CN108379597A - A kind of genophore and its gene therapy medicament for treating retinal ganglion cells degeneration - Google Patents

Abstract

A kind of gene therapy medicament the present invention relates to genophore and its for treating retinal ganglion cells degeneration, the genophore include dominant suppressive (dominant negative) mankind Fas associatedprotein with death domain (FADD DN), enhancers/promoters, the β globin intrones for increasing gene expression, human growth hormone poly (A) tail sequences etc..Application of the composition the invention further relates to said gene carrier or comprising said gene carrier in the drug for preparing treatment retinal ganglion cells degeneration.Its advantage is shown：The gene therapy vector of the present invention can be used to treat or prevent the retinal ganglial cells lesion caused by glaucoma etc..

Description

A kind of genophore and its gene for treating retinal ganglion cells degeneration are controlled Treat drug

Technical field

The present invention relates to gene engineering technology fields, specifically, being a kind of genophore and its for treating retina The gene therapy medicament of ganglion cells degeneration.

Background technology

Retinal ganglion cells degeneration is the master that the diseases such as glaucoma (glaucoma) cause vision impairment even to be blinded Reason is wanted, it is its Major Risk Factors that pathologic intraocular pressure, which increases,.Glaucoma pathomechanism is complicated, there is the factors such as h and E ginseng With, but its definite molecular mechanism is still not clear.Part glaucoma patient shows as intraocular hypertension, and drop intraocular pressure etc. pair can be taken in clinic Disease treatment means, but in the development that can not reverse or delay glaucoma for a long time.It has now realized that, glaucoma belongs to a kind of god Through degenerative disease, pathological characters are the apoptosis of retinal ganglial cells and its denaturation of aixs cylinder and incident Visual function progressive is lost.RGC is responsible for exporting the visual signal captured by retina to brain, due to intraocular hypertension, a variety of something lost RGC caused by biography factor or nerve retrograde affection etc. is persistently denaturalized or apoptosis, and papillopathy, the visual field can be caused to lack It loses, and then develops into glaucoma.

Effect of the er stress in nerve cell death is just causing more and more to pay close attention to.Cell is to endoplasmic reticulum function The response of obstacle is known as " er stress (endoplasmicreticulum stress, ERstress) ", shows as endoplasmic reticulum Intracavitary false folding assembles with unfolded protein and calcium ion Balance disorders, can activate Non-adhesion inhibition index (unfoldedprotein response,UPR).Er stress is most important to cell survival, but continues not alleviate interior Matter net stress lead to cell death.In the mammalian body, UPR is mainly activated by three kinds of er stress sensitive Proteins：Albumen Kinase RNA sample ER kinases (proteinkinase RNA-like ERkinase, PERK), inositol dependent protein -1 (inositol-requirinprotein-1, IRE1) and transcriptional factors (activatingtranscriptionfactor-6,ATF6).Wherein IRE1 has mediated the rush apoptosis effect of er stress. TRADD (tumornecrosis factorreceptortype 1R-associateddeath domain) and FADD (Fas- Associatedproteinwithdeathdomain) it is the important substance for adjusting UPR.FADD is a kind of adaptor protein, itself No signal act on, combined with Fas or TNF receptors by its death domain (deathdomain), so with TRADD phase interactions With activation caspase8 and caspase3 is to active cell apoptosis.There is research to confirm, lacks the FADD variations of death domain Body (dominantnegative FADD, FADD-DN) can by lower PERK, the related signaling molecules such as CHOP, Bip lower in Matter net stress level.In addition, the overexpression of FADD-DN can effectively block the formation of IRE1 α, so substantially reduce caspase8 according to Bad neuro-epithelial cell is dead.

Recently studies have found that, in glaucoma animal model pathology early stage, ganglion cell layer of retina occurs The raising of the er stress relevant molecule level such as FADD and CHOP, IRE1 α, XBP-1, it was demonstrated that er stress is to cause view One of the triggering factors of film ganglion cell death.And it can effectively keep retinal ganglial cells by being overexpressed FADD-DN It is horizontal and delay neuronal degeneration.In conjunction with the development of biotechnology, it is based on adeno-associated virus (adeno-associated Virus, AAV) gene therapy method for eye disease of carrier receives significant attention, and safety and validity obtain just Step is demonstrate,proved and is approved.Therefore, present invention combination AAV technologies are developed a kind of special target retinal ganglial cells and are expressed The gene therapy vector of FADD-DN.

Invention content

First purpose of the present invention is to be directed to deficiency in the prior art, provides a kind of genophore.

Second object of the present invention is to be directed to deficiency in the prior art, provides a kind of pharmaceutical composition.

Third object of the present invention is to be directed to deficiency in the prior art, provides genophore as described above or medicine group Close the pharmaceutical applications of object

Fourth object of the present invention is to be directed to deficiency in the prior art, provides and a kind for the treatment of retinal ganglial cells The method of denaturation.

To realize above-mentioned first purpose, the technical solution adopted by the present invention is that：

A kind of gland relevant viral vector described in gland relevant viral vector contains enhancer, specificity or non-specific starts The gene order of son, expression FADD-DN albumen, the sequence such as SEQ ID NO of FADD-DN albumen:Shown in 2.

Preferably, the gene order of expression FADD-DN albumen is selected from and SEQ ID NO:1 have at least 96%, 97%, 98%, the nucleotide sequence of 99%, 100% homology.

It is highly preferred that the gene order of the FADD-DN contains SEQ ID NO:1.

Most preferably, the nucleotide sequence of the FADD-DN albumen is selected from SEQ ID NO:1.

In any of the above-described preference, enhancers/promoters CMV is selected from and SEQ ID NO:4 have it is at least 95% homologous The nucleotide sequence of property.It is highly preferred that the CMV is selected from and SEQ ID NO:4 have at least 96%, 97%, 98%, 99%, The nucleotide sequence of 100% homology.Most preferably, the nucleotide sequence of the cmv enhancer/promoter is selected from SEQ ID NO:4。

In any of the above-described preference, adeno-associated virus can be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV2-AAV3, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAVHSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6 (Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2(Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAVr3.45AAV2 or AAV5.Most preferably, gland phase It closes virus and is selected from AAV2.

In any of the above-described preference, adeno-associated virus is single-stranded AAV.

In any of the above-described preference, the chimeric interon that the gland relevant viral vector also shortens containing one section, institute The chimeric intron sequence stated is selected from and SEQ ID NO:5 have the nucleotide sequence of at least 90% homology.

In any of the above-described preference, the gland relevant viral vector contains cmv enhancer/promoter, FADD-DN Gene order, β-globin intrones, hGHpA, L-ITR, R-ITR, Ampicillin, f1ori.

In any of the above-described preference, the gene order such as SEQ ID NO of FADD-DN albumen are expressed:Shown in 1, L-ITR sequences Row such as SEQ ID NO:Shown in 3, cmv enhancer/promoter sequence SEQ ID NO:Shown in 4, β-globin intron sequences are such as SEQ ID NO:Shown in 5, hGHpA sequences such as SEQ ID NO:Shown in 7, R-ITR sequences such as SEQ ID NO:Shown in 8, Ampicillin sequences such as SEQ ID NO:Shown in 9, f1ori sequences such as SEQ ID NO:Shown in 11.

In any of the above-described preference, the gland relevant viral vector sequence contains SEQ ID NO:10.

To realize above-mentioned second purpose, the technical solution adopted by the present invention is that：

A kind of pharmaceutical composition for treating retinal ganglion cells degeneration, contains above-mentioned gland relevant viral vector and pharmacy Upper acceptable carrier, the pharmaceutical composition express FADD-DN albumen.

To realize above-mentioned third purpose, the technical solution adopted by the present invention is that：

Any gland relevant viral vector as above or pharmaceutical composition are preparing treatment retinal ganglion cells degeneration Drug in application.

To realize above-mentioned 4th purpose, the technical solution adopted by the present invention is that：

A kind of method of gene therapy retinal ganglion cells degeneration inhibits gland relevant viral vector intraocular injection FADD activity.Preferably, the intraocular injection refers to subretinal space injection or intravitreal.

The invention has the advantages that：

The gene therapy vector of the present invention is for treating or preventing the retinal ganglial cells lesion caused by glaucoma.

Description of the drawings

Attached drawing 1 is the Plasmid pattern figure of AAV_FADD-DN carriers.

Attached drawing 2 is the plasmid characteristic pattern of AAV_FADD-DN.

Attached drawing 3 is C57BL/6J mouse intravitreals AAV_FADD-DN inner nuclear layer retinas after two weeks, immunofluorescence dye Chromatic graph.

Attached drawing 4 is C57BL/6J mouse intravitreal AAV_FADD-DN or AAV-GFP, and is being injected at the moment simultaneously Side's injection bead particulates cause glaucoma, the inner nuclear layer retina after 2 weeks, 4 weeks, 8 weeks, it is illustrated that are its immunofluorescence dyeing Figure.

Attached drawing 5 is C57BL/6J mouse intravitreal AAV_FADD-DN or AAV-GFP and is injecting side at the moment simultaneously Injection bead particulates cause glaucoma, the inner nuclear layer retina after 2 weeks, 4 weeks, 8 weeks, it is illustrated that laggard for its immunofluorescence dyeing Row RGC is counted and survival rate analysis result figure.

Specific implementation mode

The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after having read the content of the invention recorded, art technology Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.

The present invention provides a kind of gland relevant viral vector and its for treating retinal ganglion cells degeneration by research Related drugs, pharmaceutical composition etc..

Pharmaceutical composition and medicine box

For the ease of clinical application, pharmaceutical composition of the invention may be embodied in injection delivery device (such as pumping needle) In, in the injection delivery device, the pharmaceutical composition of single administration amount can be included.The injection administration Device can be contained in medicine box, to facilitate storage, use.It needs to place equipped with the small container of drug suspension when transport In dry ice.It should usually be stored in -80 DEG C of refrigerators.

In medicine box of the present invention or kit, it may also include operation instructions, so that those skilled in the art press It is used according to correct mode.

As used herein, the ingredient of " pharmaceutically acceptable " is suitable for people and/or mammal and without excessively bad Side reaction (such as toxicity), i.e., with rational benefit/risk than substance.Term " pharmaceutically acceptable carrier ", which refers to, to be used for The carrier of Therapeutic Administration, including various excipient and diluent.This belongs to finger some medicament carriers in this way：Themselves is not It is necessary active constituent, and does not have excessive toxicity after applying.

Suitable pharmaceutically acceptable carrier is well known to those of ordinary skill in the art.In Remington ' It can be found about pharmaceutically acceptable carrier in sPharmaceutical Sciences (Mack Pub.Co., N.J.1991) Absolutely prove.Pharmaceutically acceptable carrier can contain liquid in the composition, such as water, BBS (Balanced SaltSolution) phosphate buffer, ringer solution, physiological saline, balanced salt solution, glycerine or sorbierite etc..In addition, There is likely to be complementary substance in these carriers, as lubricant, glidant, wetting agent or emulsifier, pH buffer substance and Stabilizer etc..

Gene therapy vector

Gene therapy vector in the present invention is virus expression carrier, and according to the present invention, virus expression carrier is gland correlation Viral (AAV) carrier is such as formed selected from chimeric AAV derived from serotypes A AV1,2,3,4,5,6,7,8,9 and 10 or its Group in AAV carriers such as AAV2-AAV3, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAVHSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6 (Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2 (Y- >F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAVr3.45AAV2 or AAV5, this can be better adapted for feeling emerging Efficient transduction is carried out in the tissue of interest.Transfection when, AAV only cause in host slight immune response (if there is Words).In a preferred embodiment of the invention, gene therapy vector is AAV serotypes 2 or 5 carriers.In further preferred reality It applies in mode, gene therapy vector is AAV2 carriers.

The AAV carriers of the present invention are single-stranded AAV, can produce recombinant viral vector according to standard technique.For example, recombination gland phase Closing viral vectors can propagate in 293 cell of people (it provides trans- E1A and E1B characteristics), to reach in 10^7~10^13 disease Titre within the scope of malicious particle/mL.Before applying in vivo, viral vectors can pass through gel filtration method (such as agarose column) Desalination is carried out, and is purified by subsequent filtering.Potential illeffects in the main body of purifying reduction drug administration carrier.It is given The virus of medicine is substantially free of wild-type virus and replication competent type virus.Suitable method, such as dodecyl sulphate can be passed through Sodium-polyacrylamide gel electrophoresis (SDS-PAGE), then carries out silver staining, to prove the purity of virus.

The suitable dose of AAV for people is in the range of about 1 × 10^10~1 × 10^14 virion.

Gene order

SEQ ID NO:It is the FADD-DN sequences after optimization shown in 1.

SEQ ID NO:It is original people's FADD-DN amino acid sequences shown in 2.

SEQ ID NO:It is Left ITR sequences shown in 3.

SEQ ID NO:It is CMV sequences shown in 4.

SEQ ID NO:It is human β-globin intron sequences shown in 5.

SEQ ID NO:It is MCS sequences shown in 6.

SEQ ID NO:It is hGHpA sequences shown in 7.

SEQ ID NO:It is Right ITR sequences shown in 8.

SEQ ID NO:It is Ampicillin Resistance sequences shown in 9.

SEQ ID NO:It is full nucleotide sequence shown in 10.

SEQ ID NO:It is f1ori (2670-2976) sequence shown in 11.

SEQ ID NO:It is recombination sequence shown in 12.

Gene therapy vector is intraocular injection administration, and subretinal space or intravitreal administration may be used.Under In conjunction with specific embodiments, the present invention is further explained in face.It should be understood that these embodiments are merely to illustrate the present invention rather than limit The scope of the present invention processed.

In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition such as J. Pehanorm Brookers It writes, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to proposed by manufacturer Condition.

Experimental animal

Normal C57BL/6J mouse are purchased from Shanghai Slac Experimental Animal Co., Ltd..Periodicity of illumination 12h illumination- 12h is dark, is freely eaten, free water, what all zooscopies were issued in strict accordance with State Scientific and Technological Commission《Experiment The care of animal regulations》It carries out.

Main agents, consumptive material

Physiological saline (Zhejiang Tianrui Pharmaceutical Co., Ltd.), injection needles head (U.S. Becton Dickinson And Company companies), HA (Roche), TUJ1 (Abcam), secondary antibody goat anti-rabbit igg (JacksonImmuno), blood of goats (Sigma) clearly, Triton X-100 (Triton X-100) (Sigma), paraformaldehyde (Sigma).

Key instrument

Ophthalmology tests surgical operation microscope (Japanese Nikon companies)；Ophthalmology microinstrument (the limited public affairs of Suzhou Ming Ren medical instruments Department)；Microsyringe (Hamilton companies of the U.S.)；Laser Scanning Confocal Microscope (Leca company).

The structure of mouse glaucoma model

The injection that is prepared in advance capillary needle tubing, needle tubing top is oval, and interior to angle there are 20 degree, and prepares The good bead particulates suspension (1.6*10^beads/ul) for having removed surface epoxy group.It chooses 6-8 weeks male mice and carries out abdominal cavity Injecting anesthetic, side are placed on operation console, and modeling eye needs level upward and need to expand pupil in advance.Operation consent is magnetic by preprepared Microparticulate suspensions blow and beat mixing again, and draw 1.5ul magnetic particle suspensions in capillary needle tubing rapidly.Capillary needle tubing and corneal limbus It is in fix eyeball with plastic cement microforceps at 90 degree therewith in 45 degree of angles, and in the other side.It is careful to be pierced into capillary needle tubing, it needs to ensure Needle tubing does not enter front depths, and forbids touching iris and crystal in the process.In injection point to placing a magnetic pole at side angle Gong Yuan, Slowly injection 1.5ul magnetic particle suspensions, injection process need to ensure to complete in 15 to 30 seconds afterwards, need to ensure magnetic in injection process Pole position is constant.Rapid after the completion of injection to remove capillary needle tubing, rear position of magnetic pole remains unchanged, maintain 30 to 60 seconds to ensure There is particle to be not attached in corneal limbus to survey.Canthus Gong Yuan moves magnetic pole around eyeball later, is that magnetic particle is dispersed in angle Film, iris angle simultaneously form magnetic particle ring.Mouse needs side to be placed on hot blanket to recover after modeling, and resuscitation process needs to keep Modeling eye is upward.

It is prepared by magnetic micro-beads aaerosol solution：It because magnetic micro-beads surface is covered with epoxy group, should first remove, i.e., will before use 1ml magnetic micro-beads are mixed into rapidly the 10*Tris of the 0.02M sodium hydroxides (NaOH, MW 39.997g/mol) of the fresh configurations of 50ml In the mixed liquor of buffer solution, shaken at room temperature mixing 24 hours is collected bead particulates in test tube bottom using magnetic force.Test tube is kept flat To ensure that all particles are all adsorbed by magnetic force.Mixing 4 hours at room temperature.It is carefully gone out supernatant liquid with pipettor, carefully Magnetic bead group is mixed into 50ml 10X Tris solution, mixing simultaneously makes all particle suspensions.Bead particulates are in sterile equilibrium liquid Concentration and suspension：Experiment uses ultra-pure water oscillation cleaning particle 3 times.In super-clean bench, 500mlBBS piping and druming mixing is added 3 times.Add Enter 250mlBBS piping and druming mixings, makes particle resuspension.Ensure that solution is uniform as possible, and quickly by 25ul suspensions move into 0.5ml without In tube.Obtained Nanoparticle Solution ultimate density is 1.6*10^6/ul.4 DEG C of preservations.

Intravitreal

Using glass system capillary needle, locates inserting needle rearward in view film edge, corneosclera boundary, noted using micro syringe It penetrates, when injection should be noted needle angle and depth, pay attention to avoiding damage to crystal or other eye inner tissues.

Tissue treatment

Animal first via PBS carry out total body perfusion, after continue with 4% paraformaldehyde (PFA) perfusion, rate of flooding 5mL/ Minute.Eyeball and optic nerve impregnate about 2 hours after taking out in 4%PFA solution.It need to be in 0.1M PBS before tissue staining tile 4 DEG C of storages；Before tissue freezing section need in 30% sucrose solution soaked overnight.

Embodiment 1：It the structure of adenovirus vector and isolates and purifies

The structure of plasmid AAV_FADD-DN is as shown in Figure 1 and Figure 2, main element include cmv enhancer/promoter and FADD-DN sequences.Cmv enhancer can enhance the expression of transgene.Followed by hGHpA (SEQ ID after target gene NO:7), expression cassette both sides are inverted terminal repeat (TR), i.e., viral vectors includes L-ITR (SEQ ID NO:3), R-ITR (SEQ ID NO:8), viral vectors further includes Ampicillin (SEQ ID NO:And f1ori (SEQ ID NO 9):11).

Viral vectors is obtained by plasmid co-transfection method.AAV2 coat protein genes will be contained and AAV can be helped It is related to preliminarily form recombination gland for the helper plasmid and AAV_FADD-DN plasmids of the gene of duplication and HEK 293T cell cotransfections Viral vectors.After iodine gram butanol preliminary purification, by the fast protein liquid using 5ml-Hitrp Q Ago-Gels as filler Chromatography is further purified by ion-exchange chromatography, the use of instrument is Pharmacia AKTA FPLC system (Amersham Biosciences,Piscataway,NJ).PH8.0, the NaCl of 215mM is used to elute agarose Gel column later, The recombinant viral vector of peak value is collected.The liquid of collection by inspissator (100K concentrater, Millipore after), recombinant viral vector is concentrated with the elution inspissator containing 0.014% polysorbas20.DNase I are used again DNA other than virion is digested, and determines the titre of virus by the method for real-time fluorescence quantitative PCR.Finally use silver nitrate Dyeing-SDS polyacrylamide gel electrophoresis methods ensure that vector particles are not contaminated and are free of endotoxin, and dispense subzero 80 Degree storage.

The characteristic spectrum of viral vectors AAV_FADD-DN is as shown in Figure 1, the start-stop site of each element is as shown in Figure 2.

Embodiment 2：Adenovirus infection detects

Tissue treatment：The AAV_FADD-DN of mouse intravitreal HA label after two weeks, according to aforementioned tissues processing method Carry out the pre-treatment of eyeball, it is rear to clamp eyeball bottom using microforceps and be lifted up, with scissors cut the conjunctiva of peribulbar with Musculature keeps eyeball tissue complete, cuts off cornea, iris and crystalline lens and make eyecup, and detach retina and choroid Complex.

Immunofluorescence dyeing：Retina is chosen, 1h is closed in 20% goat serum solution, rear primary antibody is incubated overnight, and resists Body is respectively TUJ1, HA；PBS clean three times, each 10min, rear secondary antibody is incubated 1h；PBS cleanings are sealed after ten minutes three times, every time Piece.Change and take pictures using confocal microscopy retinal structure.

As a result：Mouse injection of AAV _ FADD-DN after two weeks, carries out retina immunofluorescence dyeing tile, it is seen that deposit on eyeground In the AAV_FADD-DN of a large amount of HA labels, and (TUJ1 immunofluorescence dyeings) is overlapped with RGC, show the infection of AAV_FADD-DN Excellent effect (Fig. 3).

Embodiment 3：RGC survival rates detect

Experimental method：8 week old mouse unilateral side camera vitrea bulbi injection of AAV _ FADD-DN (experimental group), AAV-GFP (controls Group), while anterior chamber's bead particulates injection is carried out in injection eye, eyeball was handled according to preceding method and regard respectively at 2 weeks, 4 weeks, 8 weeks Nethike embrane carries out immunofluorescence dyeing.Change and take pictures using confocal microscopy retinal structure.Each eyeball selects at random 5 visuals field are taken to count RGC (engineer's scales：20um), and RGC survival rates are calculated.

As a result：As illustrated in figures 4-5, RGC gradually withers in 2W, 4W, 8W after quantitative analysis proves the injection of anterior chamber's bead particulates It dies, and quantity is reduced significantly (experimental group)；In contrast, injection of AAV _ FADD-DN mouse RGC apoptosis is not notable, AAV_ is shown FADD-DN has protective effect to RGC.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as Protection scope of the present invention.

SEQUENCE LISTING

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<120>A kind of genophore and its gene therapy medicament for treating retinal ganglion cells degeneration

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ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 120

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 180

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 240

tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 300

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 360

cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 420

aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 480

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 540

tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 600

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 660

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 720

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 780

gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 840

actgattaag cattggta 858

<210> 10

<211> 4654

<212> DNA

<213>Artificial sequence

<400> 10

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggccgca cgcgtggaat tcgctagtta ttaatagtaa 180

tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 240

gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 300

tatgttccca tagtaacgtc aatagggact ttccattgac gtcaatgggt ggagtattta 360

cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 420

gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 480

tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 540

tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 600

cccattgacg tcaatgggag tttgttttgc accaaaatca acgggacttt ccaaaatgtc 660

gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 720

taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg 780

acctccatag aagacaccgg gaccgatcca gcctccgcgg attcgaatcc cggccgggaa 840

cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc 900

tataggccca caaaaaatgc tttcttcttt taatatactt ttttgtttat cttatttcta 960

atactttccc taatctcttt ctttcagggc aataatgata caatgtatca tgcctctttg 1020

caccattcta aagaataaca gtgataattt ctgggttaag gcaatagcaa tatttctgca 1080

tataaatatt tctgcatata aattgtaact gatgtaagag gtttcatatt gctaatagca 1140

gctacaatcc agctaccatt ctgcttttat tttatggttg ggataaggct ggattattct 1200

gagtccaagc taggcccttt tgctaatcat gttcatacct cttatcttcc tcccacagct 1260

cctgggcaac gtgctggtct gtgtgctggc ccatcacttt ggcaaagaat tgggattcga 1320

acatcgattg aattccccgg ggatcctcta gagtcgacct gcagaagctt gcctcgagca 1380

gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg 1440

ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca 1500

ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca 1560

aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga 1620

gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc 1680

tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt 1740

tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat 1800

ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg 1860

ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga 1920

acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 1980

gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 2040

gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 2100

ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 2160

gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 2220

cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 2280

aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 2340

cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 2400

ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 2460

aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 2520

ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 2580

acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 2640

cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 2700

tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 2760

ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 2820

ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 2880

atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 2940

taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 3000

cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 3060

aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 3120

aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 3180

tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 3240

ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 3300

catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 3360

aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 3420

ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 3480

gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 3540

aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 3600

gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 3660

gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 3720

gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 3780

gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 3840

gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 3900

atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 3960

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4020

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4080

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4140

ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4200

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4260

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4320

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4380

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4440

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4500

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 4560

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 4620

ttcctggcct tttgctggcc ttttgctcac atgt 4654

<210> 11

<211> 306

<212> DNA

<213>Artificial sequence

<400> 11

cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 60

gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 120

aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt 180

cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 240

acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc 300

tattgg 306

<210> 12

<211> 5050

<212> DNA

<213>Artificial sequence

<400> 12

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggccgca cgcgtggaat tcgctagtta ttaatagtaa 180

tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 240

gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 300

tatgttccca tagtaacgtc aatagggact ttccattgac gtcaatgggt ggagtattta 360

cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 420

gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 480

tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 540

tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 600

cccattgacg tcaatgggag tttgttttgc accaaaatca acgggacttt ccaaaatgtc 660

gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 720

taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg 780

acctccatag aagacaccgg gaccgatcca gcctccgcgg attcgaatcc cggccgggaa 840

cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc 900

tataggccca caaaaaatgc tttcttcttt taatatactt ttttgtttat cttatttcta 960

atactttccc taatctcttt ctttcagggc aataatgata caatgtatca tgcctctttg 1020

caccattcta aagaataaca gtgataattt ctgggttaag gcaatagcaa tatttctgca 1080

tataaatatt tctgcatata aattgtaact gatgtaagag gtttcatatt gctaatagca 1140

gctacaatcc agctaccatt ctgcttttat tttatggttg ggataaggct ggattattct 1200

gagtccaagc taggcccttt tgctaatcat gttcatacct cttatcttcc tcccacagct 1260

cctgggcaac gtgctggtct gtgtgctggc ccatcacttt ggcaaagaat tgggattcga 1320

acatcgattg aattccacca tgtaccccta cgacgtgcct gactacgccg acgacttcga 1380

ggccggcgcc gctgctggag ctgcacctgg agaggaggac ctgtgcgccg ccttcaacgt 1440

gatctgcgac aacgtgggca aggactggag gaggctggcc cgccagctga aagtgagtga 1500

tacaaagatt gatagcatcg aagacaggta ccctagaaac ctgaccgaga gggtgaggga 1560

gagtctgagg atctggaaga acaccgagaa ggaaaacgca accgtggcac atctggtggg 1620

cgcactgagg agctgccaga tgaacctggt ggcagacctg gtgcaggagg tgcagcaggc 1680

cagggacctg cagaacagga gcggagcaat gagccctatg agttggaaca gcgatgcttc 1740

caccagcgag gccagctgac tcgagtgact cgagcagcgc tgctcgagag atctacgggt 1800

ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc 1860

ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct 1920

ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct 1980

gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca 2040

ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg 2100

gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt 2160

tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc 2220

ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt 2280

tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac 2340

tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 2400

gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct 2460

gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa 2520

ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 2580

gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 2640

ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 2700

cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt 2760

agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 2820

aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt 2880

gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 2940

aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt 3000

acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac 3060

gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 3120

gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 3180

ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 3240

ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 3300

tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 3360

aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 3420

tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 3480

ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 3540

tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 3600

gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 3660

aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 3720

agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 3780

acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 3840

actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 3900

accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 3960

actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 4020

cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 4080

cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 4140

gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 4200

ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 4260

tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 4320

aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 4380

gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 4440

acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 4500

tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 4560

ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 4620

atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 4680

agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 4740

cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 4800

agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 4860

acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 4920

gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 4980

ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 5040

gctcacatgt 5050

Claims (10)

1. a kind of gland relevant viral vector, which is characterized in that the gland relevant viral vector contains enhancer, specificity or non- Specificity promoter, the gene order for expressing FADD-DN albumen, the sequence such as SEQ ID NO of FADD-DN albumen:Shown in 2.

2. gland relevant viral vector according to claim 1, which is characterized in that the gene order of expression FADD-DN albumen Such as SEQ ID NO:Shown in 1.

3. gland relevant viral vector according to claim 1, which is characterized in that the enhancers/promoters are CMV.

4. gland relevant viral vector according to claim 1, which is characterized in that the adeno-associated virus is AAV2.

5. gland relevant viral vector according to claim 1, which is characterized in that the adeno-associated virus is single-stranded AAV.

6. gland relevant viral vector according to claim 1, which is characterized in that the gland relevant viral vector also contains The chimeric interon of one section of shortening, the chimeric intron sequence are selected from and SEQ ID NO:5 have at least 90% homology Nucleotide sequence.

7. gland relevant viral vector according to claim 1, which is characterized in that the gland relevant viral vector contains Cmv enhancer/promoter, the gene order of FADD-DN, β-globin intrones, hGHpA, L-ITR, R-ITR, Ampicillin、f1ori。

8. gland relevant viral vector according to claim 7, which is characterized in that the gene order of expression FADD-DN albumen Such as SEQ ID NO:Shown in 1, L-ITR sequences such as SEQ ID NO:Shown in 3, cmv enhancer/promoter sequence SEQID NO:4 institutes Show, β-globin intron sequences such as SEQ ID NO:Shown in 5, hGHpA sequences such as SEQ ID NO:Shown in 7, R-ITR sequences are such as SEQ ID NO:Shown in 8, Ampicillin sequences such as SEQ ID NO:Shown in 9, f1ori sequences such as SEQ ID NO:Shown in 11.

9. a kind of pharmaceutical composition for treating retinal ganglion cells degeneration, which is characterized in that the pharmaceutical composition contains Any just like claim the 1-8 gland relevant viral vector and pharmaceutically acceptable carrier, the pharmaceutical composition Express FADD-DN albumen.

10. preparing treatment retinal ganglion cells degeneration according to any gland relevant viral vectors of claim 1-8 Drug in application.