CN113355295A

CN113355295A - Recombinant oncolytic newcastle disease virus expressing human GM-CSF and application thereof

Info

Publication number: CN113355295A
Application number: CN202110631765.5A
Authority: CN
Inventors: 边惠洁; 陈志南; 尉丁; 刘满; 张仁宇; 孔令敏; 南刚
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2021-06-07
Filing date: 2021-06-07
Publication date: 2021-09-07

Abstract

The invention relates to the field of biological virus construction, and particularly discloses a recombinant oncolytic Newcastle disease virus for expressing human GM-CSF (GM-CSF), which is obtained by inserting a human GM-CSF coding gene into a full-length genome plasmid of an Italien strain of the Newcastle disease virus and carrying out in-vitro reactivation on the virus, wherein the human GM-CSF gene is shown as SEQ ID No. 1. The invention utilizes GM-CSF molecule to recruit monocyte-derived DC cells, activate neutrophils, promote DC cell differentiation and maturation, especially have important effects on the aspect of promoting antigen processing and presentation, combines the characteristic of selective replication of tumor of oncolytic newcastle disease virus, recruits more DC cells and promotes maturation and presentation of tumor antigen while directly killing tumor cells and causing inflammatory reaction, enhances the anti-tumor immune response reaction of local and whole tumor, improves the treatment effect of treating solid tumor by singly or jointly applying oncolytic virus, and brings a new treatment idea for tumor immunotherapy.

Description

Recombinant oncolytic newcastle disease virus expressing human GM-CSF and application thereof

Technical Field

The invention belongs to the field of biological virus construction, and particularly relates to a recombinant oncolytic newcastle disease virus for expressing human GM-CSF and application thereof.

Background

Oncolytic virus (oncolytical virus) refers to a class of viruses that specifically kill tumor cells, and the use of oncolytic viruses to treat tumors is one of the hot research topics in the biological treatment of tumors. Oncolytic viruses achieve tumor killing mainly through 2 mechanisms, one is that after viral infection, dysfunction of host cells is caused or host cells directly die due to massive replication of viruses. After the oncolytic virus infects tumor cells, local and even whole-body anti-tumor immune response is enhanced through various mechanisms of enhancing presentation of tumor antigens, causing immunogenic death of the tumor cells, releasing various cytokines and chemotactic factors and the like, so that a more lasting and wide anti-tumor effect is generated. With the rise of tumor immunotherapy in recent years, the latter mechanism is considered to be more important, and particularly in combination with other anti-tumor immunotherapy strategies such as CART cell therapy, the therapeutic effect on solid tumors can be greatly enhanced.

On the other hand, the oncolytic virus can be used as a replication virus vector by itself through modification, carries a high expression of exogenous effector genes in tumor local specificity, and can further enhance the treatment effect of the oncolytic virus. The foreign molecules carried by the existing oncolytic virus mainly comprise the following types: inducing cell death-related molecules, anti-angiogenic molecules, immunomodulatory factors, and the like. Immune regulatory factors are the most major foreign effector molecules at present, such as GM-CSF, CCL5, etc. T-VEC, which is marketed as approved by the FDA, is used for expressing an immunoregulatory cytokine GM-CSF by using recombinant HSV, and the effect of the oncolytic virus is enhanced.

Newcastle Disease Virus (NDV) is a natural oncolytic virus, a single-stranded negative-strand RNA virus, whose natural host is avian, and is not pathogenic to humans. The research history of NDV used for tumor treatment is over 50 years, and research results show that NDV has good safety when being applied to a human body, can selectively kill various human tumor cells in vivo and in vitro, and achieves a certain treatment effect, but the tumor dissolving effect of natural NDV is not enough to generate enough treatment effect, so that the NDV needs to be modified, and the treatment effect is improved by carrying and expressing different effect genes and carrying out combined application with other treatment methods.

Disclosure of Invention

The invention aims to provide a recombinant oncolytic Newcastle disease virus expressing human GM-CSF and application thereof, which solve the problem of poor immunotherapy effect caused by tumor immunosuppressive microenvironment in the current tumor therapy, particularly solid tumor immunotherapy.

The invention provides a recombinant oncolytic Newcastle disease virus for expressing human GM-CSF, which is obtained by inserting a human GM-CSF gene into a genome plasmid of an Italien strain of the Newcastle disease virus and reactivating the virus in vitro, wherein the nucleotide sequence of the human GM-CSF gene is shown as SEQ ID NO. 1.

The invention also provides a construction method of the recombinant oncolytic Newcastle disease virus for expressing the human GM-CSF, which comprises the following steps:

s1, adding NDV transcription regulation sequences GS and GE at the upstream and downstream of a CDS region respectively, adding BsiWI enzyme cutting sites at two ends of a fragment, connecting to a genome plasmid pBR-rNDV site through BsiWI enzyme cutting to obtain a plasmid pBR-rNDV-hGMCSF, and transferring into engineering bacteria;

the nucleotide sequence of the GS is shown as SEQ ID NO. 3;

the nucleotide sequence of the GE is shown as SEQ ID NO. 4;

s2, transferring the digested BSR-T7/5 into a new container, adding a DMEM medium for culture, adopting a PEI transfection reagent for transfection, observing the cell growth state after the transfection is finished, indicating that the virus is successfully revived after a cell fusion phenomenon appears, and naming the recombinant virus as rNDV-hGMCSF.

Further, in S2, the DMEM medium contains 10 volume percent fetal bovine serum and 100 μ g/mL streptomycin.

Further, in S2, the concentration of PEI transfection reagent was 1 mg/mL.

Further, in S2, the mass ratio of pBR-rNDV-hGMCSF: pCI-NP: pCI-P: pCI-L is mixed 2:1:1: 1.

The invention also provides application of the recombinant oncolytic Newcastle disease virus for expressing the human GM-CSF in preparing a tumor treatment medicament.

Furthermore, the recombinant oncolytic Newcastle disease virus expressing human GM-CSF can be independently used for preparing tumor treatment medicines.

Furthermore, the recombinant oncolytic Newcastle disease virus expressing human GM-CSF can be combined with other therapeutic drugs to prepare tumor therapeutic drugs.

Further, other therapeutic agents include, but are not limited to, surgical agents for tumor, radiation therapeutic agents, chemotherapeutic agents, antibody therapeutic agents, immunocytotherapeutic agents, interventional agents, stem cell therapeutic agents.

Compared with the prior art, the recombinant oncolytic Newcastle disease virus for expressing human GM-CSF and the application thereof provided by the invention have the following beneficial effects:

1. the invention is based on an Italien strain of the Newcastle disease virus, and the strain is a virulent strain and an oncolytic strain of the Newcastle disease virus.

2. The recombinant oncolytic Newcastle disease virus constructed by the invention expresses human GM-CSF molecules which are important lymphocyte chemotactic factors of human bodies and play important roles in recruiting DC from monocyte sources, activating neutrophils and the like, promoting the differentiation and maturation of the DC and promoting the processing and presentation of antigens. GM-CSF is also currently the most common exogenous effector gene used in oncolytic virus gene engineering.

3. The invention constructs an oncolytic virus vector carrying an exogenous gene based on an oncolytic Newcastle disease virus Italien strain, and has the following characteristics:

a. the newcastle disease virus Italien strain is a virulent strain and an oncolytic strain. The maturation of newcastle disease virus (i.e. the virus has infectious activity) requires hydrolysis of the virus F protein, and the Italien strain F protein can be hydrolyzed by more proteases than the currently commonly used intermediate and low virulent strains, so the Italien strain of newcastle disease virus has a wider in vivo distribution potential and a potential to infect more tumor types.

b. Oncolytic viral vectors can replicate specifically in tumor cells compared to replication-deficient viral vectors. On one hand, the virus directly kills tumor cells, activates local inflammatory reaction and improves the anti-tumor immunoreaction level; on the other hand, the exogenous gene product is locally expressed, so that adverse reaction in whole-body application is avoided while the concentration of the local product is improved, and the safety is better.

c. The Newcastle disease virus is an RNA virus, does not enter a cell nucleus in the replication and transcription processes, and does not have the risk of gene integration; the natural host is poultry, and the poultry feed additive has better safety when being applied to a human body.

Drawings

FIG. 1 is a schematic diagram showing the structure of the genome of a recombinant oncolytic Newcastle disease virus according to the invention;

FIG. 2 is a graph of the selective killing of a variety of tumor cells by recombinant oncolytic Newcastle disease viruses of the invention in vitro;

wherein panel A shows the effect of recombinant oncolytic Newcastle disease virus on the viability of the lung cancer cell line A549;

FIG. B shows the effect of recombinant oncolytic Newcastle disease virus on the viability of the lung cancer cell line NCI-H460;

panel C shows the effect of recombinant oncolytic Newcastle disease virus on the viability of the lung cancer cell line NCI-H1299;

panel D shows the effect of recombinant oncolytic Newcastle disease virus on the viability of the normal cell line BEAS-2B;

FIG. 3 shows the GM-CSF assay of the supernatant of recombinant oncolytic Newcastle disease virus infection;

FIG. 4 is a graph showing the specific activity of human GM-CSF expressed by recombinant oncolytic viruses of the invention;

wherein, panel A shows the results of specific activity measurement of GM-CSF as a standard;

panel B shows the specific activity of GM-CSF determined in the supernatant from the recombinant virus infection.

Detailed Description

The present invention is described in detail below with reference to the drawings and the specific embodiments, but it should be understood that the scope of the present invention is not limited by the specific embodiments. The test methods in the following examples, which are not specified in specific conditions, are generally conducted under conventional conditions, and the steps thereof will not be described in detail since they do not relate to the invention.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

The synthesis of the human GM-CSF gene fragment in the examples below was done by Nanjing Kisrey and cloned in the Newcastle disease virus genomic plasmid using BsiWI cleavage, then transformed into the DH5 alpha engineered strain. BSR-T7/5 cells were offered as a gift by professor Conzelmann, university of Munich, Germany. RPMI-1640 medium, DMEM high-sugar medium, glutamine, pancreatin cell digestive juice were purchased from Sigma, fetal bovine serum was purchased from Hangzhou ilex bioengineering materials, Inc., and 100 Xcyan streptomycin solution was purchased from Biyuntian. The plasmid extraction kit and the gel recovery kit are purchased from OMEGA Bio-Tek company, and the endonuclease and the T4 DNA ligase are purchased from NEB company; CCK8 cell viability assay kit, bi yun tian biotechnology; the Human GM-CSF ELISA Kit is purchased from Strobilants, Wuhan Dreher, bioengineering, Inc.; SPF grade hatching eggs were purchased from Meiria, Beijing; the TF-1 cell line was purchased from Guangzhou Sakul Biotechnology, Inc.

The specific construction method of pBR-rNDV plasmid, auxiliary plasmids pCI-NP, pCI-P and pCI-L in the invention are disclosed in granted patent "tumor targeting recombinant Newcastle disease virus and its construction method", patent number ZL 200710018423.6.

The invention provides a recombinant oncolytic Newcastle disease virus capable of expressing human GM-CSF chemotactic factor, which is obtained by inserting human GM-CSF gene into genome plasmid of Italien strain of Newcastle disease virus and through in vitro virus revival, wherein the nucleotide sequence of the human GM-CSF gene is shown as SEQ ID No. 1.

Construction of recombinant virus genome plasmid

The expressed human GM-CSF gene is derived from the sequence of Genbank database NM-000758.4, NDV transcription regulating sequences GS (the sequence is shown as SEQ ID NO. 3) and GE (the sequence is shown as SEQ ID NO. 4) are respectively added at the upstream and downstream of the CDS region, and BsiWI enzyme cutting sites are added at both ends of the segment. The fragment is synthesized by Nanjing Kingsri and then is connected to a genome plasmid pBR-rNDV locus by BsiWI enzyme digestion to obtain a plasmid pBR-rNDV-hGMCSF, and the plasmid is transformed into DH5 alpha bacteria by Nanjing Kingsri after being confirmed to be correct by sequencing. The structure of the obtained genome plasmid is shown in figure 1, and the genome sequence of the obtained recombinant virus is shown in SEQ ID NO. 2.

SEQ ID NO.3：ACGGGTAGAA；SEQ ID NO.4：TTAGAAAAAAA；

Second, revivification and preparation of recombinant virus

1. In vitro rejuvenation of recombinant viruses

BSR-T7/5 was digested and transferred to a 10cm dish, and DMEM medium (containing fetal bovine serum with a volume fraction of 10% and streptomycin at 100. mu.g/mL) was added to the bottom of the dish and cultured until the bottom of the dish was filled with 80%. Transfection was performed with PEI (1mg/mL) transfection reagent, the procedure was as follows: the plasmid mass ratio pBR-rNDV-hGMCSF: pCI-NP: pCI-P: pCI-L ═ 2:1:1:1, prepared from 10. mu.g, 5. mu.gThe above plasmids were mixed and the final volume was adjusted to 1500. mu.l in serum-free medium. Dissolving 30 mul of PEI sterile solution with the concentration of 1mg/mL in 1500 mul of serum-free culture medium; mixing the two, incubating at room temperature for 20min, adding into a cell culture dish, and incubating at 37 ℃ for 4 hours; fourthly, after the incubation is finished, the supernatant is sucked out and DMEM complete culture medium is added to continue to carry out incubation at 37 ℃ with 5 percent CO₂Culturing under the condition.

And observing the cell growth state after transfection is finished, indicating that virus revival is successful after a cell fusion phenomenon appears, and naming the recombinant virus as rNDV-hGMCSF. The culture supernatant was collected and added to new cells to amplify the virus. Collecting culture supernatant after large-area fusion cells appear, centrifuging at 4000g and 4 deg.C for 30min to remove large-particle impurities, and determining virus Titer (TCID)₅₀/mL)。

2. Preparation of recombinant virus from chick embryo allantoic cavity

Culturing the SPF fertilized eggs till the day 9, and removing dead embryos and unfertilized eggs by using an egg lighting lamp before inoculation; viral titer was diluted to 10⁴TCID₅₀Per mL; the diluted virus suspension was aspirated by a 1mL sterile syringe, and 100. mu.L of virus diluent was injected by inserting a needle approximately 1cm vertically through a hole formed in the upper part of the air chamber. After the injection is finished, the injection hole is sealed by using sterile medical adhesive tapes, and the hatching eggs are put back into the incubator to be continuously incubated without turning over the hatching eggs. Collecting the dead chick embryos within 24-48h, and storing in a refrigerator at 4 ℃; the remaining chick embryos were sacrificed overnight in a4 ℃ freezer after 72h virus inoculation, and then chick embryo allantoic fluid was collected and stored in a4 ℃ freezer. Collecting and purifying virus in allantoic fluid by sucrose density gradient centrifugation, which comprises the following steps: firstly, allantoic fluid is roughly separated, 4000g is centrifuged for 30min at 4 ℃, impurities such as eggshell fragments are removed, and supernatant is collected. Filtering with 70 μm cell filter screen to remove impurities such as lipid which can not be precipitated. ③ adding 20 percent of sterile sucrose solution into the bottom of a centrifugal tube special for Beckman 34.5mlmL ultracentrifugation, and slowly adding the supernatant of the allantoic fluid after coarse separation by using a 10mL pipette. 25000RPM (SW32Ti horizontal rotor, same below), and 4h centrifugation at 4 ℃. Fifthly, after the centrifugation is finished, the supernatant is discarded, and 10mL of TNE buffer solution is used for resuspension and precipitation. Sixthly, sequentially adding 10mL of sterile sucrose solutions with the concentration of 50 percent and 20 percent into a centrifugal tube, finally slowly adding the virus heavy suspension,ensuring a clear boundary between the individual liquid layers. Seventhly, carrying out centrifugation at 4 ℃ for 4h at 31900 RPM. Eighthly, carefully sucking away the upper liquid, collecting floccules with the concentration of 50 percent and 20 percent of sucrose interface, and diluting the floccules to 35mL by using TNE buffer solution. Ninthly 31900RPM, centrifugation at 4 ℃ for 4h, and after completion, resuspension of the virus with 2mL of TNE buffer. The titer of the virus suspension is determined in the red, subpackaged and stored at-80 ℃ for standby.

Thirdly, functional identification of recombinant viruses

1. Killing capacity of recombinant virus to tumor cells

The in vitro killing capacity of the recombinant virus rNDV-hGMCSF on tumor cells and normal cells is detected by taking lung cancer cell lines A549, NCI-H1299, NCI-H460 and human lung normal cell line BEAS2B cells as target cells, and the specific steps are as follows:

a549 cells, H1299 cells, H460 cells and BEAS2B cells are digested, counted and inoculated on a 96-well cell culture plate until the cell confluency reaches 80-90%. ② performing multiple dilution on the virus suspension, wherein the dilution initial concentration of infected A549 cells is set according to MOI of 10, and the dilution multiple is 2; NCI-H460 starting MOI 1.11 with a dilution factor of 3; NCI-H1299 starting MOI 10 with a dilution factor of 10; the initial concentration of BEAS2B was serum-free diluted at a dilution factor of 10, set at an MOI of 100. Thirdly, replacing a fresh complete culture medium before infection, then adding rNDV-hGMCSF virus diluent with corresponding volume according to corresponding infection intensity, setting 5 multiple wells for samples with different time points of each virus, simultaneously adding equal volume of TNE buffer solution into a blank well and a negative control well, and then continuing to add cells in 5% CO₂And then the culture was continued in a 37 ℃ incubator. Fourthly, using a CCK8 detection kit to detect the cell viability rate of the infected cells 72h respectively, wherein the cell killing rate is [ (Ac-As)/(Ac-Ab)]X 100%, wherein As: absorbance values of the experimental groups; ac: average value of absorbance of the control group; ab: blank absorbance average. Fifthly, utilizing software to inhibit half of the concentration (IC) of virus infected different cells₅₀) And (6) performing calculation.

The results are shown in FIG. 2: recombinant virus rNDV-hGMCSF is most sensitive to lung cancer cell line NCI-H1299, IC₅₀0.0025, i.e. 0.0025 for initial multiplicity of infection of the virus which results in 50% of cell death; for lung cancer cell line A549 IC₅₀5.926 for lung cancer cell line NCI-H460, IC₅₀0.04; while on the IC of the normal cell line BEAS-2B₅₀30.06, 12000 times more sensitive lung cancer cell NCI-H1299, and 5.07 times more sensitive lung cancer cell A549; the rNDV-hGMCSF can effectively infect tumor cells and still has obvious tumor selective killing capability.

2. Identification of recombinant Virus expressing GM-CSF

The rNDV-hGMCSF infected BSR-T7/5 cells at MOI of 0.1, and cell supernatants were collected 48 hours after infection and diluted 50-fold with sample dilutions for testing. The ELISA kit is used for detection, and the specific steps are as follows: (ii) configuring human GMCSF standard into 1000pg/mL dilution, and then diluting 2-fold ratio into 500-15.6pg/mL solution for drawing a standard curve. Adding the diluent of the sample to be detected and the diluent of the standard product into the holes of the enzyme label plate, adding a sealing plate film into each hole with 100 mu L, and incubating for 90min at 37 ℃. ③ after the liquid is dried, 100 mu L of biotin-labeled anti-human GM-CSF antibody working solution is added with a sealing plate membrane and incubated for 60min at 37 ℃. And fourthly, after the incubation is finished, washing the plate for 3 times by using washing buffer solution, wherein each time is 1min, and then drying the liquid. Fifthly, 100 mul of ABC working solution is added into each hole, a sealing plate membrane is added, and incubation is carried out for 30min at 37 ℃. Sixthly, washing for 5 times by using the washing buffer solution, and spin-drying the liquid after 2min each time. Seventhly, adding 90 mu L of TMB color development solution into each hole, adding a sealing plate film, incubating for 15min at 37 ℃, and then adding 100 mu L of TMB stop solution into each hole. And detecting A450 by using an enzyme-labeling instrument and calculating the concentration of each group according to a standard curve.

The results are shown in FIG. 3: after rNDV-hGMCSF infects cells for 2h, the concentration of supernatant is rapidly increased until the supernatant reaches a platform 24h after infection, and after 48h, human GM-CSF is detected in the supernatant, and the concentration is (2.03 +/-0.2) multiplied by 10⁶pg/mL, which shows that the recombinant virus rNDV-hGMCSF has the function of secreting and expressing human GM-CSF after infecting cells.

3. Identification of GM-CSF functional Activity on recombinant Virus tables

According to the formula in the recombinant human granulocyte macrophage stimulating factor bioactivity determination method in the fourth part of the 'Chinese pharmacopoeia' of 2015 edition: the biological activity (IU/mL) of the test article is Pr x (Ds × Es)/(Dr × Er). Pr is the biological activity of a standard substance, IU/mL; ds is the pre-dilution multiple of the test sample; dr is the pre-dilution multiple of the standard substance; es is the dilution multiple of the half effective amount of the standard substance equivalent to the test substance; er is the dilution factor of half effective amount of the standard.

Preparation of a detection sample: ndv-hGMCSF cells BSR-T7/5 were infected at MOI of 0.1, cell culture supernatants 48 hours after infection were collected and virus in the supernatants was inactivated by uv light, and GM-CSF content thereof was measured by ELISA method and medium was added to adjust the concentration to 2 ng/. mu.l, and biological activity of human GM-CSF therein was measured using the supernatants. GM-CSF standard was prepared in 2.8 ng/. mu.L medium. ② the culture medium is used to dilute the virus culture supernatant and GM-CSF standard solution 2 times for 8-16 gradients. ③ the human erythroleukemia cell TF-1 is suspension cultured cell and depends on IL-3 or GM-CSF to grow completely, the complete culture medium is RPMI-1640 culture medium added with 10% fetal calf serum, 1% streptomycin solution, GM-CSF with final concentration of 6-8ng/mL and 1% glutamine solution (concentration 200mM), the solution is changed for 2-3 days, the cell is maintained at 3X 10⁴～5×10⁵one/mL. And washing the TF-1 cells twice by serum-free RPMI-1640, and removing GM-CSF and cytokines secreted by other cells in the original culture medium as far as possible. Count cells and adjust TF-1 cell concentration to 2X 10 with RPMI-1640 added with 10% fetal calf serum⁵one/mL. Sixthly, taking 740 mu L of TF-1 cell suspension, and sequentially adding the diluted virus infection supernatant and the GM-CSF standard solution, wherein the initial concentrations are 50ng/mL and 70ng/mL respectively; seventhly, inoculating 100ul of the culture medium into a 96-well plate, arranging 3 multiple holes, and arranging 3 zero setting holes, namely adding the RPMI-1640 culture medium which is only added with 10% fetal bovine serum without adding cells; continuously culturing for 48h in the incubator; adding CCK 810 mu L into each hole, continuously incubating for 2-4 h, and detecting A by using a microplate reader₄₅₀。

The results are shown in FIG. 4, EC of human GM-CSF recombinantly expressed in rNDV-hGMCSF₅₀It was 0.1898 ng/mL.

It should be noted that, the applicant can obtain recombinant NDV carrying an expression exogenous gene based on the recombinant NDV technical platform constructed by using the Italien strain of the virulent strain and the oncolytic strain of NDV (patent number ZL 200710018423.6).

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

<110> fourth university of military medical science

<>

<120> recombinant oncolytic newcastle disease virus expressing human GM-CSF and application thereof

<>

<160> 4

<>

<170> SIPOSequenceListing 1.0

<>

<210> 1

<211> 435

<212> DNA

<213> human

<>

<400> 1

atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc 60<>

cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg 120<>

cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc 180<>

tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag 240<>

cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac 300<>

tacaagcagc actgccctcc aaccccggaa acttcctgtg caacccagat tatcaccttt 360<>

gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag 420<>

ccagtccagg agtga 435<>

<>

<210> 2

<211> 19713

<212> DNA

<213> Artificial Synthesis

<>

<400> 2

ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa 60<>

ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg 120<>

caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt 180<>

gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata 240<>

tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg 300<>

ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac 360<>

cacacccgtc ctgtggatcc tctagagatt gtaatacgac tcactatagg gaccaaacag 420<>

agattctgtg aggtacgata aaaggcgaag gagcaatcga agtcgtacgg gtagaaggtg 480<>

tgaatctcga gtgcgaggcc gaagctcaaa ctcgagagag ccttctgctg acatgtggct 540<>

gcagagcctg ctgctcttgg gcactgtggc ctgcagcatc tctgcacccg cccgctcgcc 600<>

cagccccagc acgcagccct gggagcatgt gaatgccatc caggaggccc ggcgtctcct 660<>

gaacctgagt agagacactg ctgctgagat gaatgaaaca gtagaagtca tctcagaaat 720<>

gtttgacctc caggagccga cctgcctaca gacccgcctg gagctgtaca agcagggcct 780<>

gcggggcagc ctcaccaagc tcaagggccc cttgaccatg atggccagcc actacaagca 840<>

gcactgccct ccaaccccgg aaacttcctg tgcaacccag attatcacct ttgaaagttt 900<>

caaagagaac ctgaaggact ttctgcttgt catccccttt gactgctggg agccagtcca 960<>

ggagtgaatt agaaaaaaac gtacgggtag aaggtgtgaa tctcgagtgc gaggccgaag 1020<>

ctcaaactcg agagagcctt ctgctgacat gtcttccgta ttcgacgaat atgagcagct 1080<>

cctcgcggct cagactcgcc ctaatggagc tcacggagga ggagaaaagg ggagcacttt 1140<>

aaaagttgag gtcccggtat tcactcttaa cagtgatgat ccagaagaca gatggaattt 1200<>

tgcggtattc tgtcttcgga ttgctgttag cgaggatgcc aacaaaccac tcaggcaagg 1260<>

tgctctcata tctctcttat gctcccactc tcaagtgatg aggaaccatg ttgcccttgc 1320<>

agggaaacag aatgaggcca cactggctgt tcttgagatc gatggtttta ccaacagtgt 1380<>

gccccagttc aacaacagga gtggagtgtc tgaagagaga gcacagagat tcttgatgat 1440<>

agcaggatct ctccctcgag catgcagcaa tggtactccg ttcgtcacag ctggggttga 1500<>

agatgatgca ccagaagata tcactgatac tctggaaaga atcctatcta tccaggctca 1560<>

agtatgggtc acggtagcaa aggccatgac tgcatatgag acagcagatg agtcggaaac 1620<>

aagaagaatc aataaatata tgcagcaagg cagagtccag aagaagtaca tcctccaccc 1680<>

cgtatgcagg agtgcaattc aactcacgat cagacattcc ctggcagtcc gcattttctt 1740<>

agttagcgag cttaagagag gccgcaacac ggcaggtggg agctccacct attacaactt 1800<>

ggtaggggat gtagactcat acatcaggaa caccgggctt actgcattct tcctgacact 1860<>

caaatatggg attaatacca agacatcagc ccttgcactc agcagcctca caggcgatat 1920<>

ccaaaaaatg aagcagctca tgcgtttata tcggataaaa ggagaaaatg cgccatacat 1980<>

gacattgcta ggtgacagtg accagatgag ctttgcacca gctgagtatg cacaacttta 2040<>

ctctttcgcc atgggcatgg catcagtcct agataaagga actggcaaat accaatttgc 2100<>

cagagacttt atgagcacat cattttggag acttggagta gagtatgctc aggctcaggg 2160<>

aagtagcatc aatgaggata tggctgccga gctaaaacta accccagcag caaggagggg 2220<>

cctggcagct gctgcccaac gagtgtctga ggagaccggc agcatggata ttcctactca 2280<>

acaagccggg gtcctcaccg ggctcagcga cggaggccct cgagccccac aaggtggatc 2340<>

aaacaggccg caagggcaac cggatgccgg agatggggag acccaattcc tggatttgat 2400<>

gagagcggtg gcaaatagca tgcgagaagc gccaaactcc gcacagagca ccacccaacc 2460<>

ggagcctcct tcaactcctg ggccgtccca aggcaatgac accgactggg ggtactgacc 2520<>

gacaataccc agcctgcctc cacaggacca ccccaacccc tctgcccgca ccccaccccc 2580<>

tgatccgcag ccccgcatgg ccaaacccac aaaagaaccc ccccatctcc cctcctccct 2640<>

ccagctgcac gaccccaccc gcccaaggca acataggcac agcccgaccc accaacagtc 2700<>

tatacagagc caaagatatt agaaaaaaat acgggtagaa gagaggcatt cagagaccaa 2760<>

gacgagtcac tagggtctct gttctccctt ctacccagcg gattagggtg aagatggcca 2820<>

cctttacaga tgcggagatc gacgatatat ttgagaccag cggaactgtc attgacagca 2880<>

taattacggc ccagggtaaa tcagcagaga ctgtcgggag gagcgcaatc ccacaaggca 2940<>

agaccaaagc gttaagcgca gcatgggaga agcatgggag catccaacca ccggccagcc 3000<>

aagataccct tgaccaacag gatagatcag acaagcagcc atccacacct gaacaggcga 3060<>

ctccacataa cagcccgcca gccacatcca ccgaccagcc ccccacccag gccgcaggcg 3120<>

aggccggcga cacacagctc aagaccggag caagcaactc acttttgtcg atgctcgaca 3180<>

agcttagcaa taaatcgtct aatgctaaaa agggcccatg ggcgagtccc caggaagggc 3240<>

atcatcaacc tccggcccaa cagcagggga gtcaaccgag ccgcgggaac aatcaggaaa 3300<>

gaccgcagaa ccaggccaag gccgcccctg gagaccgggg cacagacgcg aacacagcat 3360<>

atcctggaca atggaaggag tcacaactat cagctggtgt aacccctcat gcgctccggt 3420<>

cagggcagag ccaagacaat actcctgcac ctgtggatca tgtccagcta cctgtcgact 3480<>

ttgtgcaggc gatgatgtct atgatggagg cgttatcaca gagggtaagt aaagttgact 3540<>

atcagctaga cctagtttta aaacagacat cctccatccc catgatgcgg tctgaaatcc 3600<>

aacagctaaa aacatccgtt gcggtcatgg aagccaattt gggcatgatg aaaattctgg 3660<>

accctggttg tgctaacgtt tcatctctaa gtgatttacg ggcagtcgcc cgatcccacc 3720<>

cagttttaat ttcaggtccc ggagatccat ctccttatgt gacgcaaggg ggcgaaatga 3780<>

cactcaataa actttcacaa ccagtgctac atccgtctga gttaattaaa cctgccacgg 3840<>

caagcgggcc tgacatggga gtggagaagg acactgtccg tgcattgatc acctcacgtc 3900<>

cgatgcatcc gagctcctca gctaagctcc tgagtaagct ggatgcagcc gggtcgattg 3960<>

aagagatcag gaaaatcaag cgccttgcgc tgaatggctg atcactatca caacctacaa 4020<>

caggttcccg ccttttcagt gtcacaagga ctctgccctg agctttcccc cataaaccca 4080<>

agcttcaaca ctttaggtga taaccccttc tcacctcccc taccccattg agtgatcgcg 4140<>

caactgcaat taatctagca gcattaaaga ttaagaaaaa aaacgggtag aatcggagtg 4200<>

ccccgactgc gtcaaaatgg actcatccag gacaatcggg ctgtacttcg attccaccct 4260<>

tccttccagc aacctgttag cattcccgat cgtcctacaa gacacaagag atgggaagaa 4320<>

gcaaatcacc ccgcaatata ggatccagcg tcttgactcg tggacagaca gtaaagaaga 4380<>

ttcggtattt atcaccacct atggattcat cttccaggtt gggagtgagg aagtcactgt 4440<>

cggcatgatc aatgataatc ccaagcacga gttactttcc tctgcgatgc tctgcctagg 4500<>

aagtgtcccg aatgatggag atcttgttga gctggcgagg gcctgcctta ctatggtggt 4560<>

aacatgcaag aagagtgcga ctaatactga gagaatggtc ttctcggtag tgcaagcacc 4620<>

ccgggtactg caaagctgta cggttgtggc gaacagatac tcgtcagtga atgcagttaa 4680<>

gcacgtgaaa gtaccagaga agatccctgg gagcggaacc ctagagtaca aggtgaactt 4740<>

tgtctctctg actgtggtgc cgaggaagga tgtctacagg atcccaaccg cggcattgaa 4800<>

agtgtctggc tcgagcctgt acaatcttgc gctcaatgtc actattgatg tggaggtgga 4860<>

cccgaagagc ccgttagtca aatccctttc caagtccgac agtggatact acgctaatct 4920<>

cttcttgcat atcggactta tgtccactgt agataagaag gggaagaaag tgacatttga 4980<>

caagctggag aggaagataa ggagactcga tctatccgtc gggctcagtg atgtgctcgg 5040<>

accttccgtg cttgtgaagg cgagaggtgc acggactagg ctgctggcac cttttttctc 5100<>

tagcagtggg acagcctgct atcctatagc aaatgcctct cctcaggtag ctaagatact 5160<>

ctggagtcaa actgcgcgcc tgcggagtgt aaaagtcatc atccaagcgg gcacccaacg 5220<>

cgctgtcgca gtgactgctg accatgaggt tacctctact aagatagaga agaggcatac 5280<>

cattgctaaa tacaatcctt tcaagaaata ggctgcatct ctgagattgc aatccgcccg 5340<>

ctttcccgaa tcaccatgat actagataat gatctgtctt gattgctcac agttagttta 5400<>

cctgtctatc aaattagaaa aaacacgggt agaagaattt ggatcccggt tggcacattc 5460<>

aaggtgcaag atgggctcca gatcttctac caggatcccg gtacctctaa tgctgatcat 5520<>

ccgaattgcg ctgacactga gctgtatccg tctgacaagc tctcttgatg gcaggcctct 5580<>

tgcagctgca gggatcgtgg taacaggaga taaagcagtc aacatataca cctcatccca 5640<>

gacagggtca atcatagtca agttactccc aaatatgccc aaggacaaag aggcgtgtgc 5700<>

aaaagcccca ttagaggcat acaacaggac actgactact ttgctcaccc cccttggcaa 5760<>

ttctatccgc aggatacaag agtctgtgac tacttccgga ggaaggagac agagacgctt 5820<>

tataggtgcc attatcggca gtgtagctct tggggttgca acagctgcac agataacagc 5880<>

agcctcggcc ctgatacaag ccaaccagaa tgctgccaac atcctccggc ttaaggagag 5940<>

cattgctgca accaatgaag ctgtgcacga ggtcactgac ggattatcac aactagcagt 6000<>

ggcagtaggg aagatgcagc agtttgttaa tgaccagttt aataatacag cgcaagaatt 6060<>

ggactgtata aaaattacac agcaggtcgg tgtagaactc aacttgtacc taactgaatt 6120<>

gactacagta ttcgggccac aaatcacttc ccctgcctta actccgctga ctatccaggc 6180<>

gctttacaat ttagctggtg gtaatatgga ttacttgctg actaagttag gtgtagggaa 6240<>

caaccaactc agctcattaa ttggtagcgg cttgatcacc ggcaacccta ttctgtacga 6300<>

ctcacagact cagatcttgg gtatacagat aactttgcct tcagtaggga acctaaataa 6360<>

tatgcgtgcc acctacttgg agaccttatc tgtaagcaca accaagggat ttgcctcagc 6420<>

acttgtccca aaagtggtga cacaggtcgg ttccgtgata gaagaacttg acacctcata 6480<>

ctgtatggag accgacttgg atctatactg tacaagaata gtgacattcc ctatgtctcc 6540<>

tggtatttat tcctgtctga gcggtaatac atcggcttgc atgtattcaa agactgaagg 6600<>

cgcacttact acgccatata tggctctcaa aggctcagtt atcgccaatt gcaagatgac 6660<>

aacatgtaga tgtgcagacc ccccgggtat catatcgcaa aattatggag aagctgtgtc 6720<>

cttaatagat aggcactcat gcaatgtctt atccttagac gggataactc tgaggctcag 6780<>

tggggaattt gatgcaacct atcaaaagaa tatctcaata ctagattctc aagttatagt 6840<>

gacaggcaat ctcgatatat caactgagct tgggaatgtc aacaactcaa taagtaatgc 6900<>

cctgaataag ttagaggaaa gcaacagcaa actagacaaa gtcaatgtca aactgaccag 6960<>

cacatctgct ctcattacct acatcgtttt aactgtcata tctcttgttt ttggtgtact 7020<>

tagcctggtt ctagcatgct acctgatgta caagcaaaag gcacaacaaa agaccttatt 7080<>

atggcttggg aataataccc ttgatcagat gagagccact acaaaaatat gaatgcagac 7140<>

gagaggcgga ggtatcccca atagcaattt gcgtgtaaat tctggcaacc tgttaattag 7200<>

aagaattaag aaaaagctac tggatgtaag tgacaaaaaa gcaatacacg ggtagaaccg 7260<>

gtattatcta gattaagaaa aacacgggta gaacggtcgg agaagccacc cctcaatcgg 7320<>

gaatcaggcc tcacaacgtc ctttctaccg catcaccaat agcagacttc ggtcatggac 7380<>

cgtgcagttg gcagagttgc gctagagaat gaagaaagag aagcgaagaa tacatggcgc 7440<>

tttgtattcc ggatcgcaat ctttctttta atagtaataa ccttagccat ctctgcagcc 7500<>

gccctggtat atagcatgga ggctagcacg tctggcgacc ttgttggcat accaactgtg 7560<>

atctctaggg cagaggaaaa gattacatct gcactcagtt ctaatcaaga tgtagtagat 7620<>

aggatatata agcaggtggc ccttgagtct ccgttggcgt tgctaaacac tgaatctgta 7680<>

attatgaatg caataacgtc tctctcttat caaatcaatg gagctgcaaa taatagcggg 7740<>

tgtggggcac ctgttcatga cccagattat atcgggggga taggcaaaga acttattgta 7800<>

gatgacgcta gtgatgtcac atcattctac ccctctgcgt tccaagaaca cctgaatttt 7860<>

atcccggcac ctactacagg atcaggttgc actcggatac cctcattcga cataagtgct 7920<>

acccactact gttacactca caatgtgata ttatctggtt gcagagatca ctcacactca 7980<>

catcagtact tagcacttgg tgtgcttcgg acatctgcaa cagggagggt attcttttct 8040<>

actctgcgtt ccatcaattt ggatgacaac caaaatcgga agtcttgcag tgtgagtgca 8100<>

actcccctag gttgtgatat gctgtgctct aaaatcacag agactgagga agaggattat 8160<>

agttcagtta cccccacatc tatggtgcat ggaaggttag ggtttgacgg tcaataccat 8220<>

gagaaggacc tagacgtcat aactttattt aaggattggg tggcaaatta cccaggagtg 8280<>

gggggtgggt cttttattga caaccgcgta tggttcccag tctacggagg gctaaaaccc 8340<>

aattcgccta gcgacaccgc acaagaaggg agatatgtaa tatacaagcg ctacaatgac 8400<>

acatgcccag atgaacaaga ttaccagatt cggatggcta agtcttcgta taagcctggg 8460<>

cggtttggtg gaaaacgcgt acagcaagcc atcttatcta tcaaggtgtc aacatctttg 8520<>

ggcgaggacc cggtgctgac tgtaccgcct aatacagtca cactcatggg ggccgaaggc 8580<>

agagttctca cagtagggac atctcatttc ctgtatcaac gagggtcttc atacttctct 8640<>

cccgctttat tataccccat gacagtcaac aacaaaacgg ctactcttca tagtccttac 8700<>

acattcaatg ctttcactcg gccaggtagt gtcccttgcc aggcatcagc aagatgcccc 8760<>

aactcatgtg tcactggagt ttatactgat ccgtatccct tagtcttcca taggaaccac 8820<>

accttgcggg gggtattcgg gacaatgctt gatgataaac aagcaagact taaccctgta 8880<>

tctgcagtat ttgataacat atcccgcagt cgcataaccc gggtaagttc aagcagtacc 8940<>

aaggcagcat acacgacatc gacatgtttt aaagttgtca agaccaataa aacatattgc 9000<>

ctcagcattg cagaaatatc caataccctc ttcggggaat tcaggatcgt tcctttacta 9060<>

gttgagattc tcaaggaaga tggggtttaa gaagccaggt ccggccagtt gagtcaactg 9120<>

tgggaggatt ggaaagatga cattgtgtca cctatttttt gtaatgccaa gggtcaaact 9180<>

gaataccggc gcgagcccga atcctacgct gccagtcagc cataattagc tagtgctaat 9240<>

atgattagtc ttaatcttgt cgatagtcac ttggttaaga aaaaatatga gtggtagtga 9300<>

gatacacggc taaacaactc acggaggata gcacgggtag gacatggcaa gctccggtcc 9360<>

cgaaagggca gagcatcaga tcatcctacc agagtcacac ctatcctcac cattggtcaa 9420<>

gcacaaactg ctctattact ggaaattgac tgggctaccg cttcctgacg aatgtgactt 9480<>

cgaccacctc attatcagcc gacaatggaa gaaaatactt gaatcggcca ctcctgacac 9540<>

tgagaggatg ataaaactcg ggcgggcagt acaccagact ctcaaccaca gttccaggat 9600<>

aaccggagta ctccatccca ggtgtttaga agaactggct agtattgagg tccctgattc 9660<>

aaccaacaaa tttcggaaga tcgagaagaa gatccaaatt cacaacacaa ggtatggaga 9720<>

actgtttaca aggctgtgca cgcatgtaga aaagaaattg ttggggtcgt cctggtctag 9780<>

caatgtccca cgatcagagg aattcaacag catccgtaca gatccggcat tctggtttca 9840<>

ctcaaaatgg tccagagcta agtttgcatg gctccatata aaacaggtcc aaaggcacct 9900<>

gattgtagca gcaagaacaa ggtccgcagt caacaaatta gtgacgctga cccataagat 9960<>

aggccaagtc tttgttactc ctgagcttgt cattgtgaca catacagatg agaacaagtt 10020<>

cacatgcctc acccaggaac ttgtgttgat gtatgcggat atgatggagg gcagagatat 10080<>

ggtcaacata atatcatcca cggcagcaca tctcaagagc ttatcagaga aaattgatga 10140<>

cattctgcgg ttagtagatg ctctggcaaa agatttgggc aatcaagtct acgatgttgt 10200<>

agcactaatg gagggattcg catacggcgc cgttcagctg ctggagccgt caggtacatt 10260<>

tgcaggggat ttcttcgcat tcaacctgca ggagctcaaa gatactctaa tcgagctcct 10320<>

tcccaaggat atagcagagt ccgtgactca cgcaatcgcc accgtattct ctggcttaga 10380<>

acagaatcaa gcagctgaga tgttgtgcct gctgcgtctg tggggtcacc cactgcttga 10440<>

gtcccgtatt gcagcaaaag cagtcaggag ccagatgtgc gcaccgaaga tggtagactt 10500<>

cgatatgatc ctccaggtat tatctttctt taagggaaca atcatcaatg gatacagaaa 10560<>

gaagaatgca ggtgtgtggc cacgtgtcaa gatgggtacg atatacggga aggtcattgg 10620<>

gcagctacac gcagattcag cagagatttc acatgatgtc atgttgagag agtacaagag 10680<>

tttatctgca cttgaattcg agccatgtat agaatatgac cccgtcacca atctaagcat 10740<>

gtttctaaaa gacaaggcaa tcgcacaccc aaaagataac tggctcgcct cgtttaggcg 10800<>

aaaccttctc tctgaggacc agaagaaaca tgttaaagag gcaacctcga ctaaccgcct 10860<>

cttgatagag ttcttagagt caaatgattt tgatccatat aaggagatgg aatatctgac 10920<>

gacccttgag tacctaagag atgacaacgt ggcagtatca tactcactca aagaaaagga 10980<>

ggtgaaagtt aatgggcgga ttttcgctaa gttaacaaag aaactaagga actgtcaggt 11040<>

aatggcagaa gggattctag ccgaccagat tgcacctttt ttccagggga atggggtcat 11100<>

tcaggatagc atatctttga ctaagagtat gctagcgatg agtcaactgt ccttcaacag 11160<>

caataagaaa cgtatcaccg actgcaagga aagagtatcc tcaacccgca atcacgatcc 11220<>

gaagagcaag aatcgtcgga gagttgccac ctttattaca actgacctgc aaaagtattg 11280<>

tcttaactgg agatatcaga cagtcaagct gtttgctcat gccatcaatc agctgatggg 11340<>

cctacctcac ttctttgagt ggattcatct tagactcatg gatactacga tgtttgtagg 11400<>

agacccttac aatcctccaa gtgacccgac cgactgtgat ctatcaagag tcccaaatga 11460<>

tgacatatat attgtcagtg ctagaggggg cattgaggga ttgtgccaga agctatggac 11520<>

aatgatctca attgctgcaa tccaacttgc tgcagcaaga tcacattgtc gtgttgcctg 11580<>

tatggtacaa ggtgacaatc aagtaatagc tgtaacgaga gaggtaagat cagatgattc 11640<>

cccggagatg gtgttaacac aattgcacca agccagtgat aatttcttca aggaattgat 11700<>

tcatgtcaat catttgattg gccataattt aaaagatcga gaaaccatca ggtcagacac 11760<>

attcttcata tacagcaaac gaatattcaa agatggagca attctcagtc aggtcctcaa 11820<>

gaactcatct aaattagtgc taatatcagg cgaccttagt gagaacactg taatgtcttg 11880<>

tgccaacatt gcatccacta tagcacggct gtgcgagaac gggcttccta aggatttctg 11940<>

ttactattta aactacctaa tgagttgtgt gcagacatac tttgattctg agttttccat 12000<>

cactaacaac tcgcaacccg attccaacca atcgtggatt gaggacatct ctttcgtgca 12060<>

ttcatatgtc ctgacccctg cccagctggg gggactgagt aaccttcaat actcaaggct 12120<>

ctacacaagg aacattggtg acccggggac tactgctttt gcagaggtca agcggttaga 12180<>

ggcagtgggg ttactgagtc ctagcattat gactaatatc ttaactaggc cacctggaaa 12240<>

tggagattgg gccagtctgt gcaacgatcc atattccttt aattttgaga ctgtcgcgag 12300<>

cccaaatatt gtacttaaga aacatacaca gaaagtccta tttgaaactt gctcaaatcc 12360<>

cctattatcc ggagtacata cagaggataa tgaagcagaa gagaaggcat tggctgaatt 12420<>

cttgctcaat caagaagtaa ttcacccacg cgtcgcgcat gctatcatgg aagcaagctc 12480<>

tgtaggtagg agaaagcaaa ttcaggggct tgttgacaca acaaacaccg tgattaagat 12540<>

tgcgctgact aggaggccac tcggcatcaa gaggctgatg cggatagtca attactcgag 12600<>

catgcatgca atgctattta gagatgatgt tttctcgtcc aacagatcca accacccctt 12660<>

agtctcttct aatatgtgtt cgctgacgct ggcagattat gcgcggaaca gaagctggtc 12720<>

acctttaaca ggaggtagaa aaatactggg tgtatctaat cctgatacca tagaacttgt 12780<>

agagggtgag atcctcagtg tcagcggagg gtgcacaaaa tgtgacagcg gagatgaaca 12840<>

gtttacctgg ttccatcttc caagcaatat agagctgacc gatgacacca gcaagaatcc 12900<>

tccgatgaga gtgccatatc tcggatcaaa gactcaagag aggagggccg cctcgcttgc 12960<>

gaaaatagcc catatgtcac cacatgtgaa ggcggcacta agggcatcat ctgtgttaat 13020<>

ctgggcttat ggggacaacg aagtaaattg gactgctgct cttaagattg caaagtctcg 13080<>

gtgcaacata agttcagagt atcttcgact attgtcaccc ctgccaacag ccgggaatct 13140<>

ccaacataga ttggatgatg gcataaccca gatgacgttt acccctgcat ctctctacag 13200<>

agtatcacct tacattcaca tatccaatga ttctcaaagg ctatttactg aagaaggagt 13260<>

caaggagggg aatgtggttt atcagcaaat tatgctcttg ggtttatctc taattgagtc 13320<>

actcttccca atgacaacaa ccaagacata tgatgaaatc acattgcacc tccacagtaa 13380<>

atttagctgc tgtatcaggg aagcacctgt tgcagttcct ttcgagctac tcgggttggc 13440<>

accagaacta agggcggtta cttcgaataa gtttatgtac gatcctagcc ctgtatcgga 13500<>

gggagacttt gcgagacttg atctagccat ctttaagagt tatgagctga atttagagtc 13560<>

atatcccaca atagagctaa tgaacattct ttcaatatct agtgggaagt tgattggcca 13620<>

gtctgtggtt tcctatgatg aagatacctc tataaagaat gacgctataa tagtgtatga 13680<>

caacacacga aattggatca gcgaagctca gaattcagat gtggtccgcc tattcgagta 13740<>

tgcggcactt gaagtgctcc tcgactgctc ttatcaactc tactatctga gggtaagagg 13800<>

cctaaacaat atcgtcctat atatgagtga tttatacaag aatatgccag gaattttact 13860<>

ctcgaatatt gcggctacga tatctcaccc catcatccat tcaaggttga atgcagtagg 13920<>

tctagtcaac catgacgggt cacaccaact tgcagacaca gatttcattg aaatgtctgc 13980<>

aaaactgcta gtctcttgca ctcgacgcgt ggtctcaggt ttatatgcag ggaataggta 14040<>

tgacctgctg ttcccatctg tcttagatga taacctgagt gagaagatgc ttcagttgat 14100<>

atctcggtta tgctgtctgt acacggtgct ctttgctaca acaagagaaa tcccgaaaat 14160<>

aagaggctta tctgcagaag agaaatgctc ggtacttact gaatacttac tgtcagatgc 14220<>

tgtgaaacca ttacttaggt ccgagcaagt gagctctatc atgtctccca acataattac 14280<>

attcccagcc aatctatatt acatgtctag gaagagcctt aatttggtca gggaaagaga 14340<>

ggatagggat actatcttgg cattgttgtt ccctcaagaa ccattgctcg agtttccttc 14400<>

agtacaagat attggtgctc gagtgaaaga tccatttacc cgacaacctg cggcgttctt 14460<>

acaagagtta gatttgagtg ccccagcaag gtatgacgcg tttacactta gtcaggttca 14520<>

ctctgaacac acattgccga acccagagga agactattta gtacgatact tgttcagagg 14580<>

aatagggact gcgtcctcct cttggtataa ggcatctcat cttctttctg tacccgaggt 14640<>

cagatgtgca aggcatggaa actccttata cttggcagaa ggaagtggag ccattatgag 14700<>

tcttcttgaa ttgcatgtac cgcatgaaac tatctattac aatacgcttt tttctaatga 14760<>

gatgaacccc ccacagcgac atttcggacc gaccccaaca cagtttctaa attcggtcgt 14820<>

ttataggaat ctacaggcgg aagtaccatg caaggatgga tttgttcagg agttccaccc 14880<>

attatggaga gagaatacag aagagagtga cctgacctca gataaagcgg tgggatatat 14940<>

cacatctgca gtgccttaca ggtctgtatc attattgcac tgtgacattg aaattcctcc 15000<>

aggatccaat caaagcttac tagatcaact ggctaccaat ttatccctga ttgccatgca 15060<>

ctctgtaaga gagggcgggg tcgttatcat caaagtactg tatgcaatgg ggtactactt 15120<>

ccatctactc atgaatttgt tcgctccatg ttccacgaaa ggatatattc tctctaatgg 15180<>

ctatgcatgt agaggggata tggagtgtta cttgatattt gtcatgggct atctaggcgg 15240<>

gcctacattt gtgcacgagg tggtgaggat ggcaaaaact ctagtacggc ggcacggtac 15300<>

acttctgtct aaatcagatg aaattacact gactaggtta tttacctcac agcagcatcg 15360<>

tgtaacagac atcctatcca gccctttacc gagactaatg aagttcttga gagagaatat 15420<>

tgatgctgcg ctgattgaag ccgggggaca gcctgtccgt ccattctgtg cagagagttt 15480<>

agtgagcaca ctaaaagata tgactcagat gacccagatc atcgccagcc acattgacac 15540<>

ggtcattcga tctgtgatct acatggaagc tgagggtgat cttgccgaca cagtgttctt 15600<>

atttacccct tacaatctct ctacagacgg taaaaagagg acatcactta aacagtgcac 15660<>

aagacagatc ctagaggtca caatactggg tctcagggcc aaagatctca ataaagtagg 15720<>

tgatgtaatc ggcctagtgc tcagaggtat gatttctctg gaggacctaa tcccactgag 15780<>

aacatacttg aagcgtagta cctgcccgaa gtatttgaag gctgtcctag gtattactaa 15840<>

actcaaagaa atgttcacag acacctcttt attatacttg actcgtgctc aacaaaaatt 15900<>

ctacatgaaa actataggca atgcagtcaa gggatattac agtaacggta actcttaaag 15960<>

gcaatcacat attaatatgc tttccttcta gccaattgta tccttgttga cctgattata 16020<>

ccatattaga aaaaagttga atcccgaccc tttaagactc gtattcggat tcaaataatt 16080<>

atcttaaaac agaagtgcgc atagttgttc ttgattataa tcctgtcatt caccaaatct 16140<>

ttgtttggtg ggtcggcatg gcatctccac ctcctcgcgg tccgacctgg gcatccgaag 16200<>

gaggacgcac gtccactcgg atggctaagg gagggcgtag cataacccct tggggcctct 16260<>

aaacgggtct tgaggggttt tttggtcgac cggccgacgc gctgggctac gtcttgctgg 16320<>

cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct tccggcggca 16380<>

tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac catcagggac 16440<>

agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcactgga ccgctgatcg 16500<>

tcacggcgat ttatgccgcc tcggcgagca catggaacgg gttggcatgg attgtaggcg 16560<>

ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct 16620<>

cgacctgaat ggaagccggc ggcacctcgc taacggattc accactccaa gaattggagc 16680<>

caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga acatatccat 16740<>

cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg ggtcctggcc 16800<>

acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct 16860<>

tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa 16920<>

aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct 16980<>

ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg 17040<>

ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt 17100<>

gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag 17160<>

taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg 17220<>

tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga 17280<>

aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa 17340<>

actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc 17400<>

tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca 17460<>

catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc 17520<>

ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg 17580<>

tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga 17640<>

gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg 17700<>

cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 17760<>

gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 17820<>

aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 17880<>

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 17940<>

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 18000<>

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 18060<>

ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 18120<>

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 18180<>

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 18240<>

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 18300<>

tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 18360<>

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 18420<>

ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 18480<>

cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 18540<>

ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 18600<>

tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 18660<>

agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 18720<>

gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 18780<>

ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 18840<>

gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 18900<>

cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 18960<>

gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 19020<>

cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 19080<>

cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 19140<>

ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 19200<>

tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 19260<>

acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 19320<>

tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 19380<>

actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 19440<>

aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 19500<>

ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 19560<>

ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 19620<>

cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 19680<>

aggcgtatca cgaggccctt tcgtcttcaa gaa 19713<>

<>

<210> 3

<211> 10

<212> DNA

<213> Artificial Synthesis

<>

<400> 3

acgggtagaa 10<>

<>

<210> 4

<211> 11

<212> DNA

<213> Artificial Synthesis

<>

<400> 4

ttagaaaaaa a 11<>

<>

Claims

1. The recombinant oncolytic newcastle disease virus for expressing the human GM-CSF is obtained by inserting a full-length gene of the human GM-CSF into the genome of an oncolytic strain Italien strain of the newcastle disease virus, and the nucleotide sequence of the gene of the GM-CSF is shown as SEQ ID NO. 1.

2. The method for constructing a recombinant oncolytic newcastle disease virus expressing human GM-CSF of claim 1, comprising the steps of:

the nucleotide sequence of the GS is shown as SEQ ID NO. 3;

the nucleotide sequence of the GE is shown as SEQ ID NO. 4;

3. The method of constructing a recombinant oncolytic newcastle disease virus expressing human GM-CSF of claim 2, wherein the DMEM medium comprises 10% fetal bovine serum by volume fraction and 100 μ g/mL streptomycin at S2.

4. The method of claim 2, wherein the PEI transfection reagent is present at a concentration of 1mg/mL in S2.

5. The method of claim 2, wherein in S2, the plasmid mass ratio pBR-rNDV-hGMCSF: pCI-NP: pCI-P: pCI-L is mixed 2:1:1: 1.

6. Use of the recombinant oncolytic newcastle disease virus expressing human GM-CSF of claim 1 in the preparation of a medicament for the treatment of a tumor.

7. The use of the recombinant human GM-CSF-expressing oncolytic Newcastle disease virus of claim 6 for the preparation of a medicament for the treatment of a tumor, wherein said recombinant human GM-CSF-expressing oncolytic Newcastle disease virus can be used alone for the preparation of a medicament for the treatment of a tumor.

8. The use of the recombinant oncolytic newcastle disease virus expressing human GM-CSF of claim 6 in the preparation of a medicament for the treatment of a tumor, wherein the recombinant oncolytic newcastle disease virus expressing human GM-CSF is capable of being combined with other therapeutic agents to prepare a medicament for the treatment of a tumor.

9. Use of a recombinant oncolytic newcastle disease virus expressing human GM-CSF as claimed in claim 8 in the manufacture of a medicament for the treatment of tumors, wherein other therapeutic agents include, but are not limited to, surgical agents for tumors, radiation therapeutic agents, chemotherapeutic agents, antibody therapeutic agents, immunocytotherapeutic agents, interventional agents, stem cell therapeutic agents.