CN114181972A

CN114181972A - Lentiviral vectors suitable for gene therapy of refractory angiogenic eye diseases

Info

Publication number: CN114181972A
Application number: CN202111397423.8A
Authority: CN
Inventors: 凌思凯; 汪啸渊
Original assignee: Shanghai Bendao Gene Technology Co ltd
Current assignee: Shanghai Bendao Gene Technology Co ltd
Priority date: 2021-11-23
Filing date: 2021-11-23
Publication date: 2022-03-15
Also published as: WO2023093405A1

Abstract

The invention discloses a slow virus vector suitable for gene therapy of refractory angiogenesis eye diseases; its packaging materialThe particles comprise pMD.2G for expressing envelope protein, pRSV-REV for expressing REV protein, pMDlg/pRRE-IN mut for inactivating integrase mutation and a vector plasmid for expressing VEGFA antibody gene. The vector plasmid is obtained by constructing an eGFP gene in an anti-VEGFA gene replacement plasmid pCCL-PGK-eGFP; the anti-VEGFA gene is obtained by adding Fc fragment constant region CH of human IgG to the variable regions of the heavy chain and the light chain of ranibizumab respectively₃And performing codon optimization on the gene. After the non-integrated lentivirus vector provided by the invention infects cells, the cells can stably express the vascular endothelial growth factor VEGFA antibody for a long time, and can not be integrated into a human genome, thereby avoiding the influence on the genome.

Description

Lentiviral vectors suitable for gene therapy of refractory angiogenic eye diseases

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a lentiviral vector suitable for gene therapy of refractory angiogenesis ocular diseases; in particular to a modified non-integrated lentivirus vector system suitable for gene therapy of refractory angiogenesis eye diseases.

Background

The refractory angiogenesis eye diseases are a group of diseases with choroidal or retinal angiogenesis as pathological features, and comprise angiogenesis age-related macular degeneration (nAMD), diabetic retinopathy related macular edema (DME), retinal vein occlusion related macular edema (RVO-ME) and the like. Due to structural abnormality of a new blood vessel wall, leakage and bleeding of the blood vessel are caused, a series of secondary pathological changes are caused, diseases usually involve the macular region with the most close visual function, hydrops on the upper skin and the lower skin of retinal nerves or between neuroepithelial layers in the macular region are caused, obvious vision is reduced, and finally central vision is lost. After treatment, patients may still develop fibrosis and/or RPE layer atrophy, resulting in severe permanent central vision loss. Therefore, intractable angiogenic ocular diseases become an important cause of intractable blindness in the world (Wong et al 2016), and are also important eye disease for preventing blindness in the world health organization.

At present, the pathogenesis of refractory angiogenic eye diseases is not fully elucidated, but vascular endothelial growth factor (VEGF-A) is a well-recognized important factor that promotes choroidal neovascularization. VEGF-A factor is a highly specific vascular endothelial cell growth factor, and has effects of promoting vascular permeability increase, extracellular matrix degeneration, vascular endothelial cell migration, proliferation and angiogenesis. Therefore, the existing treatment method mainly inhibits the VEGF-A factor and weakens the new vascular function of the VEGF-A factor by injecting antibody medicines of the VEGF-A factor into a vitreous cavity. Anti-vascular endothelial growth factor (Anti-VEGF) therapy is currently the standard treatment for refractory angiogenic ocular diseases. The existing medicines mainly comprise Ranibizumab (Ranibizumab), Aflibercept (Aflibercept) and combisipt (Conbercept). However, the existing anti-VEGF treatment methods generally need more injections, and each injection is uncertain in time according to the condition of a patient, and one injection is needed in about 4-8 weeks on average. This places a heavy treatment burden on the patient and the doctor. Some patients experienced fatigue from anti-VEGF therapy and chose to discontinue therapy (Boulanger-scoremama et al 2015), which may lead to disease recurrence and permanent vision loss (Essex et al 2016).

In recent years, gene therapy techniques have been gradually established and perfected. The application of gene therapy to the treatment of refractory angiogenic eye diseases is a development trend towards achieving the goal of lifelong treatment with one injection. At present, a plurality of companies develop the research and development of drugs for treating intractable angiogenesis ocular diseases through gene therapy, and even enter the clinical test stage. The principle is that the virus vector with target gene segments is injected under the retina, and the effect of treating the angiogenesis diseases can be achieved by expressing angiogenesis inhibiting factors or increasing the secretion of endogenous anti-VEGF antibodies by RPE cells and effectively controlling the concentration of the VEGF in eyes at a lower level for a long time.

However, the clinically available treatments suffer from the following problems: in the economic aspect: antibody drugs are relatively expensive. Treatment of wAMD requires an antibody injection every 4-8 weeks. Besides the direct medical expenses brought by the medicine, the direct non-medical expenses such as transportation expenses and nutrition expenses, and even the indirect losses such as the error work expenses. Side effects: 1) the long-term and frequent injection of antibody drugs into the vitreous cavity causes secondary damage to the eyes; 2) long-term injection of anti-VEGF drugs in large quantities may spread throughout the body, causing problems such as VEGF inhibition of other organs or tissues of the body.

Disclosure of Invention

The invention aims to solve the problems of safety and effectiveness of treatment of refractory angiogenic eye diseases in the prior art, and provides a lentiviral vector suitable for gene therapy of the refractory angiogenic eye diseases; in particular to a non-integration type slow virus vector suitable for gene therapy of intractable angiogenesis eye diseases.

The purpose of the invention is realized by the following technical scheme: the invention provides a non-integrative lentiviral vector, wherein a packaging plasmid of the vector comprises pMD.2G for expressing envelope protein, pRSV-REV for expressing REV protein, pMDlg/pRRE-IN mut for inactivating integrase mutation and a vector plasmid pCCL-PGK-eGFP.

Integrase mutation-inactivated pMDlg/pRRE-IN mut, including pMDlg/pRRE-D64V with integrase having D64V; other position mutations are also possible, such as N120L, W235E, Q148A, K264R, K264E, F185A, D116A, D64A, H12A, D64E, D116N.

The invention also provides a preparation method of the non-integrative lentivirus vector, which is characterized IN that the plasmid pMD.2G, pRSV-REV, pMDlg/pRRE-IN mut and the vector plasmid pCCL-PGK-eGFP are co-transfected into host cells; concentrating and purifying to obtain the non-integrated lentivirus particles.

As one embodiment, the host cell comprises a HEK293T cell.

The invention also provides a non-integrative lentivirus vector system, and the packaging plasmids of the vector system comprise pMD.2G for expressing envelope protein, pRSV-REV for expressing REV protein, pMDlg/pRRE-IN mut for inactivating integrase mutation and vector plasmids for expressing VEGFA antibody genes. The carrier plasmid can be pCCL-PGK-anti VEGFA, and is constructed by replacing eGFP genes in plasmid pCCL-PGK-eGFP with anti-VEGFA genes.

VEGFA antibodies refer to antibodies that bind to VEGFA antigen, including but not limited to ranibizumab, bevacizumab, brevacizumab, combicacept, aflibercept, and the like.

In one embodiment, the amino acid sequence of the integrase is set forth in SEQ ID NO. 2.

As one embodiment, the gene sequence encoding the integrase includes the sequence shown in SEQ ID NO. 1.

As one embodiment, the anti-VEGFA gene is obtained by adding Fc fragment constant region CH of human IgG to the variable regions of the heavy chain and the light chain of ranibizumab respectively₃And performing codon optimization on the gene. The anti-VEGFA antibody is transformed from ranibizumab and human IgG, and has stronger binding force with VEGFA. Compared with the unmodified antibody gene against VEGFA, the optimized and modified antibody gene against VEGFA has obvious improvement on the protein expression level of human cells.

As one embodiment, the anti-VEGFA gene consists of a heavy chain and a light chain, the amino acid sequence of the heavy chain being SEQ ID No. 5; the amino acid sequence of the light chain is SEQ ID NO. 7.

In one embodiment, the gene sequence of the heavy chain is shown as SEQ ID NO. 4; the gene sequence of the light chain is shown as SEQ ID NO. 6.

As one embodiment, the vector sequence of the vector plasmid pCCL-PGK-anti VEGFA is shown in SEQ ID NO. 3.

The invention also provides a preparation method of the non-integration type lentivirus vector system, which comprises the steps of co-transfecting the plasmid pMD.2G, pRSV-REV, pMDlg/pRRE-IN mut (such as pMDlg/pRRE-D64V) and the vector plasmid into host cells, concentrating and purifying to obtain the non-integration type lentivirus system capable of expressing the anti-VEGFA antibody.

As one embodiment, the host cell comprises a HEK293T cell.

The non-integrative lentiviral vector system of the invention can be used for treating refractory angiogenic eye diseases. Including angiogenesis age-related macular degeneration (nAMD), diabetic retinopathy related macular edema (DME) and retinal vein occlusion related macular edema (RVO-ME), etc.

Therefore, the invention also provides the application of the non-integrated lentivirus vector system in preparing a preparation for treating refractory angiogenesis eye diseases.

In some embodiments, a non-integrated lentivirus vector system is provided for use in preparation of a medicament for treating angiogenesis-induced age-related macular degeneration disease, diabetic retinopathy-related macular edema, or retinal vein occlusion-related macular edema.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention utilizes non-integrated slow virus vector delivery, can better target RPE cells after being injected into the eyeground through subretinal injection or suprachoroidal injection, neutralize VEGFA from the source and achieve the effect with less virus load;

2. the non-integrated lentivirus vector provided by the invention can achieve a treatment effect through one-time injection (suprachoroidal injection); the treatment burden of doctors and patients, such as eye trauma caused by multiple injections, is avoided;

3. after the non-integrated lentiviral vector provided by the invention infects cells, the cells can stably express the vascular endothelial growth factor VEGFA antibody for a long time, and can not be integrated into a human genome, so that the influence on the genome is reduced;

4. the expression level of the anti-VEGFA antibody can be regulated and controlled through dosage, so that the systemic diffusion of the anti-VEGFA antibody is reduced while the treatment effect is achieved, and the negative influence of the excessive anti-VEGFA antibody on human organs and tissues is avoided.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic composition of an anti-VEGFA antibody;

FIG. 2 is a schematic representation of in vitro expression of anti-VEGFA antibodies;

FIG. 3 is a schematic representation of the in vivo inhibition of neovascular proliferation by VEGFA antibodies.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.

The invention relates to a method for modifying a lentivirus vector into a non-integrated lentivirus vector to deliver a modified and optimized anti-VEGF antibody gene. Specific operation examples are as follows:

1. engineering the integration properties of lentiviral vectors;

common lentiviral vectors are characterized by gene integration. The packaging plasmids of the third generation lentivirus are pMD.2G, pRSV-REV, pMDlg/pRRE and third generation lentivirus vector plasmid pCCL-PGK-eGFP (the plasmid gene sequence is shown in SEQ ID NO. 12). In the invention, the 64 th amino acid aspartic acid (D) of the ordinary lentivirus integrase is mutated into valine (V), and the integrase is inactivated, so that the lentivirus loses the characteristic of gene integration. The integrase gene in the plasmid pMDlg/pRRE is subjected to base mutation to construct the plasmid pMDlg/pRRE-D64V. In the production of non-integrative lentiviruses, plasmids pMD.2G, pRSV-REV, pMDlg/pRRE-D64V and the vector plasmid pCCL-PGK-eGFP were co-transfected into HEK293T cells. Concentrating and purifying to obtain the non-integrated lentivirus particles.

2. Engineering and optimizing genes for anti-VEGFA antibodies

In the present invention, the constitution of the anti-VEGFA antibody (fig. 1) is: the variable regions of the heavy chain and the light chain of the ranibizumab are respectively added with Fc fragment constant regions CH3 of human IgG (the gene sequence of the heavy chain is shown as SEQ ID NO.4, the amino acid sequence is shown as SEQ ID NO.5, the gene sequence of the light chain is shown as SEQ ID NO.6, and the amino acid sequence is shown as SEQ ID NO. 7). And subjecting the gene to codon optimization suitable for expression in mammalian eukaryotic cells. Codon optimization was performed by sequencing tools to optimize genes to sequences with increased expression in eukaryotic cells, especially human cells, without altering the amino acid sequence of the expressed protein. The heavy chain gene is preceded by a section of Ig k-chain leader (the gene sequence is shown as SEQ ID NO.8, and the amino acid sequence is shown as SEQ ID NO. 9). A P2A sequence is added between the heavy chain and the light chain gene for the segmentation of the protein maturation (the gene sequence is shown as SEQ ID NO.10, the amino acid sequence is shown as SEQ ID NO. 11),

3. lentivirus vector packaging anti-VEGFA antibody gene

The optimized VEGFA-resistant gene is constructed into a lentiviral vector plasmid pCCL-PGK-eGFP. During construction, the anti-VEGFA gene replaces the eGFP gene in the original plasmid. The plasmid pCCL-PGK-anti VEGFA was constructed. Plasmids pMD.2G, pRSV-REV, pMDlg/pRRE-D64V and the vector plasmid pCCL-PGK-anti VEGFA were co-transfected into HEK293T cells. Concentrating and purifying to obtain the non-integrated lentivirus particle capable of expressing the anti-VEGFA antibody, namely BD 311. After BD311 infects cells, the vector gene is dissociated outside the cell genome through reverse transcription into circular DNA, and the VEGFA protein is expressed to achieve the treatment effect.

See the following examples for details:

example 1: construction of pMDlg/pRRE-D64V plasmid

1. Synthetic genes: the integrase gene with the D64V mutation was synthesized.

2. The synthesized gene was cloned into the plasmid pMDlg/pRRE, replacing the original integrase gene. Reagent for cloning

HiFi DNA Assembly was partially seamlessly cloned.

Example 2: construction of plasmid pCCL-PGK-anti VEGFA expressing VEGFA antibody

1. Design of genes to be synthesized: when synthesizing the anti-VEGFA antibody gene, restriction enzyme cleavage site BamHI, and Kozak sequence were added to the 5' end of the gene. The P2A sequence was added between the heavy and light chains for partitioning the heavy and light chains during protein maturation. The gene sequence of P2A is shown as SEQ ID NO.10, and the amino acid sequence is shown as SEQ ID NO. 11. A restriction site XhoI was added at the 3' end.

2. Genes were synthesized based on the designed gene sequences.

3. The synthesized gene and the vector pCCL-PGK-eGFP are cut by restriction enzyme BamHI/XhoI, and the gene and the vector are connected after DNA purification to construct plasmid pCCL-PGK-anti VEGFA.

Example 3: production of non-integrating Lentiviral particles expressing anti-VEGFA antibodies-BD 311

1. The first day, HEK293T cells were seeded into 15cm cell culture dishes with a cell count of 1.3X 10⁷Each dish.

2. The next day, 24 hours after cell seeding, the density was close to 85%. The cell complete medium was replaced with fresh one. Transfection was performed after 1 hour. Plasmid 9.07. mu.g pMD.2G, 7.26. mu.g pRSV-REV, 31.46. mu.g pMDlg/pRRE-D64V and 31.46. mu.g vector plasmid pCCL-PGK-anti VEGFA were co-transfected into HEK293T cells.

In this example, calcium phosphate transfection was used for transfection, and the procedure was as follows:

1) taking a 15mL centrifuge tube, sequentially adding sterile water and various plasmids to prepare DNA MIX with the total volume of 1089 mu L, and uniformly mixing;

2) 121 mu.L of 2.5M CaCl is slowly dropped on the surface of the DNA MIX liquid₂The solution is gently sucked, repeatedly dropped and uniformly mixed (about 3 times);

3) dripping 1210 μ L of 2 × HEBS solution, mixing, and standing;

4) the liquid was added dropwise to the cells over 5 minutes, mixed well and placed in an incubator.

3. On the third day, the original medium was discarded and replaced with fresh complete cell culture medium.

4. On day four, the crude virus fluid was harvested 48 hours after plasmid transfection and fresh complete cell culture medium was added.

5. On the fifth day, the crude virus fluid was collected 72 hours after plasmid transfection and combined with the crude virus fluid collected on the previous day. Adding totipotent nuclease, and treating the crude virus liquid at 37 ℃ for 1 hour. The crude virus solution was filtered into ultrafiltration tubes using a 0.45 μm disposable filter, 32mL of crude virus solution was added to each tube, and 6mL of 20% sucrose in PBS was added to the bottom of the tube to allow clear limits for sucrose and crude virus solution. The ultrafiltration tube was placed in an adapter of an ultracentrifuge, equilibrated and centrifuged in a Beckman & optima L-100XP ultracentrifuge at 25000rpm for 2 hours at 4 ℃. After centrifugation, the ultracentrifuge tube was removed, the supernatant was decanted off, the inner tube wall was wiped dry, and 100 μ L PBS was added to soak the lentiviral particles for 4-18 h.

6. After soaking completely, the lentivirus is evenly blown and sucked by a pipette, subpackaged and stored in a refrigerator at minus 80 ℃.

Example 4: in vitro infection of BD311 on 293T cells expressing anti-VEGFA antibody

The experimental method comprises the following steps:

1. the first day, HEK293T cells were plated at 4X 10⁴Density per well was seeded in 48 well plates,

2. the following day, cells were inoculated 24h before infection with BD 311. The infection amount is based on the amount of p24 which is the physical titer of the lentiviral vector. Three gradients of 100ng, 200ng and 400ng, respectively. Each gradient was 3 replicates.

3. On the third day, 500mL of fresh complete medium was changed after infection with BD 31118 h.

4. On the fourth day, cell supernatants were collected 48 hours after infection, and the antibody expression was measured by ELISA.

The blank coating protein used for detecting ELIAS is VEGF165, and the standard product is anti-VEGF antibody protein purified after in vitro expression.

5. The results show (FIG. 2) that the expression level of the anti-VEGF antibody increases with the increase in the amount of BD311 infected. The wells with the highest expression amount reach 56 ug/mL.

Example 5: BD311 inhibits angiogenesis in vivo

The effect of BD311 in slowing choroidal neovascularization was verified using a laser-induced mouse macular degeneration model.

The specific operation is as follows:

1. subretinal injection. In vivo experiments were performed in C57BL/6J, male, 6 week old mice. After anesthetizing the mice with 1.25% tribromoethanol, subretinal injections were performed under a dissecting scope using a microinjector (Hamilton). 2uL (100ng p24) BD311 injection was injected into the right eye of the mice and 2uL PBS was injected subretinally into the left eye of the mice as a control.

2. And (4) laser induction. After 7 days of subretinal injection, mice were anesthetized with 1.25% tribromoethanol, 4 Laser burns around the optic disc were performed using Laser System (IQ 532, IRIDEX), 120mW,50 μm spot and 100ms exposure time. Laser burns that produce bubbles at the film and do not bleed represent successful molding.

3. Choroidal neovascularization was detected.

1) Taking the eyeball: after anesthetizing the mouse with 1.25% tribromoethanol and removing the neck, the eyeball of the mouse was carefully removed.

2) Fixing eyeballs: a small hole is pricked at the edge of the iris by a needle, and the whole eyeball is placed in 4% paraformaldehyde solution for fixation at room temperature for 1 h. PBS was washed 3 times.

3) And (3) dissecting an eyeball: removing excess connective tissue under a dissecting microscope; circular excision of the eye wall along the equator (iris margin); removing the cornea, lens and vitreous; bowl-shaped tissues of the retina, choroid and sclera remain; four incisions were cut edgewise towards the optic disc to divide the bowl-shaped tissue evenly into four parts, spread out flat, part of the retina up, and the retina was grasped with forceps, leaving the tissue of the RPE/choroid/sclera.

4) Dyeing: after 5% goat serum was blocked at 4 ℃ and the sample washed with 0.5% Triton X-100/PBS, it was stained with the antibody Alexa Fluor 488Conjugates isonectin GS-B4, and the staining antibody dilution ratios and methods were performed as described in the specification.

5) Tabletting: the tissue was spread on a glass slide, the fluorescence anti-quencher was added dropwise, the cover slip was covered, and the slide was mounted with mounting medium.

6) Data acquisition and processing: pictures were taken with a fluorescence microscope. ImageJ software calculates the area of choroidal neovascularization. The results are shown in FIG. 3.

The foregoing describes specific embodiments of the present invention. It is to be understood that the present invention is not limited to the specific embodiments described above, nor to the VEGFA antibody described above; in fact, the non-integrated lentivirus vector can be used for delivering the genes of antibodies such as ranibizumab, bevacizumab, brevacizumab, combicacept and aflibercept, and the expressed corresponding antibodies can be combined with VEGFA to block the angiogenesis promoting effect of the VEGFA, so that the macular degeneration disease is relieved. Various changes or modifications may be made by those skilled in the art within the scope of the claims without departing from the spirit of the invention.

Sequence listing

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aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg 2700

taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc 2760

tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc 2820

gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt 2880

tctcctcttc ctcatctccg ggcctttcga cctctagcgg gatccaccgg tcgccaccat 2940

ggagaccgac accctgctgc tgtgggtgct gctgctgtgg gtgcccggca gcaccggaga 3000

ggtgcagctg gtggagagcg gcggcggact ggtgcagcct ggaggaagcc tgagactgag 3060

ttgcgccgcc agcggctatg attttaccca ctacggcatg aactgggtga gacaggcccc 3120

cggcaaaggg ctggagtggg tgggatggat caacacatat acaggagaac ccacctacgc 3180

tgctgatttc aaaaggagat tcaccttcag cctggacacc tccaagagca ccgcctacct 3240

gcagatgaac agcctgagag ccgaggacac cgccgtgtac tactgcgcca aataccccta 3300

ctactacgga acaagccact ggtacttcga cgtgtggggc cagggaaccc tggtgaccgt 3360

gagcagcgcc cagcccagag agccccaggt gtataccctg ccccctagcc gcgacgaact 3420

gaccaaaaac caggtgagcc tgagctgcgc tgtgaaaggc ttctacccct ctgacatcgc 3480

cgtggaatgg gagagcaacg gccagcctga gaacaactac aagaccactc cccccgtgct 3540

ggacagcgac ggcagcttct tcctggtgtc taaactgact gtggacaaga gcaggtggca 3600

gcagggcaac gtgttcagtt gcagcgtgat gcacgaggcc ctgcacaacc actataccca 3660

gaagtccctg tccctgagcc ccggaaaggg ctccggcgct accaacttca gcctgctgaa 3720

gcaggccggg gacgtggagg agaaccccgg accagacatc cagctgaccc agagccccag 3780

cagcctgtcc gccagcgtgg gagacagagt gaccatcaca tgcagcgcct cccaggacat 3840

cagcaactac ctgaactggt accagcagaa gcccggcaag gcccccaaag tgctgatcta 3900

cttcactagc agcctgcaca gcggcgtgcc cagcagattt tccggcagcg gctccggcac 3960

cgacttcacc ctgactatca gcagcctgca gcccgaggat ttcgccacct actactgcca 4020

gcagtactcc accgtgccct ggaccttcgg acagggcacc aaggtggaaa ttaaaagaca 4080

gcccagagaa ccccaggtgt acaccctgcc ccccagcaga gacgaactga caaagaacca 4140

ggtgtccctg tggtgcctgg tgaagggctt ttaccccagc gatatcgccg tggagtggga 4200

gagcaatggc cagcccgaga acaactataa aacaacacca cccgtgctgg atagcgacgg 4260

ctcctttttc ctgtattcta agctgaccgt ggacaagagt agatggcagc aggggaacgt 4320

gttcagctgc agcgtgatgc atgaggccct gcataaccac tatactcaga agagcctgag 4380

cctgagcccc gggaagtaaa gcggcctcga gggaattccg ataatcaacc tctggattac 4440

aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga 4500

tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc 4560

tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa 4620

cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc 4680

acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc 4740

atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc 4800

gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg 4860

attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct 4920

tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg 4980

agtcggatct ccctttgggc cgcctccccg catcgggaat tcgagctcgg tacctttaag 5040

accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact 5100

ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct 5160

ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 5220

gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 5280

tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagcatcta 5340

gctagaatta attccgtgta ttctatagtg tcacctaaat cgtatgtgta tgatacataa 5400

ggttatgtat taattgtagc cgcgttctaa cgacaatatg tacaagccta attgtgtagc 5460

atctggctta ctgaagcaga ccctatcatc tctctcgtaa actgccgtca gagtcggttt 5520

ggttggacga accttctgag tttctggtaa cgccgtcccg cacccggaaa tggtcagcga 5580

accaatcagc agggtcatcg ctagcctagg gacgtaccca attcgcccta tagtgagtcg 5640

tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 5700

acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 5760

gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc 5820

tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 5880

gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 5940

ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 6000

cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 6060

tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 6120

ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 6180

ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 6240

tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtggcacgta 6300

gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg 6360

gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg 6420

atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc 6480

ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat 6540

ctgatggcgc aggggatcaa gctctgatca agagacagga tgaggatcgt ttcgcatgat 6600

tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta 6660

tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca 6720

ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga 6780

cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga 6840

cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct 6900

cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg 6960

gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga 7020

gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca 7080

tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga 7140

ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg 7200

cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc 7260

gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt 7320

gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga 7380

gttcttctga attattaact cgagctgtca gaccaagttt actcatatat actttagatt 7440

gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 7500

atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 7560

atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 7620

aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 7680

aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 7740

ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 7800

ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 7860

tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 7920

ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 7980

acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 8040

gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 8100

cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 8160

aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 8220

atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 8280

gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 8340

gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 8400

tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 8460

tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 8520

ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag 8580

cgcgcaatta accctcacta aagggaacaa aagctggagc tgc 8623

<210> 4

<211> 690

<212> DNA

<213> Artificial Sequence

<400> 4

gaggtgcagc tggtggagag cggcggcgga ctggtgcagc ctggaggaag cctgagactg 60

agttgcgccg ccagcggcta tgattttacc cactacggca tgaactgggt gagacaggcc 120

cccggcaaag ggctggagtg ggtgggatgg atcaacacat atacaggaga acccacctac 180

gctgctgatt tcaaaaggag attcaccttc agcctggaca cctccaagag caccgcctac 240

ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caaatacccc 300

tactactacg gaacaagcca ctggtacttc gacgtgtggg gccagggaac cctggtgacc 360

gtgagcagcg cccagcccag agagccccag gtgtataccc tgccccctag ccgcgacgaa 420

ctgaccaaaa accaggtgag cctgagctgc gctgtgaaag gcttctaccc ctctgacatc 480

gccgtggaat gggagagcaa cggccagcct gagaacaact acaagaccac tccccccgtg 540

ctggacagcg acggcagctt cttcctggtg tctaaactga ctgtggacaa gagcaggtgg 600

cagcagggca acgtgttcag ttgcagcgtg atgcacgagg ccctgcacaa ccactatacc 660

cagaagtccc tgtccctgag ccccggaaag 690

<210> 5

<211> 230

<212> PRT

<213> Artificial Sequence

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gln Pro Arg Glu

115 120 125

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

130 135 140

Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile

145 150 155 160

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

165 170 175

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

195 200 205

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

210 215 220

Ser Leu Ser Pro Gly Lys

225 230

<210> 6

<211> 642

<212> DNA

<213> Artificial Sequence

<400> 6

gacatccagc tgacccagag ccccagcagc ctgtccgcca gcgtgggaga cagagtgacc 60

atcacatgca gcgcctccca ggacatcagc aactacctga actggtacca gcagaagccc 120

ggcaaggccc ccaaagtgct gatctacttc actagcagcc tgcacagcgg cgtgcccagc 180

agattttccg gcagcggctc cggcaccgac ttcaccctga ctatcagcag cctgcagccc 240

gaggatttcg ccacctacta ctgccagcag tactccaccg tgccctggac cttcggacag 300

ggcaccaagg tggaaattaa aagacagccc agagaacccc aggtgtacac cctgcccccc 360

agcagagacg aactgacaaa gaaccaggtg tccctgtggt gcctggtgaa gggcttttac 420

cccagcgata tcgccgtgga gtgggagagc aatggccagc ccgagaacaa ctataaaaca 480

acaccacccg tgctggatag cgacggctcc tttttcctgt attctaagct gaccgtggac 540

aagagtagat ggcagcaggg gaacgtgttc agctgcagcg tgatgcatga ggccctgcat 600

aaccactata ctcagaagag cctgagcctg agccccggga ag 642

<210> 7

<211> 214

<212> PRT

<213> Artificial Sequence

<400> 7

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gln Pro Arg Glu

100 105 110

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

115 120 125

Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

130 135 140

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

145 150 155 160

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

165 170 175

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

180 185 190

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

195 200 205

Ser Leu Ser Pro Gly Lys

210

<210> 8

<211> 60

<212> DNA

<213> Artificial Sequence

<400> 8

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccgga 60

<210> 9

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 9

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<210> 10

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 10

ggctccggcg ctaccaactt cagcctgctg aagcaggccg gggacgtgga ggagaacccc 60

ggacca 66

<210> 11

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 11

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 12

<211> 7751

<212> DNA

<213> Artificial Sequence

<400> 12

aagcttggcc attgcatacg ttgtatccat atcataatat gtacatttat attggctcat 60

gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 120

cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 180

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 240

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 300

ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 360

atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 420

cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 480

acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 540

acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 600

actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 660

gagctcgttt agtgaaccgg ggtctctctg gttagaccag atctgagcct gggagctctc 720

tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt 780

agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc 840

agtgtggaaa atctctagca gtggcgcccg aacagggacc tgaaagcgaa agggaaacca 900

gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg 960

cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc 1020

gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg 1080

ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa 1140

cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga 1200

cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta 1260

gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac 1320

aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt 1380

cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt 1440

agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag 1500

agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag 1560

cactatgggc gcagcctcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat 1620

agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact 1680

cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa 1740

ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt 1800

gccttggaat gctagttgga gtaataaatc tctggaacag attggaatca cacgacctgg 1860

atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa 1920

tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt 1980

ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata 2040

gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt 2100

aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac 2160

aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta 2220

gtgaacggat ctcgacggta tcggttaact tttaaaagaa aaggggggat tggggggtac 2280

agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa 2340

aaacaaatta caaaaattca aaattttatc gatcacgaga ctagcctcga cgatggtcga 2400

gtaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc 2460

gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg 2520

taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct 2580

agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt 2640

agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg 2700

cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg 2760

aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag 2820

gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc 2880

ctcttcctca tctccgggcc tttcgacctc tagcgggatc caccggtcgc caccatggtg 2940

agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac 3000

gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag 3060

ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg 3120

accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac 3180

gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag 3240

gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac 3300

cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg 3360

gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc 3420

aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac 3480

taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 3540

agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg 3600

gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc 3660

ctcgagggaa ttccgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta 3720

ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc 3780

atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt 3840

ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg 3900

ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt 3960

tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct 4020

ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt 4080

cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct 4140

acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc 4200

ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct 4260

ccccgcatcg ggaattcgag ctcggtacct ttaagaccaa tgacttacaa ggcagctgta 4320

gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga 4380

agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg 4440

gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg 4500

cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc 4560

ttttagtcag tgtggaaaat ctctagcagc atctagctag aattaattcc gtgtattcta 4620

tagtgtcacc taaatcgtat gtgtatgata cataaggtta tgtattaatt gtagccgcgt 4680

tctaacgaca atatgtacaa gcctaattgt gtagcatctg gcttactgaa gcagacccta 4740

tcatctctct cgtaaactgc cgtcagagtc ggtttggttg gacgaacctt ctgagtttct 4800

ggtaacgccg tcccgcaccc ggaaatggtc agcgaaccaa tcagcagggt catcgctagc 4860

ctaggctttt gcgtcgagac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct 4920

cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 4980

gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 5040

gcccttccca acagttgcgc agcctgaatg gcgaatggcg cgacgcgccc tgtagcggcg 5100

cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 5160

tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 5220

gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 5280

accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 5340

tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 5400

gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 5460

cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 5520

tattaacgtt tacaatttcc caggtggcac ttttcgggga aatgtgcgcg gaacccctat 5580

ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 5640

aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 5700

tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 5760

agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 5820

cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 5880

taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 5940

tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 6000

tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 6060

cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 6120

gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 6180

cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 6240

actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 6300

ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 6360

tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 6420

tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 6480

acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 6540

ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 6600

ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 6660

ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 6720

gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 6780

ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 6840

aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 6900

gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 6960

gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 7020

aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 7080

cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 7140

tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 7200

ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 7260

atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 7320

cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 7380

ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 7440

gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 7500

cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg 7560

cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 7620

ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg 7680

aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa 7740

gctggagctg c 7751

Claims

1. A non-integrative lentivirus vector system is characterized IN that a packaging plasmid of the vector system comprises pMD.2G for expressing envelope protein, pRSV-REV for expressing REV protein, pMDlg/pRRE-IN mut for inactivating integrase mutation and a vector plasmid for expressing VEGFA antibody gene.

2. The non-integrating lentiviral vector system of claim 1, wherein the amino acid sequence of the integrase comprises the sequence set forth in SEQ ID No. 2.

3. The non-integrating lentiviral vector system according to claim 1, wherein the gene sequence of the integrase comprises the sequence set forth in SEQ ID No. 1.

4. The non-integrating lentiviral vector system of claim 1, wherein the VEGFA antibody gene is inThe variable regions of the heavy chain and the light chain of the ranibizumab are respectively added with the Fc fragment constant region CH of the human IgG₃And performing codon optimization on the gene.

5. The non-integrating lentiviral vector system of claim 4, wherein the VEGFA antibody gene consists of a heavy chain and a light chain; the amino acid sequence of the heavy chain is SEQ ID NO.5, and the amino acid sequence of the light chain is SEQ ID NO. 7.

6. The non-integrative lentiviral vector system according to claim 4, wherein the gene sequence of the heavy chain of the VEGFA antibody gene is shown as SEQ ID No.4, and the gene sequence of the light chain is shown as SEQ ID No. 6.

7. The non-integrative lentiviral vector system according to claim 1, wherein the vector plasmid is pCCL-PGK-anti VEGFA, and the vector sequence is shown in SEQ ID NO. 3.

8. A method for preparing the non-integrative lentiviral vector system according to any one of claims 1 to 7, wherein the plasmid pMD.2G, pRSV-REV, integrase mutation-inactivated pMDlg/pRRE-IN mut and the vector plasmid are co-transfected into a host cell, concentrated and purified to obtain the non-integrative lentiviral system capable of expressing an anti-VEGFA antibody.

9. Use of a non-integrating lentiviral vector system according to any one of claims 1-7 in the preparation of a formulation for the treatment of a refractory angiogenic eye disease.

10. The use of claim 9, wherein the formulation comprises a formulation for treating an angiogenic age-related macular degeneration disease, diabetic retinopathy-related macular edema, or retinal vein occlusion-related macular edema.