CN103352042A - cDNA encoding recombinant Newcastle disease virus, virus rescued by cDNA, and application of cDNA in malignant tumor treatment - Google Patents

Abstract

The present invention discloses a recombinant virus, wherein a recombinant Newcastle disease virus nucleotide sequence expressing a TNF-related apoptosis inducing ligand and an application of the sequence are provided. The tumor treatment drug of the present invention has a good tumor inhibition effect. In addition, basis is provided for tumor treatment by using the recombinant Newcastle disease virus.

Description

The cDNA of coding recombinant Newcastle disease virus, by virus and the application in the treatment malignant tumour thereof of this cDNA rescue

Technical field

The cDNA rNDV-TRAIL that the present invention relates to a kind of recombinant Newcastle disease virus of encoding makes up, by virus and the application in the treatment malignant tumour thereof of this cDNA rescue.

Background technology

Malignant tumour claims again cancer, and the sickness rate of whole world cancer is just with the speed increase in every year 3%, and annual because the cancer death toll reaches more than 600 ten thousand people, the sickness rate of China's cancer and death toll are also in rising trend.Data shows that some big and medium-sized cities resident's cancer mortalities of China have occupied the first place of whole causes of the death according to statistics, therefore, strengthens the research for the treatment of malignant tumor, and seeking novel therapeutic tumour method has extremely important meaning.

Avian pneumo-encephalitis virus (Newcastle disease virus, NDV) is to be non-segmented negative sub-thread minus-stranded rna virus, in the virusology classification it is attributed to paramyxovirus section fowl Rubulavirus (Avulavirus).Studies show that in a large number, NDV is killing tumor cells effectively, and normal cell is not exerted an influence.NDV is by starting the endogenous apoptosis pathway inducing apoptosis of tumour cell do not rely on p53, and activating immune system identifies tumour and kill and wound and immunological memory, becomes emerging focus of attention of therapeutic field of tumor.

It is 200510097997.8,200610001693.1 that this institute relates to Patents.This research has following different from patent before: 1. the strain used of this institute is newcastle disease Clone30 strain, and is different from the Lasota strain of patent before; This to study applied function be treating malignant tumor, patent function before is for preventing and treating the poultry diease bivalent vaccine; 3. the insertion point of this Study of Exogenous gene is between HN and the L, and patent is between P and the M before; Therefore this patent has essence different from patent before.

Tumor necrosin relative death inducing ligand (TNF-related apoptosis inducing ligand, TRAIL) is one of member of tumour necrosis factor (tumor necrosis factor, TNF) family.TRAIL by 281 amino acid form typical

Type transmembrane protein, its a most important Biological characteristics be can the selective induction apoptosis of tumor cells and to normal cell without obvious toxic-side effects.In addition, antitumor spectrum is wide, and has obvious synergy between radiotherapy and the chemotherapeutics.These characteristics make it become a focus of anti-cancer agent research.At present, TRAIL has finished clinical abroad

The phase experiment, confirmation TRAIL toxic side effect is hanged down without immune response and can not caused any histoorgan impaired, and has entered

Clinical study stage phase.

Summary of the invention

The present invention relates to the cDNA sequence of one section coding recombinant virus, sequence contains SEQ ID NOS:1, or because insert, disappearance and sudden change cause surpasses derived dna sequence more than 90% with SEQ ID NOS:1 homology.

The invention still further relates to the reverse genetic operating system of the described recombinant virus of a kind of claim 1, this system comprises:

(1) sequence 1 claimed in claim 1;

(2) helper plasmid, the dna sequence dna of the nucleoprotein (NP) that comprising encodes expresses NDV, phosphorprotein (P), large polymerase protein (L);

(3) host cell of the virus replication of NDV virus license is such as the BHK-21 cell.

Concrete, the genome cDNA of construction expression hTRAIL recombinant Newcastle disease LaSota virus strain, saved out by this cDNA and to have specific infection tumour cell Avian pneumo-encephalitis virus, and the application in the treatment malignant tumour, make up recombinant Newcastle disease LaSota virus strain rNDV-TRAIL and mainly obtain by the following technical programs and verify its effect in the treatment malignant tumour:

Get volunteer's peripheral blood, extract the total RNA of human peripheral, reverse transcription becomes cDNA, shown in sequence 2,3, and according to the human TNF related apoptosis-inducing ligand gene of having delivered on the GenBank (sequence number GI:117606467), design PCR primer, complete human cloning trail dna.

Get that NDV Clone 30 extracts geneome RNAs behind the plaque purification, reverse transcription becomes cDNA as template, is divided into ten sections the full genome of virus cloned, and assemble at the pBluscript carrier.Between HN and L gene, introduce restriction enzyme site Hpa I, Pme I, Asc I, Mlu I restriction enzyme site.Add the T7RNA polymerase promoter at full-length cDNA fragment 5 ' end, after the full-length cDNA fragment, be connected into fourth liver ribozyme sequence (GenBank X04451) and T7 transcription termination signal with self-catalysis.The plasmid called after pBR-NDV-Clone30 that structure is finished.Take the NDV genome as template, clone respectively NP, P and L gene and be connected in pBluescript plasmid T7 promotor downstream, respectively called after pBR/NP, pBR/P, pBR/L.

The human TNF related apoptosis-inducing ligand gene that clones is inserted construction recombination plasmid called after pBR-Clone30-TRAIL in the genome of NDV-Clone30 strain with HpaI.And pBR-Clone30-TRAIL, pBR/NP, pBR/P, pBR/L cotransfection are expressed the BHK-21 cell of t7 rna polymerase, save out recombinant Newcastle disease virus rNDV-TRAIL.

On the cell levels, utilize the ELISA method to detect the expression amount of foreign gene TRAIL; After utilizing MTT to detect recombinant virus infection, the apoptosis situation of tumour cell is analyzed by AnnexinV/PI flow cytometer method, and the ability of recombinant Newcastle disease virus cell death inducing reaches the impact on the growth of tumour cell cycle.

Set up the subcutaneous solid tumor models of kunming mice H22, set up C57BL/6 murine melanoma model, on the animal level, detect recombinant Newcastle disease virus rNDV-TRAIL, to result for the treatment of and the security of tumour.　

But recombinant Newcastle disease virus rNDV-TRAIL establishment animal tumor growth provided by the invention has the double dominant that trail protein and Avian pneumo-encephalitis virus are treated tumour, can be used as the pharmacological agent tumor disease.

Description of drawings

Fig. 1 is clone hTRAIL(apoptosis induction ligand related to human tumor necrosis factor among the embodiment 1) the gene sequencing result

Fig. 2 is the positive recombinant plasmid PCR of rNDV-TRAIL qualification result among the embodiment 1: 1 is the result of recombinant plasmid rNDV-TRAIL pcr amplification trail dna; M is DL2000 DNA marker;

Fig. 3 is rNDV-TRAIL recombinant plasmid recombinant plasmid among the embodiment 1 HpaThe I enzyme is cut evaluation: M is λ T14 digest DNA marker; 1 is the rNDV-TRAIL recombinant plasmid; 2 is rNDV-TRAIL HpaThe I enzyme is cut evaluation; M is DL2000 DNA Marker

Fig. 4 is blood clotting (HA) experimental result

Fig. 5 utilizes the ELISA method at B16F10 among the embodiment 2, HepG2, the expression amount of detection trail dna on A549 and the CT26 cell levels

Fig. 6 be among the embodiment 3 rNDV-TRAIL and rNDV empty carrier to the inhibition of HepG2 cell

Fig. 7 be among the embodiment 3 rNDV-TRAIL and rNDV empty carrier to the inhibition of B16F10 cell

Fig. 8 be among the embodiment 3 rNDV-TRAIL and rNDV empty carrier to the inhibition of A549 cell

Fig. 9 be among the embodiment 3 RNDV-TRAIL and rNDV empty carrier to the inhibition of CT26 cell

Figure 10 is rNDV-TRAIL and rNDV empty carrier effect B16F10 and the two flow cytometers detection apoptosis rates that dye of HepG2 cell Annexin V/PI among the embodiment 3

Figure 11 is the effect of rNDV-TRAIL virus and rNDV empty carrier viral therapy murine melanoma among the embodiment 4

Figure 12 is the effect of rNDV-TRAIL virus and rNDV empty carrier viral therapy mouse H22 liver cancer among the embodiment 4.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.

The structure of embodiment 1 recombinant Newcastle disease virus rNDV-TRAIL

1. the structure of recombinant Newcastle disease virus rNDV-TRAIL: the total RNA of human peripheral extracts, reverse transcription cDNA,

According to the apoptosis induction ligand related to human tumor necrosis factor of having delivered on the GenBank (TRAIL) gene order (sequence number GI:117606467), the complete human cloning trail dna of design PCR primer.

2. the structure of NDV Clone30 pnca gene group cDNA and helper plasmid:

With the 30 inoculation SPF chick embryo allantoic cavities of NDV Clone behind the plaque purification, every piece of 200ml.Hatch after 3～4 days, the chicken embryo is collected allantoic fluid after-20 ℃ of refrigerators are placed 1 hour, with 9 age in days SPF chicken embryo continuous passages twice, 3 centrifuging and taking supernatants of gained allantoic fluid multigelation, behind the ultracentrifugation (37000rpm) ,-70 ℃ frozen for subsequent use.Get virus behind the 450ml purifying, add Proteinase K (final concentration 500mg/ml and 10%SDS 50ml, 55 ℃ of water-bath 2h, add the 500ml precooling the phenol chloroform (phenol: chloroform=1: 1), thermal agitation mixing 30 sec, 12000 r/min, 4 ℃ of centrifugal 10min.Get supernatant, repeat once.Get supernatant, add the equal-volume chloroform, abundant mixing, 12000 r/min, 4 ℃ of centrifugal 10 min.Get supernatant, add the 3M NaAc (PH5.2) of 1/10 volume and the dehydrated alcohol of 2.5 times of volumes ,-70 ℃ of sedimentation 12h.12000r/min, 4 ℃ of centrifugal 30min.With 1ml precooling 75% ethanol, 8000r/min, 4 ℃ of centrifugal 5min washing RNA precipitations are abandoned supernatant, repeat drying at room temperature 3 times.With 30 μ l sterilization DEPC water dissolution RNA ,-70 ℃ save backup.Take this RNA as template, be that 10 fragments are carried out global cDNA clone with gene element, utilize the restriction enzyme site of the lap of each fragment, assemble at transcription vector plasmid pBluescript plasmid, obtain the global cDNA clone of 15186bp, between HN and L gene, introduce restriction enzyme site Hpa I, Pme I, Hand III, Asc I, Mlu I restriction enzyme site.Add the T7RNA polymerase promoter at full-length cDNA fragment 5 ' end, after the full-length cDNA fragment, be connected into fourth liver ribozyme sequence (GenBank X04451) and T7 transcription termination signal with self-catalysis.The plasmid called after pBR-NDV-Clone30 that structure is finished.Take the NDV genome as template, clone respectively NP, P and L gene and be connected in pBluescript plasmid T7 promotor downstream, respectively called after pBR/NP, pBR/P, pBR/L.

3. will make up restructuring matter in the genome of human TNF related apoptosis-inducing ligand gene with HpaI insertion NDV-Clone30 strain that clone

Grain called after pBR-Clone30-TRAIL.

The result as shown in Figure 1, 2, 3, the trail dna of successfully cloning people, and successfully inserting in the genome of NDV-Clone30 strain.

4. save recombinant Newcastle disease virus rNDV-TRAIL: the BHK-21 cell of expressing t7 rna polymerase is inoculated in and grows to the 70-80% individual layer in six orifice plates, go endotoxic plasmid pBR-Clone30-TRAIL, pBR/NP, pBR/P, pBR/L to adopt the liposome transfection method after getting 1 μ g, 0.5 μ g, 0.25 μ g and 0.1 μ g purifying, with Lipofe-ctmine 2000 transfection reagent cotransfection BHK-21 cells, concrete operation step carries out with reference to Invitrogen company transfection reagent specification sheets.Discard nutrient solution behind transfection 4 h, add perfect medium and continue to cultivate, discard again nutrient solution behind the 5h, add the substratum Opti-MEM of serum-free and add TPCK(1 μ gml ^-1), continue to hatch behind 72 h cell-70 ℃ freezing, then multigelation is 3 times, 4 ℃ of low-speed centrifugals (1 500 rpm/min) are collected supernatant again, get 100 ~ 200 μ L and are inoculated in 9 age in days SPF chicken embryos.Behind 3-4 d, collect and the mixing allantoic fluid, carry out according to a conventional method blood clotting (HA) test and blood clotting and suppress (HI) test.Results HA and the positive allantoic fluid of HI test-results, frozen in-70 ℃ after the packing.

The result as shown in Figure 4, for successfully saving out the blood coagulation tests of recombinant Newcastle disease virus rNDV-TRAIL.

On embodiment 2 cell levelss, utilize the ELISA method to detect the expression amount of foreign gene human TNF related apoptosis-inducing ligand

To be in the people B16F10 of logarithmic phase, HepG2, A549 and CT26 tumour cell are collected behind tryptic digestion, are prepared into 1～10 * 10 ⁵The cell suspension of/mL adds 6 orifice plates after the piping and druming evenly, treat cell length to 80% experimental port with 0.1 MOI recombinant Newcastle disease virus rNDV-TRAIL and NDV empty carrier virus infection.After infecting 48h, the harvested cell supernatant detects the exogenous gene expression amount with human TNF related apoptosis-inducing ligand ELISA test kit.

The result as shown in Figure 5, prove insert recombinant Newcastle disease virus rNDV-TRAIL can be at expression in tumor cells human TNF related apoptosis-inducing ligand albumen, and be secreted in the supernatant substratum.

Embodiment 3 recombinant Newcastle disease virus rNDV-TRAIL reach the impact on the tumour cell cell cycle to restraining effect, the lethal effect of tumour

1.MTT method is measured recombinant Newcastle disease virus rNDV-TRAIL to the restraining effect of tumour cell: will be in the people's of logarithmic phase B16F10, HepG2, A549, the CT26 cell is collected behind tryptic digestion, is diluted to 2

10 ⁴The tumour cell suspension of individual/mL blows adding 200 μ L tumour cell suspensions in every hole of sparing backward 96 orifice plates, at 37 ℃, 5% CO ₂Continue overnight incubation in the incubator, discard substratum, add the viral dilution liquid 200 μ L that are diluted to 0.1 MOI with the DMEM substratum in the experimental port, add the NDV empty carrier virus 200 μ L that are diluted to 0.1 MOI with the DMEM substratum in the control group, add 200 μ L DMEM substratum in the control wells.Behind virus infection 1 h, once add afterwards cell maintenance medium (containing 5% serum) with the PBS washing, continue to cultivate.After cultivating respectively 24,48,72,96 h, add 20 μ L MTT solution/holes (5 mg/mL) in every hole and hatch 4 h, discard nutrient solution, every hole adds 150uL DMSO again, behind the concussion mixing 10min, is 490nm mensuration OD value with the enzyme linked immunological instrument in measuring wavelength.Calculate the inhibiting rate of Growth of Cells.

The result shows recombinant Newcastle disease virus rNDV-TRAIL to malignant cell B16F10 shown in Fig. 6,7,8,9, HepG2, A549, and inhibiting rate and the time of CT26 are dependency relationships, reach maximal percentage inhibition to tumour cell at 96h.

2.Annexin the V/PI method is analyzed recombinant Newcastle disease virus rNDV-TRAIL to the fragmentation effect of tumour cell: the B16F10 that will be in logarithmic phase, HepG2, A549 and CT26 cell are collected behind tryptic digestion, and are diluted to and contain 5 * 10 of 0.001% pancreatin ⁵The cell suspension of/mL, blow even rear 6 orifice plates that add, every hole 1mL, treat that cell density reaches 50-80%, grouping is tested: it is 0.1 MOI recombinant Newcastle disease virus rNDV-TRAIL that the every hole of experimental group adds dosage, the every hole of control group adds respectively the NDV empty carrier virus that dosage is 0.1 MOI, adds 1mL DMEM in the blank group culture hole.With adding cell maintenance medium behind virus infection 1 h.Cultivate 48 h, the trypsin digestion cell with 0.25%, cold PBS washing 2 times, adjusting cell concn is 1 * 10 ⁶/ mL, add 200 μ L Binding Buffer suspension cells, the Annexin-V that adds again 5 μ L FITC marks, the mixing lucifuge is reacted 30 min under the room temperature, add at last 5 μ L PI and 300 μ L Binding Buffer, use immediately flow cytometry detection by quantitative (generally in 1 h) behind lucifuge reaction 5～15 min, simultaneously with cell one pipe that do not add AnnexinV-FITC and PI as negative control.

The result as shown in figure 10, recombinant Newcastle disease virus rNDV-TRAIL and NDV empty carrier virus all can be induced HepG2 and B16F10 tumour cell apoptosis, but the cell effect that the apoptosis situation of the HepG2 that processed via recombinant Newcastle disease virus rNDV-TRAIL and B16F10 tumour cell is crossed than the NDV empty carrier virus treated under the same terms is more obvious.

Embodiment 4 animal level detection, recombinant Newcastle disease virus rNDV-TRAIL detects therapeutic action and the Viral safety of tumour

1. newcastle disease recombinant virus rNDV-TRAIL is to the therapeutic action of kunming mice H22 liver cancer model tumour:

Set up kunming mice H22 liver cancer animal model, get 6 age in week kunming mice test, the H22 murine hepatocarcinoma cell in the kunming mice intraperitoneal inoculation, is treated after 7 days that ascites grows disconnected neck and puts to death.Extract the ascites that contains the H22 cell under the aseptic condition, add an amount of PBS, be made into the cancer cells suspension, cell counting and mensuration cell viability, living cell rate reaches 95 %.Adjusting cell density is 10 ⁶Individual cell/mL is for subsequent use.Every subcutaneous implantation dosage 0.2mL of right side of mice inguinal region approximately contains 2 * 10 ⁵Individual tumour cell.Form the solid tumor diameter after 8-12 days at 5-8mm, the modeling success can be carried out subsequent experimental.The cut-off footpath will remain tumor-bearing mice by body weight, knurl volume averaging distribution principle at the mouse removal form of 5-8 mm, the individuality that difference in size is large, be divided at random the PBS control group, NDV empty carrier virus control group, recombinant Newcastle disease virus rNDV-TRAIL experimental group.Experimental group is from first day, and per two days intratumor injection 0.2 mL approximately 5 * 10 ⁷The recombinant virus of pfu is injected four times continuously.NDV empty carrier control group injection 0.2 mL identical with experimental group approximately 5 * 10 ⁷The NDV empty carrier virus of pfu, the PBS control group is from first day, and the PBS of per two days intratumor injection 0.2 mL injects four times continuously.

The result proves that the effect of recombinant Newcastle disease virus rNDV-TRAIL check melanin knurl growth is extremely obvious as shown in figure 11.

2. newcastle disease recombinant virus rNDV-TRAIL is to the therapeutic action of C57BL/6 murine melanoma model tumour:

Set up C57BL/6 murine melanoma animal model, get 6 age in week the C57BL/6 mouse test, the B16F10 mouse melanin tumor cell is added an amount of PBS, be made into the cancer cells suspension, cell counting and measure cell viability, living cell rate reaches 95 %.Adjusting cell density is 10 ⁶Individual cell/mL is for subsequent use.Every right side of mice sole back implantation dosage 0.2mL approximately contains 2 * 10 ⁵Individual tumour cell.Form the melanoma diameter after 8-12 days at 5mm, the modeling success can be carried out subsequent experimental.Modeling success when treating that visible tumour appears in mouse, removal form, the individuality that difference in size is large, to remain tumor-bearing mice by body weight, knurl volume averaging distribution principle, be divided at random the PBS control group, NDV empty carrier virus control group, recombinant Newcastle disease virus rNDV-TRAIL experimental group.Experimental group is from first day, and per two days intratumor injection 0.2 mL approximately 1 * 10 ⁷The recombinant virus of pfu is injected four times continuously.NDV empty carrier control group injection 0.2 mL identical with experimental group approximately 1 * 10 ⁷The NDV empty carrier virus of pfu, the PBS control group is from first day, and the PBS of per two days intratumor injection 0.2 mL injects four times continuously.

The result proves that the effect of recombinant Newcastle disease virus rNDV-TRAIL inhibition H22 liver cancer growth is extremely obvious as shown in figure 12.

3. the recombinant virus security detects: carry out acute toxicity test, subacute toxicity test:

The results show recombinant Newcastle disease virus rNDV-TRAIL has no adverse effects to the normal growth of normal mouse, this recombinant virus is used as the security for the treatment of mouse tumor reliable.

Sequence 1

1?CTGGCGTAAT?AGCGAAGAGG?CCCGCACCGA?TCGCCCTTCC?CAACAGTTGC

51?GTAGCCTGAA?TGGCGAATGG?GACGCGCCCT?GTAGCGGCGC?ATTAAGCGCG

101?GCGGGTGTGG?TGGTTACGCG?CAGCGTGACC?GCTACACTTG?CCAGCGCCCT

151?AGCGCCCGCT?CCTTTCGCTT?TCTTCCCTTC?CTTTCTCGCC?ACGTTCGCCG

201?GCTTTCCCCG?TCAAGCTCTA?AATCGGGGGC?TCCCTTTAGG?GTTCCGATTT

251?AGTGCTTTAC?GGCACCTCGA?CCCCAAAAAA?CTTGATTAGG?GTGATGGTTC

301?ACGTAGTGGG?CCATCGCCCT?GATAGACGGT?TTTTCGCCCT?TTGACGTTGG

351?AGTCCACGTT?CTTTAATAGT?GGACTCTTGT?TCCAAACTGG?AACAACACTC

401?AACCCTATCT?CGGTCTATTC?TTTTGATTTA?TAAGGGATTT?TGCCGATTTC

451?GGCCTATTGG?TTAAAAAATG?AGCTGATTTA?ACAAAAATTT?AACGCGAATT

501?TTAACAAAAT?ATTAACGTTT?ACAATTTCAG?GTGGCACTTT?TCGGGGAAAT

551?GTGCGCGGAA?CCCCTATTTG?TTTATTTTTC?TAAATACATT?CAAATATGTA

601?TCCGCTCATG?AGACAATAAC?CCTGATAAAT?GCTTCAATAA?TATTGAAAAA

651?GGAAGAGTAT?GAGTATTCAA?CATTTCCGTG?TCGCCCTTAT?TCCCTTTTTT

701?GCGGCATTTT?GCCTTCCTGT?TTTTGCTCAC?CCAGAAACGC?TGGTGAAAGT

751?AAAAGATGCT?GAAGATCAGT?TGGGTGCACG?AGTGGGTTAC?ATCGAACTGG

801?ATCTCAACAG?CGGTAAGATC?CTTGAGAGTT?TTCGCCCCGA?AGAACGTTTT

851?CCAATGATGA?GCACTTTTAA?AGTTCTGCTA?TGTGGCGCGG?TATTATCCCG

901?TATTGACGCC?GGGCAAGAGC?AACTCGGTCG?CCGCATACAC?TATTCTCAGA

951?ATGACTTGGT?TGAGTACTCA?CCAGTCACAG?AAAAGCATCT?TACGGATGGC

1001?ATGACAGTAA?GAGAATTATG?CAGTGCTGCC?ATAAGCATGA?GTGATAACAC

1051?TGCGGCCAAC?TTACTTCTGA?CAACGATCGG?AGGACCGAAG?GAGCTAACCG

1101?CTTTTTTTCA?CAACATGGGG?GATCATGTAA?CTCGCCTTGA?TCGTTGGGAA

1151?CCGGAGCTGA?ATGAAGCCAT?ACCAAACGAC?GAGCGTGACA?CCACGATGCC

1201?TGTAGCAATG?GCAACAACGT?TGCGCAAACT?ATTAACTGGC?GAACTACTTA

1251?CTCTAGCTTC?CCGGCAACAA?TTAATAGACT?GGATGGAGGC?GGATAAAGTT

1301?GCAGGACCAC?TTCTGCGCTC?GGCCCTTCCG?GCTGGCTGGT?TTATTGCTGA

1351?TAAATCTGGA?GCCGGTGAGC?GTGGGTCTCG?CGGTATCATT?GCAGCACTGG

1401?GGCCAGATGG?TAAGCCCTCC?CGTATCGTAG?TTATCTACAC?GACGGGCAGT

1451?CAGGCAACTA?TGGATGAACG?AAATAGACAG?ATCGCTGAGA?TAGGTGCCTC

1501?ACTGATTAAG?CATTGGTAAC?TGTCAGACCA?AGTTTACTCA?TATATACTTT

1551?AGATTGATTT?AAAACTTCAT?TTTTAATTTA?AAAGGATCTA?GGTGAAGATC

1601?CTTTTTGATA?ATCTCATGAC?CAAAATCCCT?TAACGTGAGT?TTTCGTTCCA

1651?CTGAGCGTCA?GACCCCGTAG?AAAAGATCAA?AGGATCTTCT?TGAGATCCTT

1701?TTTTTCTGCG?CGTAATCTGC?TGCTTGCAAA?CAAAAAAACC?ACCGCTACCA

1751?GCGGTGGTTT?GTTTGCCGGA?TCAAGAGCTA?CCAACTCTTT?TTCCGAAGGT

1801?AACTGGCTTC?AGCAGAGCGC?AGATACCAAA?TACTGTCCTT?CTAGTGTAGC

1851?CGTAGTTAGG?CCACCACTTC?AAGAACTCTG?TAGCACCGCC?TACATACCTC

1901?GCTCTGCTAA?TCCTGTTACC?AGTGGCTGCT?GCCAGTGGCG?ATAAGTCGTG

1951?TCTTACCGGG?TTGGACTCAA?GACGATAGTT?ACCGGATAAG?GCGCAGCGGT

2001?CGGGCTGAAC?GGGGGGTTCG?TGCACACAGC?CCAGCTTGGA?GCGAACGACC

2051?TACACCGAAC?TGAGATACCT?ACAGCGTGAG?CATTGAGAAA?GCGCCACGCT

2101?TCCCGAAGGG?AGAAAGGCGG?ACAGGTATCC?GGTAAGCGGC?AGGGTCGGAA

2151?CAGGAGAGCG?CACGAGGGAG?CTTCCAGGGG?GGAACGCCTG?GTATCTTTAT

2201?AGTCCTGTCG?GGTTTCGCCA?CCTCTGACTT?GAGCGTCGAT?TTTTGTGATG

2251?CTCGTCAGGG?GGGCCGAGCC?TATGGAAAAA?CGCCAGCAAC?GCGGCCTTTT

2301?TACGGTTCCT?GGCCTTTTGC?TGGCCTTTTG?CTCACATGTT?CTTTCCTGCG

2351?TTATCCCCTG?ATTCTGTGGA?TAACCGTATT?ACCGCCTTTG?AGTGAGCTGA

2401?TACCGCTCGC?CGCAGCCGAA?CGACCGAGCG?CAGCGAGTCA?GTGAGCGAGG

2451?AAGCGGAAGA?GCGCCCAATA?CGCAAACCGC?CTCTCCCCGC?GCGTTGGCCG

2501?ATTCATTAAT?GCAGCTGGCA?CGACAGGTTT?CCCGACTGGA?AAGCGGGCAG

2551?TGAGCGCAAC?GCAATTAATG?TGAGTTACCT?CACTCATTAG?GCACCCCAGG

2601?CTTTACACTT?TATGCTTCCG?GCTCCTATGT?TGTGTGGAAT?TGTGAGCGGA

2651?TAACAATTTC?ACACAGGAAA?CAGCTATGAC?CATGATTACG?CCAAGCTCGG

2701?AAGCGGCCGC?TAATACGACT?CACTATAGGG?ACCAAACAGA?GAATCCGTAA

2751?GTTACGATAA?AAGGCGAAGG?AGCAATTGAA?GTCGCACGGG?TAGAAGGTGT

2801?GAATCTCGAG?TGCGAGCCCG?AAGCACAAAC?TCGAGAAAGC?CTTCTGCCAA

2851?CATGTCTTCC?GTATTTGATG?AGTACGAACA?GCTCCTCGCG?GCTCAGACTC

2901?GCCCCAATGG?AGCTCATGGA?GGGGGAGAAA?AAGGGAGTAC?CTTAAAAGTA

2951?GACGTCCCGG?TATTCACTCT?TAACAGTGAT?GACCCAGAAG?ATAGATGGAG

3001?CTTTGTGGTA?TTCTGCCTCC?GGATTGCTGT?TAGCGAAGAT?GCCAACAAAC

3051?CACTCAGGCA?AGGTGCTCTC?ATATCTCTTT?TATGCTCCCA?CTCACAGGTA

3101?ATGAGGAACC?ATGTTGCCCT?TGCAGGGAAA?CAGAATGAAG?CCACATTGGC

3151?CGTGCTTGAG?ATTGATGGCT?TTGCCAACGG?CACGCCCCAG?TTCAACAATA

3201?GGAGTGGAGT?GTCTGAAGAG?AGAGCACAGA?GATTTGCGAT?GATAGCAGGA

3251?TCTCTCCCTC?GGGCATGCAG?CAACGGAACC?CCGTTCGTCA?CAGCCGGGGC

3301?CGAAGATGAT?GCACCAGAAG?ACATCACCGA?TACCCTGGAG?AGGATCCTCT

3351?CTATCCAGGC?TCAAGTATGG?GTCACAGTAG?CAAAAGCCAT?GACTGCGTAT

3401?GAGACTGCAG?ATGAGTCGGA?AACAAGGCGA?ATCAATAAGT?ATATGCAGCA

3451?AGGCAGGGTC?CAAAAGAAAT?ACATCCTCTA?CCCCGTATGC?AGGAGCACAA

3501?TCCAACTCAC?GATCAGACAG?TCTCTTGCAG?TCCGCATCTT?TTTGGTTAGC

3551?GAGCTCAAGA?GAGGCCGCAA?CACGGCAGGT?GGTACCTCTA?CTTATTATAA

3601?CCTGGTAGGG?GACGTAGACT?CATACATCAG?GAATACCGGG?CTTACTGCAT

3651?TCTTCTTGAC?ACTCAAGTAC?GGAATCAACA?CCAAGACATC?AGCCCTTGCA

3701?CTTAGTAGCC?TCTCAGGCGA?CATCCAGAAG?ATGAAGCAGC?TCATGCGTTT

3751?GTATCGGATG?AAAGGAGATA?ATGCGCCGTA?CATGACATTA?CTTGGTGATA

3801?GTGACCAGAT?GAGCTTTGCG?CCTGCCGAGT?ATGCACAACT?TTACTCCTTT

3851?GCCATGGGTA?TGGCATCAGT?CCTAGATAAA?GGTACTGGGA?AATACCAATT

3901?TGCCAGGGAC?TTTATGAGCA?CATCATTCTG?GAGACTTGGA?GTAGAGTACG

3951?CTCAGGCTCA?GGGAAGTAGC?ATTAACGAGG?ATATGGCTGC?CGAGCTAAAG

4001?CTAACCCCAG?CAGCAAGGAG?GGGCCTGGCA?GCTGCTGCCC?AACGGGTCTC

4051?CGAGGAGACC?AGCAGCATAG?ACATGCCTAC?TCAACAAGTC?GGAGTCCTCA

4101?CTGGGCTTAG?CGAGGGGGGG?TCCCAAGCTC?TACAAGGCGG?ATCGAATAGA

4151?TCGCAAGGGC?AACCAGAAGC?CGGGGATGGG?GAGACCCAAT?TCCTGGATCT

4201?GATGAGAGCG?GTAGCAAATA?GCATGAGGGA?GGCGCCAAAC?TCTGCACAGG

4251?GCACTCCCCA?ATCGGGGCCT?CCCCCAACTC?CTGGGCCATC?CCAAGATAAC

4301?GACACCGACT?GGGGGTATTG?ATGGACAAAA?CCCAGCCTGC?TTCCACAAAA

4351?ACATCCCAAT?GCCCTCACCC?GTAGTCGACC?CCTCGATTTG?CGGCTCTATA

4401?TGACCACACC?CTCAAACAAA?CATCCCCCTC?TTTCCTCCCT?CCCCCTGCTG

4451?TACAACTCCG?CACGCCCTAG?ATACCACAGG?CACAATGCGG?CTCACTAACA

4501?ATCAAAACAG?AGCCGAGGGA?ATTAGAAAAA?AGTACGGGTA?GAAGAGGGAT

4551?ATTCAGAGAT?CAGGGCAAGT?CTCCCGAGTC?TCTGCTCTCT?CCTCTACCTG

4601?ATAGACCAGG?ACAAACATGG?CCACCTTTAC?AGATGCAGAG?ATCGACGAGC

4651?TATTTGAGAC?AAGTGGAACT?GTCATTGACA?ACATAATTAC?AGCCCAGGGT

4701?AAACCAGCAG?AGACTGTTGG?AAGGAGTGCA?ATCCCACAAG?GCAAGACCAA

4751?GGTGCTGAGC?GCAGCATGGG?AGAAGCATGG?GAGCATCCAG?CCACCGGCCA

4801?GTCAAGACAA?CCCCGATCGA?CAGGACAGAT?CTGACAAACA?ACCATCCACA

4851?CCCGAGCAAA?CGACCCCGCA?TGACAGCCCG?CCGGCCACAT?CCGCCGACCA

4901?GCCCCCCACC?CAGGCCACAG?ACGAAGCCGT?CGACACACAG?CTCAGGACCG

4951?GAGCAAGCAA?CTCTCTGCTG?TTGATGCTTG?ACAAGCTCAG?CAATAAATCG

5001?TCCAATGCTA?AAAAGGGCCC?ATGGTCGAGC?CCCCAAGAGG?GGAATCACCA

5051?ACGTCCGACT?CAACAGCAGG?GGAGTCAACC?CAGTCGCGGA?AACAGTCAGG

5101?AAAGACCGCA?GAACCAAGTC?AAGGCCGCCC?CTGGAAACCA?GGGCACAGAC

5151?GTGAACACAG?CATATCATGG?ACAATGGGAG?GAGTCACAAC?TATCAGCTGG

5201?TGCAACCCCT?CATGCTCTCC?GATCAAGGCA?GAGCCAAGAC?AATACCCTTG

5251?TATCTGCGGA?TCATGTCCAG?CCACCTGTAG?ACTTTGTGCA?AGCGATGATG

5301?TCTATGATGG?AGGCGATATC?ACAGAGAGTA?AGTAAGGTTG?ACTATCAGCT

5351?AGATCTTGTC?TTGAAACAGA?CATCCTCCAT?CCCTATGATG?CGGTCCGAAA

5401?TCCAACAGCT?GAAAACATCT?GTTGCAGTCA?TGGAAGCCAA?CTTGGGAATG

5451?ATGAAGATTC?TGGATCCCGG?TTGTGCCAAC?ATTTCATCTC?TGAGTGATCT

5501?ACGGGCAGTT?GCCCGATCTC?ACCCGGTTTT?AGTTTCAGGC?CCTGGAGACC

5551?CCTCTCCCTA?TGTGACACAA?GGAGGCGAAA?TGGCACTTAA?TAAACTTTCG

5601?CAACCAGTGC?CACATCCATC?TGAATTGATT?AAACCCGCCA?CTGCATGCGG

5651?GCCTGATATA?GGAGTGGAAA?AGGACACTGT?CCGTGCATTG?ATCATGTCAC

5701?GCCCAATGCA?CCCGAGTTCT?TCAGCCAAGC?TCCTAAGCAA?GTTAGATGCA

5751?GCCGGGTCGA?TCGAGGAAAT?CAGGAAAATC?AAGCGCCTTG?CTCTAAATGG

5801?CTAATTACTA?CTGCCACACG?TAGCGGGTCC?CTGTCCACTC?GGCATCACAC

5851?GGAATCTGCA?CCGAGTTCCC?CCCCGCAGAC?CCAAGGTCCA?ACTCTCCAAG

5901?CGGCAATCCT?CTCTCGCTTC?CTCAGCCCCA?CTGAATGATC?GCGTAACCGT

5951?AATTAATCTA?GCTACATTTA?AGATTAAGAA?AAAATACGGG?TAGAATTGGA

6001?GTGCCCCAAT?TGTGCCAAGA?TGGACTCATC?TAGGACAATT?GGGCTGTACT

6051?TTGATTCTGC?CCATTCTTCT?AGCAACCTGT?TAGCATTTCC?GATCGTCCTA

6101?CAAGACACAG?GAGATGGGAA?GAAGCAAATC?GCCCCGCAAT?ATAGGATCCA

6151?GCGCCTTGAC?TTGTGGACTG?ATAGTAAGGA?GGACTCAGTA?TTCATCACCA

6201?CCTATGGATT?CATCTTTCAA?GTTGGGAATG?AAGAAGCCAC?TGTCGGCATG

6251?ATCGATGATA?AACCCAAGCG?CGAGTTACTT?TCCGCTGCGA?TGCTCTGCCT

6301?AGGAAGCGTC?CCAAATACCG?GAGACCTTAT?TGAGCTGGCA?AGGGCCTGTC

6351?TCACTATGAT?AGTCACATGC?AAGAAGAGTG?CAACTAATAC?TGAGAGAATG

6401?GTTTTCTCAG?TAGTGCAGGC?ACCCCAAGTG?CTGCAAAGCT?GTAGGGTTGT

6451?GGCAAACAAA?TACTCATCAG?TGAATGCAGT?CAAGCACGTG?AAAGCGCCAG

6501?AGAAGATTCC?CGGGAGTGGA?ACCCTAGAAT?ACAAGGTGAA?CTTTGTCTCC

6551?TTGACTGTGG?TACCGAAGAA?GGATGTCTAC?AAGATCCCAG?CTGCAGTATT

6601?GAAGGTTTCT?GGCTCGAGTC?TGTACAATCT?TGCGCTCAAT?GTCACTATTA

6651?ATGTGGAGGT?AGACCCGAGG?AGTCCTTTGG?TTAAATCTCT?GTCTAAGTCT

6701?GACAGCGGAT?ACTATGCTAA?CCTCTTCTTG?CATATTGGAC?TTATGACCAC

6751?CGTAGATAGG?AAGGGGAAGA?AAGTGACATT?TGACAAGCTG?GAAAAGAAAA

6801?TAAGGAGCCT?TGATCTATCT?GTCGGGCTCA?GTGATGTGCT?CGGGCCTTCC

6851?GTGTTGGTAA?AAGCAAGAGG?TGCACGGACT?AAGCTTTTGG?CACCTTTCTT

6901?CTCTAGCAGT?GGGACAGCCT?GCTATCCCAT?AGCAAATGCT?TCTCCTCAGG

6951?TGGCCAAGAT?ACTCTGGAGT?CAAACCGCGT?GCCTGCGGAG?CGTTAAAATC

7001?ATTATCCAAG?CAGGTACCCA?ACGCGCTGTC?GCAGTGACCG?CCGACCACGA

7051?GGTTACCTCT?ACTAAGCTGG?AGAAGGGGCA?CACCCTTGCC?AAATACAATC

7101?CTTTTAAGAA?ATAAGCTGCG?TCTCTGAGAT?TGCGCTCCGC?CCACTCACCC

7151?AGATCATCAT?GACACAAAAA?ACTAATCTGT?CTTGATTATT?TACAGTTAGT

7201?TTACCTGTCT?ATCAAGTTAG?AAAAAACACG?GGTAGAAGAT?TCTGGATCCC

7251?GGTTGGCGCC?CTCCAGGTGC?AAGATGGGCT?CCAGACCTTC?TACCAAGAAC

7301?CCAGCACCTA?TGATGCTGAC?TATCCGGGTT?GCGCTGGTAC?TGAGTTGCAT

7351?CTGTCCGGCA?AACTCCATTG?ATGGCAGGCC?TCTTGCAGCT?GCAGGAATTG

7401?TGGTTACAGG?AGACAAAGCC?GTCAACATAT?ACACCTCATC?CCAGACAGGA

7451?TCAATCATAG?TTAAGCTCCT?CCCGAATCTG?CCCAAGGATA?AGGAGGCATG

7501?TGCGAAAGCC?CCCTTGGATG?CATACAACAG?GACATTGACC?ACTTTGCTCA

7551?CCCCCCTTGG?TGACTCTATC?CGTAGGATAC?AAGAGTCTGT?GACTACATCT

7601?GGAGGGGGGA?GACAGGGGCG?CCTTATAGGC?GCCATTATTG?GCGGTGTGGC

7651?TCTTGGGGTT?GCAACTGCCG?CACAAATAAC?AGCGGCCGCA?GCTCTGATAC

7701?AAGCCAAACA?AAATGCTGCC?AACATCCTCC?GACTTAAAGA?GAGCATTGCC

7751?GCAACCAATG?AGGCTGTGCA?TGAGGTCACT?GACGGATTAT?CGCAACTAGC

7801?AGTGGCAGTT?GGGAAGATGC?AGCAGTTTGT?TAATGACCAA?TTTAATAAAA

7851?CAGCTCAGGA?ATTAGACTGC?ATCAAAATTG?CACAGCAAGT?TGGTGTAGAG

7901?CTCAACCTGT?ACCTAACCGA?ATTGACTACA?GTATTCGGAC?CACAAATCAC

7951?TTCACCTGCT?TTAAACAAGC?TGACTATTCA?GGCACTTTAC?AATCTAGCTG

8001?GTGGAAATAT?GGATTACTTA?TTGACTAAGT?TAGGTGTAGG?GAACAATCAA

8051?CTCAGCTCAT?TAATCGGTAG?CGGCTTAATC?ACCGGTAACC?CTATTCTATA

8101?CGACTCACAG?ACTCAACTCT?TGGGTATACA?GGTAACTCTA?CCTTCAGTCG

8151?GGAACCTAAA?TAATATGCGT?GCCACCTACT?TGGAAACCTT?ATCCGTAAGC

8201?ACAACCAGGG?GATTTGCCTC?GGCACTTGTC?CCAAAAGTGG?TGACACAGGT

8251?CGGTTCTGTG?ATAGAAGAAC?TTGACACCTC?ATACTGTATA?GAAACTGACT

8301?TAGATTTATA?TTGTACAAGA?ATAGTAACGT?TCCCTATGTC?CCCTGGTATT

8351?TATTCCTGCT?TGAGCGGCAA?TACGTCGGCC?TGTATGTACT?CAAAGACCGA

8401?AGGCGCACTT?ACTACACCAT?ACATGACTAT?CAAAGGTTCA?GTCATCGCCA

8451?ACTGCAAGAT?GACAACATGT?AGATGTGTAA?ACCCCCCGGG?TATCATATCG

8501?CAAAACTATG?GAGAAGCCGT?GTCTCTAATA?GATAAACAAT?CATGCAATGT

8551?TTTATCCTTA?GGCGGGATAA?CTTTAAGGCT?CAGTGGGGAA?TTCGATGTAA

8601?CTTATCAGAA?GAATATCTCA?ATACAAGATT?CTCAAGTAAT?AATAACAGGC

8651?AATCTTGATA?TCTCAACTGA?GCTTGGGAAT?GTCAACAACT?CGATCAGTAA

8701?TGCTTTGAAT?AAGTTAGAGG?AAAGCAACAG?AAAACTAGAC?AAAGTCAATG

8751?TCAAACTGAC?TAGCACATCT?GCTCTCATTA?CCTATATCGT?TTTGACTATC

8801?ATATCTCTTG?TTTTTGGTAT?ACTTAGCCTG?ATTCTAGCAT?GCTACCTAAT

8851?GTACAAGCAA?AAGGCGCAAC?AAAAGACCTT?ATTATGGCTT?GGGAATAATA

8901?CTCTAGACCA?GATGAGAGCC?ACTACAAAAA?TGTGAACACA?GATGAGGAAC

8951?GAAGGTTTCC?CTAATAGTAA?TTTGTGTGAA?AGTTCTGGTA?GTCTGTCAGT

9001?TCAGAGAGTT?AAGAAAAAAC?TACCGGTTGT?AGATGACCAA?AGGACGATAT

9051?ACGGGTAGAA?CGGTAAGAGA?GGCCGCCCCT?CAATTGCGAG?CCAGGCTTCA

9101?CAACCTCCGT?TCTACCGCTT?CACCGACAAC?AGTCCTCAAT?CATGGACCGC

9151?GCCGTTAGCC?AAGTTGCGTT?AGAGAATGAT?GAAAGAGAGG?CAAAAAATAC

9201?ATGGCGCTTG?ATATTCCGGA?TTGCAATCTT?ATTCTTAACA?GTAGTGACCT

9251?TGGCTATATC?TGTAGCCTCC?CTTTTATATA?GCATGGGGGC?TAGCACACCT

9301?AGCGATCTTG?TAGGCATACC?GACTAGGATT?TCCAGGGCAG?AAGAAAAGAT

9351?TACATCTACA?CTTGGTTCCA?ATCAAGATGT?AGTAGATAGG?ATATATAAGC

9401?AAGTGGCCCT?TGAGTCTCCG?TTGGCATTGT?TAAATACTGA?GACCACAATT

9451?ATGAACGCAA?TAACATCTCT?CTCTTATCAG?ATTAATGGAG?CTGCAAACAA

9501?CAGTGGGTGG?GGGGCACCTA?TCCATGACCC?AGATTATATA?GGGGGGATAG

9551?GCAAAGAACT?CATTGTAGAT?GATGCTAGTG?ATGTCACATC?ATTCTATCCC

9601?TCTGCATTTC?AAGAACATCT?GAATTTTATC?CCGGCGCCTA?CTACAGGATC

9651?AGGTTGCACT?CGAATACCCT?CATTTGACAT?GAGTGCTACC?CATTACTGCT

9701?ACACCCATAA?TGTAATATTG?TCTGGATGCA?GAGATCACTC?ACATTCATAT

9751?CAGTATTTAG?CACTTGGTGT?GCTCCGGACA?TCTGCAACAG?GGAGGGTATT

9801?CTTTTCTACT?CTGCGTTCCA?TCAACCTGGA?CGACACCCAA?AATCGGAAGT

9851?CTTGCAGTGT?GAGTGCAACT?CCCCTGGGTT?GTGATATGCT?GTGCTCGAAA

9901?GTCACGGAGA?CAGAGGAAGA?AGATTATAAC?TCAGCTGTCC?CTACGCGGAT

9951?GGTACATGGG?AGGTTAGGGT?TCGACGGCCA?GTACCACGAA?AAGGACCTAG

10001?ATGTCACAAC?ATTATTCGGG?GACTGGGTGG?CCAACTACCC?AGGAGTAGGG

10051?GGTGGATCTT?TTATTGACAG?CCGCGTATGG?TTCTCAGTCT?ACGGAGGGTT

10101?AAAACCCAAT?TCACCCAGTG?ACACTGTACA?GGAAGGGAAA?TATGTGATAT

10151?ACAAGCGATA?CAATGACACA?TGCCCAGATG?AGCAAGACTA?CCAGATTCGA

10201?ATGGCCAAGT?CTTCGTATAA?GCCTGGACGG?TTTGGTGGGA?AACGCATACA

10251?GCAGGCTATC?TTATCTATCA?AGGTGTCAAC?ATCCTTAGGC?GAAGACCCGG

10301?TACTGACTGT?ACCGCCCAAC?ACAGTCACAC?TCATGGGGGC?CGAAGGCAGA

10351?ATTCTCACAG?TAGGGACATC?TCATTTCTTG?TATCAACGAG?GGTCATCATA

10401?CTTCTCTCCC?GCGTTATTAT?ATCCTATGAC?AGTCAGCAAC?AAAACAGCCA

10451?CTCTTCATAG?TCCTTATACA?TTCAATGCCT?TCACTCGGCC?AGGTAGTATC

10501?CCTTGCCAGG?CTTCAGCAAG?ATGCCCCAAC?TCGTGTGTTA?CTGGAGTCTA

10551?TACAGATCCA?TATCCCCTAA?TCTTCTATAG?AAACCACACC?TTGCGAGGGG

10601?TATTCGGGAC?AATGCTTGAT?GGTGTACAAG?CAAGACTTAA?CCCTGCGTCT

10651?GCAGTATTCG?ATAGCACATC?CCGCAGTCGC?ATTACTCGAG?TGAGTTCAAG

10701?CAGTACCAAA?GCAGCATACA?CAACATCAAC?TTGTTTTAAA?GTGGTCAAGA

10751?CTAATAAGAC?CTATTGTCTC?AGCATTGCTG?AAATATCTAA?TACTCTCTTC

10801?GGAGAATTCA?GAATCGTCCC?GTTACTAGTT?GAGATCCTCA?AAGATGACGG

10851?GGTTAGAGAA?GCCAGGTCTG?GCTAGTTGAG?TCAATTATAA?AGGAGTTGGA

10901?AAGATGGCAT?TGTATCACCT?ATCTTCTGCG?ACATCAAGAA?TCAAACCGAA

10951?TGCCGGCGCG?TGCTCGAATT?CCATGTTGCC?AGTTGACCAC?AATCAGCCAG

11001?TGCTCATGCG?ATCAGATTAA?GCCTTGTCAA?TAGTCTCGTT?AACTTAAGAA

11051?AAAATACGGG?TAGAACCGCC?ACCATGGAGA?CAGACACACT?CCTGCTATGG

11101?GTACTGCTGC?TCTGGGTTCC?AGGATCCACT?GGTACCTCTG?AGGAAACCAT

11151?TTCTACAGTT?CAAGAAAAGC?AACAAAATAT?TTCTCCCCTA?GTGAGAGAAA

11201?GAGGTCCTCA?GAGAGTAGCA?GCTCACATAA?CTGGGACCAG?AGGAAGAAGC

11251?AACACATTGT?CTTCTCCAAA?CTCCAAGAAT?GAAAAGGCTC?TGGGCCGCAA

11301?AATAAACTCC?TGGGAATCAT?CAAGGAGTGG?GCATTCATTC?CTGAGCAACT

11351?TGCACTTGAG?GAATGGTGAA?CTGGTCATCC?ATGAAAAAGG?GTTTTACTAC

11401?ATCTATTCCC?AAACATACTT?TCGATTTCAG?GAGGAAATAA?AAGAAAACAC

11451?AAAGAACGAC?AAACAAATGG?TCCAATATAT?TTACAAATAC?ACAAGTTATC

11501?CTGACCCTAT?ATTGTTGATG?AAAAGTGCTA?GAAATAGTTG?TTGGTCTAAA

11551?GATGCAGAAT?ATGGACTCTA?TTCCATCTAT?CAAGGGGGAA?TATTTGAGCT

11601?TAAGGAAAAT?GACAGAATTT?TTGTTTCTGT?AACAAATGAG?CACTTGATAG

11651?ACATGGACCA?TGAAGCCAGT?TTTTTCGGGG?CCTTTTTAGT?TGGCTAAGTT

11701?AACGTTTAAA?CAAGCTTGGC?GCGCCCGACG?CGTTTGATTA?AGAAAAAATG

11751?TAAGTGGCAA?TGAGATACAA?GGCAAAACAG?CTCATGGTAA?ATAATACGGG

11801?TAGGACATGG?CGAGCTCCGG?TCCTGAAAGG?GCAGAGCATC?AGATTATCCT

11851?ACCAGAGTCA?CACCTGTCTT?CACCATTGGT?CAAGCACAAA?CTACTCTATT

11901?ACTGGAAATT?AACTGGGCTA?CCGCTTCCTG?ATGAATGTGA?CTTCGACCAC

11951?CTCATTCTCA?GCCGACAATG?GAAAAAAATA?CTTGAATCGG?CCTCTCCTGA

12001?TACTGAGAGA?ATGATAAAAC?TCGGAAGGGC?AGTACACCAA?ACTCTTAACC

12051?ACAATTCCAG?AATAACCGGA?GTGCTCCACC?CCAGGTGTTT?AGAAGAACTG

12101?GCTAATATTG?AGGTCCCAGA?TTCAACCAAC?AAATTTCGGA?AGATTGAGAA

12151?GAAGATCCAA?ATTCACAACA?CGAGATATGG?AGAACTGTTC?ACAAGGCTGT

12201?GTACGCATAT?AGAGAAGAAA?CTGCTGGGGT?CATCTTGGTC?TAACAATGTC

12251?CCCCGGTCAG?AGGAGTTCAG?CAGCATTCGT?ACGGATCCGG?CATTCTGGTT

12301?TCACTCAAAA?TGGTCCACAG?CCAAGTTTGC?ATGGCTCCAT?ATAAAACAGA

12351?TCCAGAGGCA?TCTGATGGTG?GCAGCTAGGA?CAAGGTCTGC?GGCCAACAAA

12401?TTGGTGATGC?TAACCCATAA?GGTAGGCCAA?GTCTTTGTCA?CTCCTGAACT

12451?TGTCGTTGTG?ACGCATACGA?ATGAGAACAA?GTTCACATGT?CTTACCCAGG

12501?AACTTGTATT?GATGTATGCA?GATATGATGG?AGGGCAGAGA?TATGGTCAAC

12551?ATAATATCAA?CCACGGCGGT?GCATCTCAGA?AGCTTATCAG?AGAAAATTGA

12601?TGACATTTTG?CGGTTAATAG?ACGCTCTGGC?AAAAGACTTG?GGTAATCAAG

12651?TCTACGATGT?TGTATCACTA?ATGGAGGGAT?TTGCATACGG?AGCTGTCCAG

12701?CTACTCGAGC?CGTCAGGTAC?ATTTGCAGGA?GATTTCTTCG?CATTCAACCT

12751?GCAGGAGCTT?AAAGACATTC?TAATTGGCCT?CCTCCCCAAT?GATATAGCAG

12801?AATCCGTGAC?TCATGCAATC?GCTACTGTAT?TCTCTGGTTT?AGAACAGAAT

12851?CAAGCAGCTG?AGATGTTGTG?TCTGTTGCGT?CTGTGGGGTC?ACCCACTGCT

12901?TGAGTCCCGT?ATTGCAGCAA?AGGCAGTCAG?GAGCCAAATG?TGCGCACCGA

12951?AAATGGTAGA?CTTTGATATG?ATCCTTCAGG?TACTGTCTTT?CTTCAAGGGA

13001?ACAATCATCA?ACGGGTACAG?AAAGAAGAAT?GCAGGTGTGT?GGCCGCGAGT

13051?CAAAGTGGAT?ACAATATATG?GGAAGGTCAT?TGGGCAACTA?CATGCAGATT

13101?CAGCAGAGAT?TTCACACGAT?ATCATGTTGA?GAGAGTATAA?GAGTTTATCT

13151?GCACTTGAAT?TTGAGCCATG?TATAGAATAT?GACCCTGTCA?CCAACCTGAG

13201?CATGTTCCTA?AAAGACAAGG?CAATCGCACA?CCCCAACGAT?AATTGGCTTG

13251?CCTCGTTTAG?GCGGAACCTT?CTCTCCGAAG?ACCAGAAGAA?ACATGTAAAA

13301?GAAGCAACTT?CGACTAATCG?CCTCTTGATA?GAGTTTTTAG?AGTCAAATGA

13351?TTTTGATCCA?TATAAAGAGA?TGGAATATCT?GACGACCCTT?GAGTACCTTA

13401?GAGATGACAA?TGTGGCAGTA?TCATACTCGC?TCAAGGAGAA?GGAAGTGAAA

13451?GTTAATGGAC?GGATCTTCGC?TAAGCTGACA?AAGAAGTTAA?GGAACTGTCA

13501?GGTGATGGCG?GAAGGGATCC?TAGCCGATCA?GATTGCACCT?TTCTTTCAGG

13551?GAAATGGAGT?CATTCAGGAT?AGCATATCCT?TGACCAAGAG?TATGCTAGCG

13601?ATGAGTCAAC?TGTCTTTTAA?CAGCAATAAG?AAACGTATCA?CTGACTGTAA

13651?AGAAAGAGTA?TCTTCAAACC?GCAATCATGA?TCCGAAAAGC?AAGAACCGTC

13701?GGAGAGTTGC?AACCTTCATA?ACAACTGACC?TGCAAAAGTA?CTGTCTTAAT

13751?TGGAGATATC?AGACAATCAA?ATTGTTCGCT?CATGCCATCA?ATCAGTTGAT

13801?GGGCCTACCT?CACTTCTTCG?AATGGATTCA?CCTAAGACTG?ATGGACACTA

13851?CGATGTTCGT?AGGAGACCCT?TTCAATCCTC?CAAGTGACCC?TACTGACTGT

13901?GACCTCTCAA?GAGTCCCTAA?TGATGACATA?TATATTGTCA?GTGCCAGAGG

13951?GGGTATCGAA?GGATTATGCC?AGAAGCTATG?GACAATGATC?TCAATTGCTG

14001?CAATCCAACT?TGCTGCAGCT?AGATCGCATT?GTCGTGTTGC?CTGTATGGTA

14051?CAGGGTGATA?ATCAAGTAAT?AGCAGTAACG?AGAGAGGTAA?GATCAGACGA

14101?CTCTCCGGAG?ATGGTGTTGA?CACAGTTGCA?TCAAGCCAGT?GATAATTTCT

14151?TCAAGGAATT?AATTCATGTC?AATCATTTGA?TTGGCCATAA?TTTGAAGGAT

14201?CGTGAAACCA?TCAGGTCAGA?CACATTCTTC?ATATACAGCA?AACGAATCTT

14251?CAAAGATGGA?GCAATCCTCA?GTCAAGTCCT?CAAAAATTCA?TCTAAATTAG

14301?TGCTAGTGTC?AGGTGATCTC?AGTGAAAACA?CCGTAATGTC?CTGTGCCAAC

14351?ATTGCCTCTA?CTGTAGCACG?GCTATGCGAG?AACGGGCTTC?CCAAAGACTT

14401?CTGTTACTAT?TTAAACTATA?TAATGAGTTG?TGTGCAGACA?TACTTTGACT

14451?CTGAGTTCTC?CATCACCAAC?AATTCGCACC?CCGATCTTAA?TCAGTCGTGG

14501?ATTGAGGACA?TCTCTTTTGT?GCACTCATAT?GTTCTGACTC?CTGCCCAATT

14551?AGGGGGACTG?AGTAACCTTC?AATACTCAAG?GCTCTACACT?AGAAATATCG

14601?GTGACCCGGG?GACTACTGCT?TTTGCAGAGA?TCAAGCGACT?AGAAGCAGTG

14651?GGATTACTGA?GTCCTAACAT?TATGACTAAT?ATCTTAACTA?GGCCGCCTGG

14701?GAATGGAGAT?TGGGCCAGTC?TGTGCAACGA?CCCATACTCT?TTCAATTTTG

14751?AGACTGTTGC?AAGCCCAAAT?ATTGTTCTTA?AGAAACATAC?GCAAAGAGTC

14801?CTATTTGAAA?CTTGTTCAAA?TCCCTTATTG?TCTGGAGTGC?ACACAGAGGA

14851?TAATGAGGCA?GAAGAGAAGG?CATTGGCTGA?ATTCTTGCTT?AATCAAGAGG

14901?TGATTCATCC?CCGCGTTGCG?CATGCCATCA?TGGAGGCAAG?CTCTGTAGGT

14951?AGGAGAAAGC?AAATTCAAGG?GCTTGTTGAC?ACAACAAACA?CCGTAATTAA

15001?GATTGCGCTT?ACTAGGAGGC?CATTAGGCAT?CAAGAGGCTG?ATGCGGATAG

15051?TCAATTATTC?TAGCATGCAT?GCAATGCTGT?TTAGAGACGA?TGTTTTTTCC

15101?TCCAGTAGAT?CCAACCACCC?CTTAGTCTCT?TCTAATATGT?GTTCTCTGAC

15151?ACTGGCAGAC?TATGCACGGA?ATAGAAGCTG?GTCACCTTTG?ACGGGAGGCA

15201?GGAAAATACT?GGGTGTATCT?AATCCTGATA?CGATAGAACT?CGTAGAGGGT

15251?GAGATTCTTA?GTGTAAGCGG?AGGGTGTACA?AGATGTGACA?GCGGAGATGA

15301?ACAATTTACT?TGGTTCCATC?TTCCAAGCAA?TATAGAATTG?ACCGATGACA

15351?CCAGCAAGAA?TCCTCCGATG?AGGGTACCAT?ATCTCGGGTC?AAAGACACAG

15401?GAGAGGAGAG?CTGCCTCACT?TGCAAAAATA?GCTCATATGT?CGCCACATGT

15451?AAAGGCTGCC?CTAAGGGCAT?CATCCGTGTT?GATCTGGGCT?TATGGGGATA

15501?ATGAAGTAAA?TTGGACTGCT?GCTCTTACGA?TTGCAAAATC?TCGGTGTAAT

15551?GTAAACTTAG?AGTATCTTCG?GTTACTGTCC?CCTTTACCCA?CGGCTGGGAA

15601?TCTTCAACAT?AGACTAGATG?ATGGTATAAC?TCAGATGACA?TTCACCCCTG

15651?CATCTCTCTA?CAGGGTGTCA?CCTTACATTC?ACATATCCAA?TGATTCTCAA

15701?AGGCTGTTCA?CTGAAGAAGG?AGTCAAAGAG?GGGAATGTGG?TTTACCAACA

15751?GATCATGCTC?TTGGGTTTAT?CTCTAATCGA?ATCGATCTTT?CCAATAACAA

15801?CAACCAGGAC?ATATGATGAG?ATCACACTGC?ACCTACATAG?TAAATTTAGT

15851?TGCTGTATCA?GAGAAGCACC?TGTTGCGGTT?CCTTTCGAGC?TACTTGGGGT

15901?GGTACCGGAA?CTGAGGACAG?TGACCTCAAA?TAAGTTTATG?TATGATCCTA

15951?GCCCTGTATC?GGAGGGAGAC?TTTGCGAGAC?TTGACTTAGC?TACTTTCAAG

16001?AGTTATGAGC?TTAATCTGGA?GTCATATCCC?ACGATAGAGC?TAATGAACAT

16051?TCTTTCAATA?TCCAGCGGGA?AGTTGATTGG?CCAGTCTGTG?GTTTCTTATG

16101?ATGAAGATAC?CTCCATAAAG?AATGACGCCA?TAATAGTGTA?TGACAATACC

16151?CGAAATTGGA?TCAGTGAAGC?TCAGAATTCA?GATGTGGTCC?GCCTATTTGA

16201?ATATGCAGCA?CTTGAAGTGC?TCCTCGACTG?TTCTTACCAA?CTCTATTACC

16251?TGAGAGTAAG?AGGCCTAGAC?AATATTGTCT?TATATATGGG?TGATTTATAC

16301?AAGAATATGC?CAGGAATTCT?ACTTTCCAAC?ATTGCAGCTA?CAATATCTCA

16351?TCCCGTCATT?CATTCAAGGT?TACATGCAGT?GGGCCTGGTC?AACCATGACG

16401?GATCACACCA?ACTTGCAGAT?ACGGATTTTA?TCGAAATGTC?TGCAAAACTA

16451?TTAGTATCTT?GCACCCGACG?TGTGATCTCC?GGCTTATATT?CAGGAAATAA

16501?GTATGATCTG?CTGTTCCCAT?CTGTCTTAGA?TGATAACCTG?AATGAGAAGA

16551?TGCTTCAGCT?GATATCCCGG?TTATGCTGTC?TGTACACGGT?ACTCTTTGCT

16601?ACAACAAGAG?AAATCCCGAA?AATAAGAGGC?TTAACTGCAG?AAGAGAAATG

16651?TTCAATACTC?ACTGAGTATT?TACTGTCGGA?TGCTGTGAAA?CCATTACTTA

16701?GTCCCGATCA?AGTGAGCTCT?ATCATGTCTC?CTAACATAAT?TACATTCCCA

16751?GCTAATCTGT?ACTACATGTC?TCGGAAGAGC?CTCAATTTGA?TCAGGGAAAG

16801?GGAGGACAGG?GATACTATCC?TGGCGTTGTT?GTTCCCCCAA?GAGCCATTAT

16851?TAGAGTTCCC?TTCTGTGCAA?GATATTGGTG?CTCGAGTGAA?AGATCCATTC

16901?ACCCGACAAC?CTGCGGCATT?TTTGCAAGAG?TTAGATTTGA?GTGCTCCAGC

16951?AAGGTATGAC?GCATTCACAC?TTAGTCAGAT?TCATCCTGAA?CTCACATCTC

17001?CAAATCCGGA?GGAAGACTAC?TTAGTACGAT?ACTTGTTCAG?AGGGATAGGG

17051?ACTGCATCTT?CCTCTTGGTA?TAAGGCATCT?CATCTCCTTT?CTGTACCCGA

17101?GGTAAGATGT?GCAAGACACG?GGAACTCCTT?ATACTTAGCT?GAAGGGAGCG

17151?GAGCCATCAT?GAGTCTTCTC?GAACTGCATG?TACCACATGA?AACTATCTAT

17201?TACAATACGC?TCTTTTCAAA?TGAGATGAAC?CCCCCGCAAC?GACATTTCGG

17251?GCCGACCCCA?ACTCAGTTTT?TGAATTCGGT?TGTTTATAGG?AATCTACAGG

17301?CGGAGGTAAC?ATGCAAAGAT?GGATTTGTCC?AAGAGTTCCG?TCCATTATGG

17351?AGAGAAAATA?CAGAGGAAAG?TGACCTGACC?TCAGATAAAG?TAGTGGGGTA

17401?TATTACATCT?GCAGTGCCCT?ACAGATCTGT?ATCATTGCTG?CATTGTGACA

17451?TTGAAATTCC?TCCAGGGTCC?AATCAAAGCT?TACTAGATCA?ACTAGCTATC

17501?AATTTATCTC?TGATTGCCAT?GCATTCTGTA?AGGGAGGGCG?GGGTAGTAAT

17551?CATCAAAGTG?TTGTATGCAA?TGGGATACTA?CTTTCATCTA?CTCATGAACT

17601?TGTTTGCTCC?GTGTTCCACA?AAAGGATATA?TTCTCTCTAA?TGGTTATGCA

17651?TGTCGAGGAG?ATATGGAGTG?TTACCTGGTA?TTTGTCATGG?GTTACCTGGG

17701?CGGGCCTACA?TTTGTACATG?AGGTGGTGAG?GATGGCGAAA?ACTCTGGTGC

17751?AGCGGCACGG?TACGCTTTTG?TCTAAATCAG?ATGAGATCAC?ACTGACCAGG

17801?TTATTCACCT?CACAGCGGCA?GCGTGTGACA?GACATCCTAT?CCAGTCCTTT

17851?ACCAAGATTA?ATAAAGTACT?TGAGGAAGAA?TATTGACACT?GCGCTGATTG

17901?AAGCCGGGGG?ACAGCCCGTC?CGTCCATTCT?GTGCGGAGAG?TCTGGTGAGC

17951?ACGCTAGCGA?ACATAACTCA?GATAACCCAG?ATCATCGCTA?GTCACATTGA

18001?CACAGTTATC?CGGTCTGTGA?TATATATGGA?AGCTGAGGGT?GATCTCGCTG

18051?ACACAGTATT?TCTATTTACC?CCTTACAATC?TCTCTACTGA?CGGGAAAAAG

18101?AGGACATCAC?TTAAACAGTG?CACGAGACAG?ATCCTAGAGG?TTACAATACT

18151?AGGTCTTAGA?GTCGAAAATC?TCAATAAAAT?AGGCGATATA?ATCAGCCTAG

18201?TGCTTAAAGG?CATGATCTCC?ATGGAGGACC?TTATCCCACT?AAGGACATAC

18251?TTGAAGCATA?GTACCTGCCC?TAAATATTTG?AAGGCTGTCC?TAGGTATTAC

18301?CAAACTCAAA?GAAATGTTTA?CAGACACTTC?TGTACTGTAC?TTGACTCGTG

18351?CTCAACAAAA?ATTCTACATG?AAAACTATAG?GCAATGCAGT?CAAAGGATAT

18401?TACAGTAACT?GTGACTCTTA?ACGAAAATCA?CATATTAATA?GGCTCCTTTT

18451?TTGGCCAATT?GTATTCTTGT?TGATTTAATC?ATATTATGTT?AGAAAAAAGT

18501?TGAACCCTGA?CTCCTTAGGA?CTCGAATTCG?AACTCAAATA?AATGTCTTAA

18551?AAAAAGGTTG?CGCACAATTA?TTCTTGAGTG?TAGTCTCGTC?ATTCACCAAA

18601?TCTTTGTTTG?GTTTGGTGGC?CGGCATGGTC?CCAGCCTCCT?CGCTGGCGCC

18651?GGCTGGGCAA?CATTCCGAGG?GGACCGTCCC?CTCGGTAATG?GCGAATGGGA

18701?CGCGGCCGAT?CCGGCTGCTA?ACAAAGCCCG?AAAGGAAGCT?GAGTTGGCTG

18751?CTGCCACCGC?TGAGCAATAA?CTAGCATAAC?CCCTTGGGGC?CTCTAAACGG

18801?GTCTTGAGGG?GTTTTTTGCT?GAAAGGAGGA?ACTATATCCG?GATCGGCCGA

18851?TCCGGCTGCT?AACAAAGCCC?GAAAGGAAGC?TGAGTTGGCT?GCTGCCACCG

18901?CTGAGCAATA?ACTAGCATAA?CCCCTTGGGG?CCTCTAAACG?GGTCTTGAGG

18951?GGTTTTTTGC?TGAAAGGAGG?AACTATATCC?GGATGGCCGC?CACCGGTGGG

19001?CCTTGCAGCA?CATCCCCCCT?TCGCCAG

 

 

 

Sequence 1 explanation:

11038-11043 Hpa I restriction enzyme site 11044-11054 GE sequence 11055 IG sequences

11056-11067 GS sequence 11068-11073 Kozak sequence 11074-11697 TRAIL open reading frame

11698-11703 Hpa I restriction enzyme site 11704-11711 Pme I restriction enzyme site 11712-11717 Hind III restriction enzyme site

11718-11725 Asc I restriction enzyme site 11728-11733 Mlu I restriction enzyme site

 

 

Sequence 2

1?ATGGAGACAG?ACACACTCCT?GCTATGGGTA?CTGCTGCTCT?GGGTTCCAGG

51?ATCCACTGGT?ACCTCTGAGG?AAACCATTTC?TACAGTTCAA?GAAAAGCAAC

101?AAAATATTTC?TCCCCTAGTG?AGAGAAAGAG?GTCCTCAGAG?AGTAGCAGCT

151?CACATAACTG?GGACCAGAGG?AAGAAGCAAC?ACATTGTCTT?CTCCAAACTC

201?CAAGAATGAA?AAGGCTCTGG?GCCGCAAAAT?AAACTCCTGG?GAATCATCAA

251?GGAGTGGGCA?TTCATTCCTG?AGCAACTTGC?ACTTGAGGAA?TGGTGAACTG

301?GTCATCCATG?AAAAAGGGTT?TTACTACATC?TATTCCCAAA?CATACTTTCG

351?ATTTCAGGAG?GAAATAAAAG?AAAACACAAA?GAACGACAAA?CAAATGGTCC

401?AATATATTTA?CAAATACACA?AGTTATCCTG?ACCCTATATT?GTTGATGAAA

451?AGTGCTAGAA?ATAGTTGTTG?GTCTAAAGAT?GCAGAATATG?GACTCTATTC

501?CATCTATCAA?GGGGGAATAT?TTGAGCTTAA?GGAAAATGAC?AGAATTTTTG

551?TTTCTGTAAC?AAATGAGCAC?TTGATAGACA?TGGACCATGA?AGCCAGTTTT

601?TTCGGGGCCT?TTTTAGTTGG?CTAA

Claims (5)

1. the cDNA sequence of one section coding recombinant virus, sequence contains SEQ ID NOS:1, or because insert, disappearance and sudden change cause surpasses derived dna sequence more than 90% with SEQ ID NOS:1 homology.

2. the reverse genetic operating system of the described recombinant virus of claim 1, this system comprises:

(1) sequence 1 claimed in claim 1;

(2) helper plasmid, the dna sequence dna of the nucleoprotein (NP) that comprising encodes expresses NDV, phosphorprotein (P), large polymerase protein (L);

(3) host cell of the virus replication of NDV virus license is as expressing the BHK-21 cell of t7 rna polymerase.

3. the recombinant Newcastle disease virus of saving out by claim 1 sequence.

4. the application of recombinant Newcastle disease virus claimed in claim 3 in the medicine for the treatment of malignant tumour.

5. application as claimed in claim 4, wherein malignant tumour is liver tumor or melanochrome tumour.

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