the vaccinia virus recombinant the Temple of Heaven strain that tracer target practice plasmid of the present invention is prepared, can analyze oncolytic vaccinia virus and exist Internal distribution situation, the distribution especially in tumour, it is that vaccinia virus researches and develops live vaccine and tumor biotherapy as carrier Provide good tool.

Brief description of the drawings

Fig. 1 is the schematic diagram of the tracer target practice plasmid for vaccinia virus Tiantan strain TK genes of the present invention；

Fig. 2 is promoter and polyclonal position in the tracer target practice plasmid for vaccinia virus Tiantan strain TK genes of the invention The schematic diagram of point；

Fig. 3 is the construction step schematic diagram of the tracer target practice plasmid for vaccinia virus Tiantan strain TK genes of the present invention；

Fig. 4 is the digestion qualification figure of the tracer target practice plasmid for vaccinia virus Tiantan strain TK genes of the present invention.

Embodiment

Below in conjunction with the accompanying drawings and embodiment the present invention is described in detail.It is it should be appreciated that described herein Specific embodiment only to explain the present invention, be not intended to limit the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

Material, reagent used in following embodiments, unless otherwise specified, are commercially obtained.

The genomic dna sequence of wild vaccinia virus Tiantan strain is No. GenBank sequence for being AF095689.1 in the present invention Arrange (VRL 14-FEB-2000).

Wild-type vaccinia strain the Temple of Heaven strain in following examples is real in Xi'an Medical University's molecule virus and virological immunology Test room (this laboratory, similarly hereinafter) preservation.Plasmid p-EGFP-Luc is by this Laboratories Accession.Plasmid pMV is carried by Huada gene company For.

The invention provides a kind of tracer target practice plasmid for vaccinia virus Tiantan strain TK genes, its ideograph such as Fig. 1 Shown, its sequence is shown in sequence 1, including the carrier pMV that sets out, and it includes Escherichia coli replication origin pUC ori and ammonia benzyl is blue or green Chloramphenicol resistance selection markers, the upstream homologous recombination of propagation and screening, vaccinia virus Tiantan strain TK genes for recombinant plasmid Arm VTT-TKL and the TK genes both sides of downstream homologous recombination arm VTT-TKR, VTT-TKL and VTT-TKR and vaccinia virus Tiantan strain Sequence homology, can target remove vaccinia virus Tiantan strain TK genes, have vaccinia virus between VTT-TKL and VTT-TKR Early late phase promoter P11 and late promoter P7.5, the catenation sequence between promoter P11 and promoter P7.5 have two digestions Site (MCS1 and MCS2), new expression cassette, promoter P11 downstream and promoter P7.5 downstream Jun Bao can be added here Containing 1 multiple cloning sites, tracer gene Luciferase, Luciferase base is inserted at the multiple cloning sites after promoter P7.5 Reason P7.5 starts expression, recombinant virus is had traceability, other foreign genes is can be inserted into after promoter P11, for carrying The Suppressive effect of high vaccinia virus Tiantan strain, Luciferase genetic fragments can be plugged under late promoter P7.5, can also insert Enter under early late phase P11 promoters, line replacement can be entered according to different foreign gene demands difference.

Promoter P11 and the order of promoter P7.5 promotor genes expression are on the contrary, promoter P11 be reverse starting, startup Sub- P7.5 starts to be positive；The restriction enzyme site that 2 multiple cloning sites in above-mentioned promoter downstream include is different.

The present invention is used for the tracer target practice plasmid pMV-VTTTK-Luc building process of vaccinia virus Tiantan strain TK genes：People Promoter P11, P7.5 and multiple cloning sites sequence (i.e. the 1-417 positions of sequence 2, name that work synthesis tracer target practice plasmid includes For P＆MCS) and be inserted into the carrier pMV that sets out, form pMV-PRO plasmids；By the DNA sequence dna of wild vaccinia virus Tiantan strain 80725-81559 positions (i.e. the 1-835 positions of sequence 3, be named as VTT-TKR) and 79441-80319 positions (the i.e. 1- of sequence 4 879, it is named as VTT-TKL) pMV-PRO plasmids are successively cloned into, obtain including vaccinia virus Tiantan strain TK gene left and right sides The target practice plasmid pMV-VTTTK of homologous recombination arm；Luciferase genes are inserted between multiple cloning sites after P7.5 promoters Sequence (i.e. the 1-1659 positions of sequence 5, be named as Lucifefase, abbreviation Luc), obtain tracer target practice plasmid pMV-VTTTK- Luc。

Concrete operations are as follows：

1. engineer and synthetic promoter and multiple cloning sites sequence

Vaccinia virus early late phase promoter P11 and late promoter P7.5 is selected, and screens suitable restriction endonuclease identification sequence Row form multiple cloning sites (MCS).Promoter P11 is reverse starting, and promoter P7.5 starts to be positive, respectively positioned at people's process The both ends of row, MCS1 are located at P11 downstream, and MCS2 is located at P7.5 downstream, designs catenation sequence between two promoters, make two Kept at a distance between promoter, restriction enzyme site SacI and SpeI are designed with catenation sequence, send the artificial sequence of design to Hua Da base Because company is synthesized, and it is cloned into pMV carriers, forms pMV-PRO plasmids.What initiator sequence and multiple cloning sites included Restriction enzyme site is as shown in Figure 2.

2. design of primers and synthesis

(1) VTT-TKR primer pair is expanded：

Forward primer VTT-TKR-F：5’-CGGGATCCThe GGCTTCCTTTTCTAAACGATTGG-3 ' (front portions of underscore It is divided into protection base, underscore part is BamHI endonuclease recognition sequences, and sequence is the 1-23 positions of sequence 3 thereafter)

Reverse primer VTT-TKR-R：5’-GGGGTACCThe GAGTCAGTCTCATGTTCTCACCG-3 ' (front portions of underscore It is divided into protection base, underscore part is KpnI endonuclease recognition sequences, and sequence is the anti-of the 813-835 positions of sequence 3 thereafter To complementary series)

(2) VTT-TKL primer pair is expanded：

Forward primer VTT-TKL-F：5’-AACTGCAGThe ACAGATATTCCGACAAAGGATTG-3 ' (front portions of underscore It is divided into protection base, underscore part is PstI endonuclease recognition sequences, and sequence is the 1-23 positions of sequence 4 thereafter)

Reverse primer VTT-TKL-R：5’-ACGCGTCGACThe ATCAACTGAATATGTCCGCCG-3 ' (front portions of underscore It is divided into protection base, underscore part is SalI endonuclease recognition sequences, and sequence is the anti-of the 759-879 positions of sequence 4 thereafter To complementary series)

(3) primer pair of Luciferase genes is expanded：

Luc-F：5’-GGAATTC(forward part of underscore is protection base to GCCACCATGGAAGACGCCA-3 ', lower stroke Line part is EcoRI endonuclease recognition sequences, and sequence is the 1-19 positions of sequence 5 thereafter)

Luc-R：5’-CGGGATCC(forward part of underscore is protection alkali to CGGGTTACACGGCGATCTTTCCGC-3 ' Base, underscore part are BamHI endonuclease recognition sequences, and sequence is the reverse complemental sequence of the 1640-1659 positions of sequence 5 thereafter Row)

3. the acquisition of homologous recombination arm VTT-TKR and VTT-TKL and tracer gene Luciferase DNA fragmentation

(1) wild type VACV-VTT genomic DNAs are extracted：Using the viral genome extracts kit of Biomiga companies Viral DNA is extracted, is operated by the specification provided in kit；

(2) using VACV-VTT genomic DNAs template, carried by primer PCR of VTT-TKR-F/VTT-TKR-R The VTT-TKR of BamHI/KpnI restriction enzyme sites, obtained using VTT-TKL-F/VTT-TKL-R as primer PCR and carry PstI/SalI enzymes The VTT-TKL of enzyme site；Using p-EGFP-Luc plasmids as template, obtained using Luc-F/Luc-R as primer PCR and carry EcoRI/ The Luciferase genetic fragments of BamHI restriction enzyme sites；

PCR reaction systems are：DNA 1 μ l, dNTP mix (2.5mM) 1 μ l, the μ l of forward primer (10 μM) 2, reverse primer The μ l of 10 μ l, PrimeSTAR HS archaeal dna polymerases of (10 μM) 2 μ l, 5 × PrimeSTAR buffer 0.5, add DEPC water to totality 50 μ l of product.

PCR reaction conditions are：98 DEG C of pre-degeneration 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；

(3) PCR fragment reclaims：For PCR primer through 1.5% agarose gel electrophoresis, it is 851bp, VTT- to see figure VTT-TKR TKL is 897bp, Luciferase 1678bp, gel extraction purpose band, obtains VTT-TKR, VTT-TKL and Luciferase DNA fragmentation.

4. tracer target practice plasmid pMV-VTTTK-Luc structure, flow are as shown in Figure 3.

(1) pMV-PRO plasmids and VTT-TKR fragments purify recovery after BamHI/KpnI double digestions respectively, by recovery Fragment is attached, converted, interstitial granules pMV-VTTTKR in acquisition；

(2) pMV-VTTTKR plasmids and VTT-TKL fragments purify recovery after PstI/SalI double digestions respectively, will reclaim Fragment be attached, convert, obtain target practice plasmid pMV-VTTTK；

(3) pMV-VTTTK plasmids and Luciferase fragments purify recovery after EcoRI/BamHI double digestions respectively, will The fragment of recovery is attached, converted, and obtains tracer target practice plasmid pMV-VTTTK-Luc.

Each of the above walks plasmid construction, carries out corresponding digestion identification, digestion qualification figure as shown in Figure 4, wherein M is MarkerIII.A：PMV-VTTTKR is through BamHI/KpnI digestion qualification figures；B：PMV-VTTTK identifies through PstI/SalI digestions Figure；C：PMV-VTTTK-Luc is through EcoRI/BamHI digestion qualification figures.PMV-VTTTKR should occur through BamHI/KpnI double digestions 845bp and the bar segments of 2511bp two, digestion result is with expected consistent, and digestion result is as shown in Figure 4 A；PMV-VTTTK is through PstI/ SalI double digestions, 881bp and the bar segments of 3348bp two should occur, digestion result is with expected consistent, and digestion result is as shown in Figure 4 B； PMV-VTTTK-Luc 1669bp and the bar segments of 4193bp two, digestion result and expection should occur through EcoRI/BamHI double digestions Unanimously, digestion result as shown in Figure 4 C, finally, the tracer target practice plasmid pMV-VTTTK- for vaccinia virus Tiantan strain TK genes Luc is successfully constructed.

Other foreign genes are can be inserted into after promoter P11, for improving the Suppressive effect of vaccinia virus Tiantan strain, are opened There are two restriction enzyme sites SacI and SpeI on catenation sequence between mover P11 and P7.5, can add here according to demand new Expression cassette to express more foreign genes.

During present invention work, homology arm VTT-TKL and VTT-TKR and Wild-type vaccinia strain the Temple of Heaven strain TK genes both sides Homologous recombination occurs for sequence, the vaccinia virus recombinant the Temple of Heaven strain formed and knock out TK genes, expressed Lucifefase genes, The reaction of Luciferase produces chemiluminescences produces bioluminescence, plays tracking function.

<110>Xi'an Medical University

<120>Tracer target practice plasmid for vaccinia virus Tiantan strain TK genes and preparation method thereof

<130>Nothing

<210>1

<211>5862

<212>DNA

<213>pMV-VTTTK-Luc

<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 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 ATCGCCGTGT AACCCGGGAT CCGGCTTCCT TTTCTAAACG ATTGGGTGAG 3120

GAAACCGAGA TAGAAATAAT AGGAGGTAAT GATATGTATC AATCGGTGTG TAGAAAGTGT 3180

TACATCGACT CATAATATTA TATTTTTTAT CTAAAAAACT AAAAATAAAC ATTGATTAAA 3240

TTTTAATATA ATACTTAAAA ATGGATGTTG TGTCGTTAGA TAAACCGTTT ATGTATTTTG 3300

AGGAAATTGA TAATGAGTTA GATTACGAAC CAGAAAGTGC AAATGAGGTC GCAAAAAAAC 3360

TGCCGTATCA AGGACAGTTA AAACTATTAC TAGGAGAATT ATTTTTTCTT AGTAAGTTAC 3420

AGCGACACGG TATATTAGAT GGTGCCACCG TAGTGTATAT AGGATCTGCT CCCGGTACAC 3480

ATATACGTTA TTTGAGAGAT CATTTCTATA ATTTAGGAGT GATCATCAAA TGGATGCTAA 3540

TTGACGGCCG CCATCATGAT CCTATTTTAA ATGGATTGCG TGATGTGACT CTAGTGACTC 3600

GGTTCGTTGA TGAGGAATAT CTACGATCCA TCAAAAAACA ACTGCATCCT TCTAAGATTA 3660

TTTTAATTTC TGATGTGAGA TCCAAACGAG GAGGAAATGA ACCTAGTACG GCGGATTTAC 3720

TAAGTAATTA CGCTCTACAA AATGTCATGA TTAGTATTTT AAACCCCGTG GCATCTAGTC 3780

TTAAATGGAG ATGCCCGTTT CCAGATCAAT GGATCAAGGA CTTTTATATC CCACACGGTA 3840

ATAAAATGTT ACAACCTTTT GCTCCTTCAT ATTCAGCTGA AATGAGATTA TTAAGTATTT 3900

ATACCGGTGA GAACATGAGA CTGACTCGGT ACCTAAAGAG ACGGAGTCAC TGCCAACCGA 3960

GACGGTCATA GCTGTTTCCT GTGTGCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC 4020

GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT ACCCACAGAA 4080

TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT 4140

AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA 4200

AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT 4260

CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG 4320

TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GCTCACGCTG TAGGTATCTC 4380

AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC 4440

GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA 4500

TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT 4560

ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC 4620

TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA 4680

CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA 4740

AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA 4800

AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT 4860

TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC 4920

AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC 4980

ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC 5040

CCCAGTGCTG CAATAATACC GCGGGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA 5100

AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC 5160

CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC 5220

AACGTTGTTG CCATCGCTAC AGGCATCGTG GTATCACGCT CGTCGTTTGG TATGGCTTCA 5280

TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT GCGCAAAAAA 5340

GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC CGTGTTATCA 5400

CTCATGGTTA TGGCAGCACT ACATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT 5460

TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT 5520

TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG 5580

CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA 5640

TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC 5700

AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG 5760

ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG 5820

GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AG 5862

<210>2

<211>417

<212>DNA

<213>P&MCS

<400>2

TTACTGCAGG CATGCGTCGA CAAGCTTCAA CTGTCCATGG GCCCGCGGCC GCTCGAGCAT 60

CTGGTAATTT ATAGCATAGA AAAAAACAAA ATGAAATTCT ACTATATTTT TACATACATA 120

TATTCTAAAT ATGAAAGTGG TGATTGTGAC TAGCGTAGCA TCGCTCATCT ATATACTATA 180

TAGTAATACC AATAGAGCTC AAGACTACGA CTAGTAAACT GATACAATCT CTTATATGTG 240

GGTAATGTTC TCGATGTCGA TAGCCATATG CCCGGTAGTT GCGATATACA TAAACTGATC 300

ACTAATTCCA AACCCACCCG CTTTTTATAG TAAGTTTTTC ACCCATAAAT AATAAATACA 360

ATGGAATTCG CGCGCGATAT CGGCGCCTAT TCGTCTAGAG GATCCCCGGG TACCTAA 417

<210>3

<211>835

<212>DNA

<213>VTT-TKR

<400>3

GGCTTCCTTT TCTAAACGAT TGGGTGAGGA AACCGAGATA GAAATAATAG GAGGTAATGA 60

TATGTATCAA TCGGTGTGTA GAAAGTGTTA CATCGACTCA TAATATTATA TTTTTTATCT 120

AAAAAACTAA AAATAAACAT TGATTAAATT TTAATATAAT ACTTAAAAAT GGATGTTGTG 180

TCGTTAGATA AACCGTTTAT GTATTTTGAG GAAATTGATA ATGAGTTAGA TTACGAACCA 240

GAAAGTGCAA ATGAGGTCGC AAAAAAACTG CCGTATCAAG GACAGTTAAA ACTATTACTA 300

GGAGAATTAT TTTTTCTTAG TAAGTTACAG CGACACGGTA TATTAGATGG TGCCACCGTA 360

GTGTATATAG GATCTGCTCC CGGTACACAT ATACGTTATT TGAGAGATCA TTTCTATAAT 420

TTAGGAGTGA TCATCAAATG GATGCTAATT GACGGCCGCC ATCATGATCC TATTTTAAAT 480

GGATTGCGTG ATGTGACTCT AGTGACTCGG TTCGTTGATG AGGAATATCT ACGATCCATC 540

AAAAAACAAC TGCATCCTTC TAAGATTATT TTAATTTCTG ATGTGAGATC CAAACGAGGA 600

GGAAATGAAC CTAGTACGGC GGATTTACTA AGTAATTACG CTCTACAAAA TGTCATGATT 660

AGTATTTTAA ACCCCGTGGC ATCTAGTCTT AAATGGAGAT GCCCGTTTCC AGATCAATGG 720

ATCAAGGACT TTTATATCCC ACACGGTAAT AAAATGTTAC AACCTTTTGC TCCTTCATAT 780

TCAGCTGAAA TGAGATTATT AAGTATTTAT ACCGGTGAGA ACATGAGACT GACTC 835

<210>4

<211>879

<212>DNA

<213>VTT-TKL

<400>4

ACAGATATTC CGACAAAGGA TTGATTACTA TAAATGGAGA ATGTTCCTAA TGTATACTTT 60

AATCCTGTGT TTATAGAGCC CACGTTTAAA CATTCTTTAT TAAGTGTTTA TAAACACAGA 120

TTAATAGTTT TATTTGAAGT ATTCGTTGTA TTCATTCTAA TATATGTATT TTTTAGATCT 180

GAATTAAATA TGTTCTTCAT GCCTAAACGA AAAATACCCG ATCCTATTGA TAGATTACGA 240

CGTGCTAATC TAGCGTGTGA AGACGATAAA TTAATGATCT ATGGATTACC ATGGATGACA 300

ACTCAAACAT CTGCGTTATC AATAAATAGT AAACCGATAG TGTATAAAGA TTGTGCAAAG 360

CTTTTGCGAT CAATAAATGG ATCACAACCA GTATCTCTTA ACGATGTTCT TCGCAGATGA 420

TGATTCATTT TTTAAGTATT TGGCTAGTCA AGATGATGAA TCTTCATTAT CTGATATATT 480

GCAAATCACT CAATATCTAG ACTTTCTGTT ATTATTATTG ATCCAATCAA AAAATAAATT 540

AGAAGCCGTG GGTCATTGTT ATCAATCTCT TTCAGAGGAA TACAGACAAT TGACAAAATT 600

CACAGACTCT CAAGATTTTA AAAAACTGTT TAACAAGGTC CCTATTGTTA CAGATGGAAG 660

GGTCAAACTT AATAAAGGAT ATTTGTTCGA CTTTGTGATT AGTTTGATGC GATTCAAAAA 720

AGAATCCTCT CTAGCTACCA CCGCAATAGA TCCTATTAGA TACATAGATC CTCGTCGCGA 780

TATCGCATTT TCTAACGTGA TGGATATATT AAAGTCGAAT AAAGTGAACA ATAATTAATT 840

CTTTATTGTC ATCATGAACG GCGGACATAT TCAGTTGAT 879

<210>5

<211>1659

<212>DNA

<213>Luciferase

<400>5

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 GCCGTGTAA 1659

Claims (6)

1. the tracer target practice plasmid for vaccinia virus Tiantan strain TK genes, it is characterised in that including carrier pMV, the bovine vaccine of setting out The upstream homologous recombination arm VTT-TKL and downstream homologous recombination arm VTT-TKR of viral the Temple of Heaven strain TK genes, VTT-TKL with There is vaccinia virus early late phase promoter P11 and late promoter P7.5 between VTT-TKR, between promoter P11 and promoter P7.5 Catenation sequence have two restriction enzyme sites, promoter P11 downstream and promoter P7.5 downstream include 1 polyclonal position Point, tracer gene Luciferase, the nucleotides sequence of the tracer target practice plasmid are inserted at the multiple cloning sites after promoter P7.5 Row are as shown in sequence 1.

2. it is used for the tracer target practice plasmid of vaccinia virus Tiantan strain TK genes as claimed in claim 1, it is characterised in that described The carrier pMV that sets out includes Escherichia coli replication origin pUC ori and amicillin resistance selection markers.

3. it is used for the tracer target practice plasmid of vaccinia virus Tiantan strain TK genes as claimed in claim 1 or 2, it is characterised in that The order of promoter P11 and promoter P7.5 promotor gene expression is on the contrary, promoter P11 is reverse starting, promoter P7.5 starts to be positive.

4. being used for the preparation method of the tracer target practice plasmid of vaccinia virus Tiantan strain TK genes as claimed in claim 1, it is special Sign is, specifically implements according to following steps：

Step 1：Engineer and synthetic promoter P7.5 and promoter P11 sequences and two multiple cloning sites sequences, and clone Into pMV carriers, pMV-PRO plasmids are formed；Synthetic primer is designed, PCR obtains upstream homologous recombination arm VTT-TKL and downstream is same Source recombinates arm VTT-TKR and tracer gene Luciferase fragments；

Step 2：The pMV-PRO plasmids obtained in step 1 and VTT-TKR fragments are purified after BamHI/KpnI double digestions respectively Recovery, the fragment of recovery is attached, converted, interstitial granules pMV-VTTTKR in acquisition；

Step 3：By the VTT-TKL fragments obtained in the pMV-VTTTKR plasmids and step 1 that are obtained in step 2 respectively through PstI/ Recovery is purified after SalI double digestions, the fragment of recovery is attached, converted, obtains target practice plasmid pMV-VTTTK；

Step 4：The Luciferase fragments obtained in the pMV-VTTTK plasmids and step 1 that are obtained in step 3 are passed through respectively Recovery is purified after EcoRI/BamHI double digestions, the fragment of recovery is attached, converted, obtains tracer target practice plasmid pMV- VTTTK-Luc。

5. being used for the preparation method of the tracer target practice plasmid of vaccinia virus Tiantan strain TK genes as claimed in claim 4, it is special Sign is that the primer that synthesis is designed in the step 1 specifically includes following three pairs of primers：

Expand VTT-TKR primer pair：

Forward primer VTT-TKR-F：5’-CGGGATCCGGCTTCCTTTTCTAAACGATTGG-3’；

Reverse primer VTT-TKR-R：5’-GGGGTACCGAGTCAGTCTCATGTTCTCACCG-3’；

Expand VTT-TKL primer pair：

Forward primer VTT-TKL-F：5’-AACTGCAGACAGATATTCCGACAAAGGATTG-3’；

Reverse primer VTT-TKL-R：5’-ACGCGTCGACATCAACTGAATATGTCCGCCG-3’；

Expand the primer pair of Luciferase genes：

Luc-F：5’-GGAATTCGCCACCATGGAAGACGCCA-3’；

Luc-R：5’-CGGGATCCTTACACGGCGATCTTTCCGC-3’。

6. being used for the preparation method of the tracer target practice plasmid of vaccinia virus Tiantan strain TK genes as claimed in claim 5, it is special Sign is, upstream homologous recombination arm VTT-TKL and downstream homologous recombination arm VTT-TKR and tracer gene are obtained in the step 1 Luciferase's comprises the following steps that：

1) wild type VACV-VTT genomic DNAs are extracted；

2) using VACV-VTT genomic DNAs template, obtained using VTT-TKR-F/VTT-TKR-R as primer PCR and carry BamHI/ The VTT-TKR of KpnI restriction enzyme sites, obtained using VTT-TKL-F/VTT-TKL-R as primer PCR and carry PstI/SalI restriction enzyme sites VTT-TKL；Using p-EGFP-Luc plasmids as template, obtained using Luc-F/Luc-R as primer PCR and carry EcoRI/BamHI enzymes The Luciferase genetic fragments of enzyme site；

3) PCR primer obtains upstream homologous recombination arm VTT-TKL respectively and downstream is homologous through agarose gel electrophoresis and glue reclaim Recombinate arm VTT-TKR and tracer gene Luciferase fragments.