CN105111314B - A kind of new fusion protein, pharmaceutical composition and its preparation method and application - Google Patents

Abstract

Disclosed by the invention is the pharmaceutical composition of a kind of new fusion protein and new fusion protein composition and the preparation method and purposes of the new fusion protein.The general structure of the new fusion protein is as follows in the present invention: X- link peptide-Y- link peptide-IgG Fc segment or Y- link peptide-X- link peptide-IgG Fc segment；Wherein, X is VEGF-Trap and its derivative；Y is PD-1 antibody fragment and its derivative or PD-1 antagonist segment and its derivative.The present invention has new vessels inhibition and T cell Immunestimulatory effect, but also has the effect of the collaboration promotion there are two types of function, makes to inhibit the function of tumour growth more significant.

Description

A kind of new fusion protein, pharmaceutical composition and its preparation method and application

The present invention relates to a kind of fusion proteins, and in particular to be it is a kind of inhibit tumour growth new fusion protein and medicine The preparation method and purposes of compositions and the new fusion protein.

Background technique

Immunologic escape is a very important step in the deterioration process of tumour.Tumour cell is by avoiding human immune system Lethal effect be able to fast-growth, to promote the generation of cancer.T cell surface important negative regulatory molecule PD-1, CTLA- 4 and TIM-3 is able to suppress the immunologic cytotoxicity effect of T cell in development of cancer.Currently, clinically have for PD-1 and The drug of CTLA-4 target spot, these drugs clinically therapeutic effect highly significant.

Programmed cell death 1 (PD-1) receptor is mainly expressed in the T cell of activation and B cell, and major function is suppression The activation of immune system cell processed, this is also a kind of normal homeostasis of immune system, because excessive T/B cell-stimulating can draw Autoimmunity disease is played, so PD-1 is one of amulet of our human bodies.In pathological conditions, tumor microenvironment can induce infiltration T cell height express PD-1 molecule, tumour cell can high expression PD-1 ligand PD-L1 and PD-L2, cause in tumor microenvironment PD-1 signal path sustained activation, T cell function be suppressed, can not killing tumor cell, so as to cause the generation of cancer.It blocks The PD-1 antibody of this signal path can partially restore the function of T cell, and T cell is made to can continue to killing tumor cell.At present For PD-1 antibody there are two drug list, be Nivolumab and Pembrolizumab respectively, the two is clinically right Kinds cancer shows significant therapeutic effect.

VEGF-A is the generation factor of a kind of angiogenesis promoting of tumor cells expression, its main function is to provide immune The microenvironment of inhibition creates favorable conditions for the proliferation and cancer of tumour cell.Scientist Magali Terme (Magali T, Thibault V, et al, 2015, J. Exp. Med.212:139-148) research discovery VEGF-A is built Tumor microenvironment can cause the up-regulation of CD8+T cell surface PD-1 molecule, act on so as to cause the immunologic cytotoxicity of CD8+T cell It reduces.Find that the stimulation of VEGF-A can cause TIM-3 simultaneously, CTLA-4, LAG-3 etc. inhibit the expression of molecule.

Has the report using VEGF-A antibody and PD-1 antibody URIN Treatment tumour in the prior art, in clinical application often It is often single use one of antibody or is treated simultaneously using two kinds of antibodies on tumor, when simultaneously using above two anti- It then needs daily multiple injection to be administered when body is treated, not only makes troubles to operator, but also also brought more to subject It is mostly painful.

Summary of the invention

It is an object of the invention to overcome in the prior art VEGF-Trap and PD-1 antibody need in clinical application daily The problem of multiple injection is administered, providing not only has VEGF TRAP and PD-1 antibody physiological activity, but also one kind with long-acting function New fusion protein, and disclose the pharmaceutical composition of new fusion protein composition and the preparation method of the new fusion protein And purposes.

In order to achieve the above objectives, technical scheme is as follows:

A kind of novel fusion protein, general structure are as follows:

X- link peptide-Y- link peptide-IgG Fc segment or Y- link peptide-X- link peptide-IgG Fc segment；

Wherein, X is VEGF-Trap and its derivative；Y is PD-1 antibody fragment and its derivative or PD-1 antagonist piece Section and its derivative.

The present invention is not simply to mix two kinds of antibody proteins, but form one after being merged using two kinds of antibody The novel fusion protein of kind, the albumen can inhibit the activity of PD-1 and VEGF-A simultaneously, to can inhibit tumor vascular new It is raw, but the generation and activity of energy double inhibition PD-1, a plurality of pathway activation T cell；It has can hungry tumour cell and Activate the effect of T cell killing tumor cell.

PD-1 antibody and VEGF-A antibody is applied in combination to PD-1 antibody and VEGF-A antibody is used alone in the present invention, with And the effect of the fusion protein VNI combined using PD-1 antibody and VEGF-A antibody is carried out tumour growth situation in mouse and detected, Testing result is as shown in table 7 and Fig. 3.By table 7: using new fusion protein of the invention, be not only only capable of having simultaneously There are two types of the functions of the hungry tumour cell of antibody difference and activation T cell killing tumor cell, and can also reach two kinds of function The collaboration facilitation of energy makes to inhibit the function of tumour growth more significant；Also, by shown in Fig. 3, using of the invention new Type fusion protein also has the function of effectively extending each antibody units physiological active functions effect.

Preferably, the link peptide is amino acid sequence shown in formula (Gly-Gly-Gly-Gly-Ser) n, which is 1~4 Integer.The amino acid sequence of the X is SEQ ID NO:1 or SEQ ID NO:3 or its nucleotide coding sequence is SEQ ID NO:2 or SEQ ID NO:4.The amino acid sequence of the Y is SEQ ID NO:5 or SEQ ID NO:7 or its nucleotide coding Sequence is SEQ ID NO:6 or SEQ ID NO:8.The IgG Fc segment is IgG1 Fc segment, IgG2 Fc segment, IgG3 Fc segment or IgG4 Fc segment, amino acid sequence are respectively SEQ ID NO:9,11,13,15 or its nucleotide coding sequence Respectively SEQ ID NO:10,12,14,16.

A method of new fusion protein being prepared, by the coding of any one of the claim 1-6 new fusion protein Nucleotide sequence is inserted into expression vector, and this expression vector is introduced corresponding expression system and then carries out the table of fusion protein It reaches.The expression vector of fusion protein of the present invention can be recombinant eukaryon expression vector, preferred mammal cell expression vector；? It can be recombinant virus expression vector, preferably adeno-associated virus or adenovirus vector.

The host cell of fusion protein in above-mentioned expression system can be Chinese hamster ovary celI and its subbreed or 293 cells and its Asia System.

A kind of pharmaceutical composition, by new fusion protein described in any one of claims 1-6 with it is pharmaceutically acceptable Carrier or excipient composition.

Further, the dosage form of described pharmaceutical composition is injection, injection freeze-dried powder.

A kind of purposes of new fusion protein, the new fusion protein described in any one of claims 1-6 is for swelling The treatment of tumor；The tumour is melanoma, lung cancer, liver cancer, lymthoma, colon cancer, colorectal cancer, breast cancer, oophoroma, uterine neck One in cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, the cancer of the esophagus, kidney, glioma, melanoma, cancer of pancreas and prostate cancer Kind is a variety of.

Compared with prior art, the present invention have the following advantages that and the utility model has the advantages that

1, fusion protein of the invention can not only have there are two types of the hungry tumour cell of antibody difference simultaneously and activate T thin The function of born of the same parents' killing tumor cell, and the collaboration facilitation of two kinds of functions can also be reached, make the function of inhibiting tumour growth It is more significant；

2, new fusion protein of the invention can effectively extend each antibody units physiological active functions effect, and then reduce one Total dosage of a course for the treatment of reduces drug cost；

3, the present invention can effectively reduce the number of drug administration by injection, mitigate administration pain.

Detailed description of the invention

Fig. 1 is the EC50 value curve synoptic diagram that VNI influences HUVEC cell Proliferation.

Fig. 2 is the curve synoptic diagram that VNI stimulates PBMC generation IFN γ.

Fig. 3 is the comparative situation schematic diagram that source of mouse VNI inhibits tumour growth in vivo.

Specific embodiment

Below with reference to embodiment, the present invention is described in further detail, embodiments of the present invention are not limited thereto.

The preparation of 1 albumen of embodiment

A kind of new fusion protein, general structure are as follows:

X- link peptide-Y- link peptide-IgG Fc segment；Wherein, X is VEGF-Trap and its derivative；Y is PD-1 antibody Segment and its derivative or PD-1 antagonist segment and its derivative.

The X is VEGF-Trap in the present embodiment, and nucleotide sequence is as shown in SEQ ID NO:4；Y is PD-1 antibody piece Section, nucleotide sequence is as shown in SEQ ID NO:6；The link peptide is ammonia shown in formula (Gly-Gly-Gly-Gly-Ser) n Base acid sequence, n is 3 in the link peptide；The nucleotide sequence of IgG Fc segment is as shown in SEQ ID NO:16；That is, the present embodiment The nucleotide sequencing of middle synthesis fusion protein is as shown in SEQ ID NO:18.

The specific synthetic route of above-mentioned new fusion protein is as follows in the present embodiment:

1. construction recombination plasmid

1.1 according to the logical of X- link peptide-Y- link peptide-IgG Fc segment or Y- link peptide-X- link peptide-IgG Fc segment Formula obtains the genetic fragment and primer of synthesis fusion protein, and the genetic fragment for synthesizing fusion protein and primer is recombinated to matter Puc57-VNI plasmid is obtained in grain carrier puc57.The nucleotide sequencing of the synthesis fusion protein such as SEQ ID NO: Shown in 13, the gene and primer are synthesized by Beijing Jin Weizhi company of specialized company.

Puc57-VNI plasmid and pCHO1.0 plasmid are carried out double digestion processing by 1.2 respectively, the foundation of the digestion system:

Following ingredient: 40 μ l, Buffer4 10 of pCHO1.0 plasmid or puc57-VNI plasmid is added in 1.5ml EP pipe Each 5 μ l of μ l, Avr II and BstZ17I, 45 μ l of aqua sterilisa, reacts 5h at 37 DEG C after mixing, utilizes QIAGEN product purification reagent Box recycling, obtains genetic fragment pCHO1.0 and VNI respectively.

1.3 under the action of T4 ligase, will be with the genetic fragment pCHO1.0 and VNI for recycling acquisition after identical digestion Genetic fragment connection；Coupled reaction system is as follows:

It is added the pCHO1.0 of following 2 μ l of ingredient in 1.5ml EP pipe, the VNI of 6 μ l, 10 × T4 Buffer 1 μ l, 1 The T4 DNA ligase of μ l, reacts 4h or more under room temperature (20 DEG C or so) after mixing, connection product is converted to Top10 large intestine bar In bacterium competence cell, it is coated on 2YT (KANA) plating medium and stands overnight for 37 DEG C, plate number pCHO1.0-VNI.

1.4 from the several recombination single colonies of picking each in pCHO1.0-VNI plate after culture as pcr template, respectively Carry out PCR screening and identification；

Bacterium solution pcr amplification reaction system (20 μ L of total volume): 2 × Taq HS, 10 μ L, 2 μ L of bacterium solution template, upstream and downstream are drawn Each 1 μ L of object P1 and P2 (0.3 μm of ol/L of final concentration), is finally mended with distilled water to 20 μ L；Reaction condition: 95 DEG C of 2min, one is followed Ring；94 DEG C of 60s, 53 DEG C of 60s, 72 DEG C of 120s, 30 circulations；Last 72 DEG C of 5min.It is analyzed by agarose gel electrophoresis As a result, selecting the correct bacterium colony with target product.

1.5 carry out digestion identification by extracting after the colony inoculation of PCR screening and identification respectively again；Recombinant bacterium is carried out first Plasmid extract, then carry out restriction analysis；

Digestion system: it is added following ingredient in 1.5ml EP pipe: plasmid 2 μ l, Buffer1 1 μ l, BSA 0.1 μ l, Avr Each 1 μ l of II and BstZ17I mends aqua sterilisa to 10 μ l, reacts 4h at 37 DEG C after mixing.It is analyzed and is tied by agarose gel electrophoresis Fruit selects digestion and identifies correct bacterium colony.

1.6 will identify that correct bacterium colony is inoculated with several bacterium colonies at random and carries out sequencing identification again by bacterium colony PCR and digestion, select Expression plasmid is taken out to save backup.

2. plasmid transfection and cell screening

Using host cell CHO-S or DG44, plasmid is carried out respectively according to Freedom CHO-S Kit kit specification Transfection, the cell for being transferred to plasmid are respectively placed in shaking flask culture 8h, the condition of shaking flask culture are as follows: 37 DEG C, 8%CO₂、110rpm/min。 Cell viability and cell number are detected by cell counter.

Two steps pressurization screening: the μ g/mL of 10P/100M, 20P/200M(P=10 Puromycin, M=nM is carried out after transfection 48h MTX)；30P/500M, 50P/1000M obtain initial screening cell.Monoclonal cell screening is carried out with limiting dilution assay, passes through detection Expressing quantity and purity are therefrom preferably cloned expands culture step by step.

3. Protein expression and purification

Screening High producing clones cell, which expands from 96 orifice plates to 24 orifice plates, to be grown, and is then expanded to 6 orifice plates and is grown, expands later to 50mL Shake flask grown.

7 days cell culture supernatants of culture are collected, cell fragment is centrifuged off, supernatant is adjusted with 0.45 μm of membrane filtration PH to 7.4 is saved, with Protein A sepharose affinity column chromatography (HiTrap Protein-A Sepharose affinity Column the deionized water of) purified fusion albumen, 5 times of bed volumes rinses pillar, then the PBS buffer solution with 5 times of bed volumes (20mM phosphate, pH 7.4) balances pillar, and loading collects efflux detection, with 10 times of volume PBS buffer solution (0.02 Mol/L phosphate, pH 7.4) washing removes foreigh protein removing, then using 0.1M glycine buffer (pH3) by destination protein from column Upper elution, new fusion protein VNI as of the invention.It is detected by SDS-PAGE, the purity of new fusion protein VNI reaches To 90% or more.

The analysis of embodiment 2.VNI binding kinetics

(1) the binding kinetics analysis of VNI and VEGF-A

25 μ g/ml Biotin-hVEGF-A are incorporated on SA sensor by Loading；It is arranged according to preliminary experiment dense It spends range: being respectively 10000,5000,1000,500,100nM, using Sample Diluent buffer as blank control.In Dynamic analysis in Octet QK detection platform carries out the kinetic parameter of hVEGF-A and VNI, VEGF-Trap and PD1 antibody Detection, test procedure setting are as shown in table 1:

Table 1

Detect step number	Solution	Step type	Detection time (s)
				1	Sample Diluent buffer	Baseline	120
2	Biotin-hVEGF-A	Loading	360
				3	Sample Diluent buffer	Baseline	120
4	Sample	Association	720
				5	Sample Diluent buffer	Dissociation	1800

The experimental result detected by above-mentioned test procedure is as shown in table 2:

2 binding kinetics parameter of table compares

KD (M)

kon(1/Ms)

kon Error

kdis(1/s)

kdis Error

Full R^2

VNI

1.33E-10

4.34E+05

1.73E+04

5.79E-05

1.70E-05

0.99448

VEGF-Trap

0.83E-10

9.51E+05

6.11E+04

7.93E-05

3.23E-05

0.99368

PD-1 antibody

4.36E-03

1.20E+01

1.25E+01

5.25E-02

1.13E-03

0.91908

Table 2 as above shows that VNI still keeps the high-affinity with VEGF-A, remains to combine VEGF-A well in vitro；But Binding force is weaker than VEGF-TRAP.And PD-1 antibody and VEGF-A are almost without combination.

(2) the binding kinetics analysis of VNI and PD-1

25 μ g/ml Biotin-h PD-1 are incorporated on SA sensor by Loading；It is arranged according to preliminary experiment dense It spends range: being respectively 10000,5000,1000,500,100nM, using Sample Diluent buffer as blank control.In Dynamic analysis in Octet QK detection platform carries out the kinetic parameter of h PD-1 and VNI, VEGF-Trap and PD1 antibody Detection, test procedure setting are as shown in table 3:

Table 3

Detect step number	Solution	Step type	Detection time (s)
				1	Sample Diluent buffer	Baseline	120
2	Biotin-hPD-1	Loading	360
				3	SampleDiluent buffer	Baseline	120
4	Sample	Association	720
				5	Sample Diluent buffer	Dissociation	1800

The experimental result detected by above-mentioned test procedure is as shown in table 4:

4 binding kinetics parameter of table compares

KD (M)

kon(1/Ms)

kon Error

kdis(1/s)

kdis Error

Full R^2

VNI

5.5E-10

1.32E+05

7.74E+04

7.32E-05

2.37E-05

0.99646

VEGF-Trap

6.24E-3

3.07E+01

3.29E+02

1.92E-01

1.79E-06

0.89421

PD1 antibody

2.17E-10

9.71E+05

1.19E+03

2.11E-04

1.80E-06

0.99911

Table 4 as above shows that VNI is in conjunction with PD-1 holding high-affinity, but binding force is weaker than PD-1 antibody.And PD-1 antibody Segment or antagonist and PD-1 binding force are very weak.

In conclusion VNI is still able to maintain and VEGF-A and PD-1 high affine combination in vitro, thus it is speculated that the molecule has active isomer Interior activity.

The In vitro biological activity of 3. VNI of embodiment detects.

(1) the influence comparative studies to HUVEC cell Proliferation

The HUVEC cell inoculation of logarithmic growth phase is in 96 well culture plates, and 3 × 10³The hole cells/, 100 holes μ l/, 37 DEG C, 5%CO₂Cultivate 20h；With Base culture medium prepare different molar concentrations VNI, PD-1 antibody and VEGF-Trap(9.7~ It 10000ng/ml) is mixed respectively with the VEGF of 40ng/ml, 37 DEG C of incubation 2h；It is inoculated with 96 orifice plates of HUVEC cell in addition On, 100 holes μ l/, every group of 3 multiple holes, 37 DEG C, 5%CO₂Continue to be incubated for 96h, negative control group bonus point does not add Base culture medium and complete Full culture medium；Add CCK-8 reagent, 20 μ l/well, 37 DEG C are protected from light incubation 2h, and 450nm detects light absorption value, and VNI is to HUVEC cell The EC50 value curve synoptic diagram of proliferative effect is as shown in Figure 1, experimental result is as shown in table 5.

Table 5

sample	EC50 (ng/ml)	standard Error	R-Square
				VNI	229.74	20.19	0.983
VEGF-Trap	197.62	17.59	0.979
				PD1 antibody	1.23E12	7.11E15	0.608

It is shown by table 5 and Fig. 1, the In vitro biological activity of VNI is slightly below VEGF-Trap, and PD-1 antibody pair VEGF stimulates HUVEC cell-proliferation activity substantially without inhibiting effect.

(2) to the influence of the cytokine secretion of cytomegalovirus lysate stimulation PBMC cell

From the donor separating peripheral blood mononuclear cells (PBMC) of CMV positive (once infecting cytomegalovirus), CMV is split Solution object can stimulate this kind of cell to generate IFN γ, and anti-PD-1 antibody can enhance CMV lysate stimulation PBMC and generate IFN γ.

105 people PMBC from CMV positive donor are cultivated in 200 μ l of total volume, and are added in each hole CMV lysate.Anti- PD-1 antibody, VNI and PD-1 antibody fragment or the antagonist of various concentration are added in each hole.Often Rule take 100 μ l culture mediums to measure for cell factor IFN γ after CMC model cell the 4th day from every part of culture, VNI thorn Swash PBMC and generates the curve synoptic diagram of IFN γ as shown in Fig. 2, measurement result is as shown in table 6.

Table 6

sample	EC50 (ng/ml)	standard Error	R-Square
				VNI	112.42	16.75	0.951
VEGF-Trap	--	--	--
				PD1 antibody	82.95	15.82	0.978

It is shown by Fig. 2 and table 6, under the effect of CMV lysate, VNI generates IFN in concentration dependant stimulation PBMC cell γ, in low concentration, its ability to function is lower than PD-1 antibody, but suitable with PD-1 antibody effect at high concentrations.VEGF-TRAP PBMC cannot be stimulated to generate IFN γ substantially.

4. source of mouse VNI of embodiment inhibits Growth of Tumors Transplanted Effect study in Mice Body

Since mouse PD-1 and humanization source of people PD-1 antibody do not have cross action, and then source of people PD-1 antibody is caused to exist PD-1 can not be blocked to act in Mice Body, PD-1 antibody inhibiting tumor growth, thus this hair cannot be played in Mice Body It is bright to use the internal influence that processing is implanted with the mouse of cancerous tumour to examine antibodies on tumor to grow in source of mouse VNI body.

The preparation of source of mouse PD-1 antibody and source of mouse VNI: first use mouse PD-1 immune rat, referring to " tissue cultures and point Daughter cell technology " (E Zheng chief editor) hybridoma preparation method, anti-mouse PD-1 antibody gene is obtained, by the genetic recombination Into carrier for expression of eukaryon, source of mouse PD-1 antibody is obtained, the antibody is spare as compareing；Then, egg will be separately merged in the present invention The anti-PD-1 antibody fab sequence of the source of people of white VNI is substituted for the fab sequence of mouse PD-1 antibody gene, and it is anti-to form anti-mouse PD-1 The fusion protein source of mouse VNI of body and VEGF-TRAP, which is recombinated into carrier for expression of eukaryon, through transfection, expression and purifying Obtain recombination source of mouse VNI.

The female AJ mouse between 6-8 week old is randomly divided into 6 groups according to weight, every group includes 10 animals.At the 0th day 2 × 106 SA1/N fibrosarcoma cells for being dissolved in 200 μ l DMEM culture mediums are subcutaneously implanted mouse on right side.With PBS pairs According to or 10mg/kg drug-treated mouse.Led at the 1st, 4,8 and 11 day with the about 200 μ l PBS for containing drug or control Intraperitoneal injection is crossed to be administered to animal dosage.Each group composition is as follows: (1) PBS control group, (2) source of mouse VNI group, (3) source of mouse PD- 1 antibody group, (4) VEGF-TRAP group.Tumour growth is monitored to mouse twice a week, continues about 8 weeks.Use electronic caliper Three-dimensional measurement (height × width × length) is carried out to tumour and calculates gross tumor volume.When tumour reaches tumour terminal (1500mm3) or display put to death mouse when being greater than 15% weight loss, account for percentage such as table without tumor mouse after drug-treated Shown in 7, source of mouse VNI inhibits the phenomenon that tumour growth is as shown in Figure 3 in vivo.

Table 7

Group	Study mouse number of elements	Without tumour number of elements (only)	Without tumour percentage (%)
				PBS group	10	0	0
Source of mouse VNI group	10	7	70
				Source of mouse PD-1 antibody group	10	3	30
VEGF-TRAP group	10	0	0

In above-mentioned comparative experiments, 9 mouse about reached tumour terminal at 28 days in PBS group, had 1 mouse tumour to fester；Source of mouse 3 mouse of VNI group reached tumour terminal at 56 days, remaining 7 without tumour；7 mouse of source of mouse PD-1 antibody group reached tumour at 56 days Terminal, remaining 3 mouse is without tumour；VEGF-TRAP group has 6 mouse to reach tumour terminal at 42 days, remaining 4 mouse reached at 49 days Tumour terminal.

By Fig. 3, table 7 and above-mentioned experimental result describe: new fusion protein VNI of the invention, not only Can have there are two types of the hungry tumour cell of antibody difference and activate the function of T cell killing tumor cell simultaneously, and can also reach To the collaboration facilitation of two kinds of functions, make to inhibit the function of tumour growth more significant；Also, pass through shown in Fig. 3, using this The new fusion protein of invention also has the function of effectively extending each antibody units physiological active functions effect.

Embodiment 5

The nucleotide serialization of X arranges, the nucleosides of the present embodiment described in X different with the nucleotide serialization of Y column in the present embodiment Coding sequences are SEQ ID NO:4, and the nucleotide coding sequence of the Y is SEQ ID NO:8.The IgG Fc segment is IgG3 Fc segment, the IgG3 Fc segment nucleotide coding sequence are SEQ ID NO:14.

Embodiment 6

The present embodiment the difference from embodiment 1 is that: the segment of IgG Fc described in the present embodiment be IgG2 Fc segment, should IgG2 Fc segment nucleotide coding sequence is SEQ ID NO:12.

Above-described embodiment is merely a preferred embodiment of the present invention, and it is not intended to limit the protection scope of the present invention, as long as using Design principle of the invention, and the non-creative variation worked and made is carried out on this basis, it should belong to of the invention Within protection scope.

SEQUENCE LISTING

<110>Chengdu all generations wins Bioisystech Co., Ltd

<120>a kind of new fusion protein, pharmaceutical composition and its preparation method and application

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50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

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

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Ile Val

115 120 125

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

130 135 140

Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp

145 150 155 160

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala

165 170 175

Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Phe

180 185 190

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

195 200 205

Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg Thr Phe Gly

210 215 220

Gln Gly Thr Lys Val Glu Ile Lys

225 230

<210> 6

<211> 696

<212> DNA

<213>artificial sequence

<400> 6

caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ctggccggtc cctgcggctg 60

gactgcaagg cctccggcat caccttctcc aactccggca tgcactgggt gcggcaggcc 120

cctggcaagg gcctggagtg ggtggcctgg atctggtacg acggctccaa gcggtactac 180

gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgttc 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc caccaacgac 300

gactactggg gccagggcac cctggtgacc gtgtcctccg gcgggtccgg cgggtccggc 360

gggtccggcg ggtccgagat cgtgctgacc cagtcccctg ccaccctgtc cctgtcccct 420

ggcgagcggg ccaccctgtc ctgccgggcc tcccagtccg tgtcctccta cctggcctgg 480

taccagcaga agcctggcca ggcccctcgg ctgctgatct acgacgcctc caaccgggcc 540

accggcatcc ctgcccggtt ctccggctcc ggcttcggca ccgacttcac cctgaccatc 600

tcctccctgg agcctgagga cttcgccgtg tactactgcc agcagtcctc caactggcct 660

cggaccttcg gccagggcac caaggtggag atcaag 696

<210> 7

<211> 232

<212> PRT

<213>unknown

<400> 7

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

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

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

35 40 45

Ala Trp Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

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

85 90 95

Ala Ser Asn Glu Asp Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Ile Val

115 120 125

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

130 135 140

Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp

145 150 155 160

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala

165 170 175

Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Phe

180 185 190

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

195 200 205

Ala Val Tyr Tyr Cys Gln Gln Ser Thr Asn Phe Pro Arg Thr Phe Gly

210 215 220

Gln Gly Thr Lys Val Glu Ile Lys

225 230

<210> 8

<211> 696

<212> DNA

<213>artificial sequence

<400> 8

caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ctggccggtc cctgcggctg 60

gactgcaagg cctccggcat caccttctcc aactccggca tgcactgggt gcggcaggcc 120

cctggcaagg gcctggagtg ggtggcctgg atctggtacg acggctccaa gcggtactac 180

gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgttc 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc ctccaacgag 300

gacttctggg gccagggcac cctggtgacc gtgtcctccg gcgggtccgg cgggtccggc 360

gggtccggcg ggtccgagat cgtgctgacc cagtcccctg ccaccctgtc cctgtcccct 420

ggcgagcggg ccaccctgtc ctgccgggcc tcccagtccg tgtcctccta cctggcctgg 480

taccagcaga agcctggcca ggcccctcgg ctgctgatct acgacgcctc caaccgggcc 540

accggcatcc ctgcccggtt ctccggctcc ggcttcggca ccgacttcac cctgaccatc 600

tcctccctgg agcctgagga cttcgccgtg tactactgcc agcagtccac caacttccct 660

cggaccttcg gccagggcac caaggtggag atcaag 696

<210> 9

<211> 227

<212> PRT

<213>unknown

<400> 9

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 10

<211> 681

<212> DNA

<213>artificial sequence

<400> 10

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 11

<211> 227

<212> PRT

<213>unknown

<400> 11

Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 12

<211> 684

<212> DNA

<213>artificial sequence

<400> 12

gagcgcaaat gttgtgtcga gtgcccaccg tgcccagcac cacctgtggc aggaccgtca 60

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120

acgtgcgtgg tggtggacgt gagccacgaa gaccccgagg tccagttcaa ctggtacgtg 180

gacggcgtgg aggtgcataa tgccaagaca aagccacggg aggagcagtt caacagcacg 240

ttccgtgtgg tcagcgtcct caccgttgtg caccaggact ggctgaacgg caaggagtac 300

aagtgcaagg tctccaacaa aggcctccca gcccccatcg agaaaaccat ctccaaaacc 360

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 420

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 480

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacacctcc catgctggac 540

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660

agcctctccc tgtctccggg taaa 684

<210> 13

<211> 264

<212> PRT

<213>unknown

<400> 13

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala

35 40 45

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

50 55 60

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

65 70 75 80

Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val

85 90 95

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

100 105 110

Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln

115 120 125

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

130 135 140

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro

145 150 155 160

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

165 170 175

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

180 185 190

Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr

195 200 205

Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

210 215 220

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

225 230 235 240

Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys

245 250 255

Ser Leu Ser Leu Ser Pro Gly Lys

260

<210> 14

<211> 792

<212> DNA

<213>artificial sequence

<400> 14

gagctcaaaa ccccacttgg tgacacaact cacacatgcc cacggtgccc agagcccaaa 60

tcttgtgaca cacctccccc gtgcccacgg tgcccagagc ccaaatcttg tgacacacct 120

cccccatgcc cacggtgccc agcacctgaa ctcctgggag gaccgtcagt cttcctcttc 180

cccccaaaac ccaaggatac ccttatgatt tcccggaccc ctgaggtcac gtgcgtggtg 240

gtggacgtga gccacgaaga ccccgaggtc cagttcaagt ggtacgtgga cggcgtggag 300

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgtt ccgtgtggtc 360

agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 420

tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaaaccaa aggacagccc 480

cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 540

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 600

agcgggcagc cggagaacaa ctacaacacc acgcctccca tgctggactc cgacggctcc 660

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacatcttc 720

tcatgctccg tgatgcatga ggctctgcac aaccgcttca cgcagaagag cctctccctg 780

tctccgggta aa 792

<210> 15

<211> 229

<212> PRT

<213>unknown

<400> 15

Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

1 5 10 15

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

20 25 30

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

35 40 45

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

50 55 60

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

65 70 75 80

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

85 90 95

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

100 105 110

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

115 120 125

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

130 135 140

Gln Val Ser Leu Thr Cys Leu 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 Tyr Ser Arg

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 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 Leu Gly

225

<210> 16

<211> 687

<212> DNA

<213>artificial sequence

<400> 16

gctgagtcca agtatggccc tccctgccct ccttgccctg ctcctgaggc tgctggaggc 60

cctagcgtgt tcctgttccc ccctaagcct aaggacaccc tgatgatttc ccggaccccc 120

gaggtgacct gtgtggtggt ggatgtgtcc caggaggacc ctgaagtgca gttcaactgg 180

tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggaaga gcagttcaac 240

agcacctaca gggtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 300

gagtacaagt gcaaggtgag caataagggc ctgccctcct ccatcgagaa gaccatttcc 360

aaggccaagg gccagcccag ggaaccccag gtgtacaccc tccctcccag ccaggaggag 420

atgaccaaga accaggtgtc cctgacctgc ctggtgaaag gcttctaccc ctccgacatt 480

gccgtcgagt gggaaagcaa cggccagccc gagaacaatt acaagaccac accccccgtg 540

ctggacagcg atggcagctt tttcctgtac tccaggctga ccgtcgacaa gtccaggtgg 600

caggagggca acgtcttctc ctgctccgtg atgcatgagg ccctgcacaa ccactacacc 660

cagaagtccc tgtccctgag cctgggc 687

<210> 17

<211> 684

<212> PRT

<213>unknown

<400> 17

Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His

1 5 10 15

Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro

20 25 30

Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro

35 40 45

Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser

50 55 60

Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val

65 70 75 80

Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn

85 90 95

Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser

100 105 110

Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn

115 120 125

Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His

130 135 140

Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met

145 150 155 160

Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp

165 170 175

Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys

180 185 190

Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Ser Gly Gly Ser

195 200 205

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

210 215 220

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

225 230 235 240

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

245 250 255

Ala Trp Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

260 265 270

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

275 280 285

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

290 295 300

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

305 310 315 320

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Ile Val

325 330 335

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

340 345 350

Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp

355 360 365

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala

370 375 380

Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Phe

385 390 395 400

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

405 410 415

Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg Thr Phe Gly

420 425 430

Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys

450 455 460

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

465 470 475 480

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

485 490 495

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

500 505 510

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

515 520 525

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

530 535 540

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

545 550 555 560

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

565 570 575

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

580 585 590

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

595 600 605

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

610 615 620

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

625 630 635 640

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

645 650 655

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

660 665 670

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

675 680

<210> 18

<211> 2159

<212> DNA

<213>artificial sequence

<400> 18

agatctccta gggccaccat ggtcagctac tgggacaccg gggtcctgct gtgcgcgctg 60

ctcagctgtc tgcttctcac aggatctagt tccggaggta gacctttcgt agagatgtac 120

agtgaaatcc ccgaaattat acacatgact gaaggaaggg agctcgtcat tccctgccgg 180

gttacgtcac ctaacatcac tgttacttta aaaaagtttc cacttgacac tttgatccct 240

gatggaaaac gcataatctg ggacagtaga aagggcttca tcatatcaaa tgcaacgtac 300

aaagaaatag ggcttctgac ctgtgaagca acagtcaatg ggcatttgta taagacaaac 360

tatctcacac atcgacaaac caatacaatc atagatgtgg ttctgagtcc gtctcatgga 420

attgaactat ctgttggaga aaagcttgtc ttaaattgta cagcaagaac tgaactaaat 480

gtggggattg acttcaactg ggaataccct tcttcgaagc atcagcataa gaaacttgta 540

aaccgagacc taaaaaccca gtctgggagt gagatgaaga aatttttgag caccttaact 600

atagatggtg taacccggag tgaccaagga ttgtacacct gtgcagcatc cagtgggctg 660

atgaccaaga agaacagcac atttgtcagg gtccatgaaa agggcgggtc cggcgggtcc 720

caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ctggccggtc cctgcggctg 780

gactgcaagg cctccggcat caccttctcc aactccggca tgcactgggt gcggcaggcc 840

cctggcaagg gcctggagtg ggtggcctgg atctggtacg acggctccaa gcggtactac 900

gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgttc 960

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc caccaacgac 1020

gactactggg gccagggcac cctggtgacc gtgtcctccg gcgggtccgg cgggtccggc 1080

gggtccggcg ggtccgagat cgtgctgacc cagtcccctg ccaccctgtc cctgtcccct 1140

ggcgagcggg ccaccctgtc ctgccgggcc tcccagtccg tgtcctccta cctggcctgg 1200

taccagcaga agcctggcca ggcccctcgg ctgctgatct acgacgcctc caaccgggcc 1260

accggcatcc ctgcccggtt ctccggctcc ggcttcggca ccgacttcac cctgaccatc 1320

tcctccctgg agcctgagga cttcgccgtg tactactgcc agcagtcctc caactggcct 1380

cggaccttcg gccagggcac caaggtggag atcaagggcg gcggaggctc cggcggaggc 1440

ggctccggcg gcggcggctc cgctgagtcc aagtatggcc ctccctgccc tccttgccct 1500

gctcctgagg ctgctggagg ccctagcgtg ttcctgttcc cccctaagcc taaggacacc 1560

ctgatgattt cccggacccc cgaggtgacc tgtgtggtgg tggatgtgtc ccaggaggac 1620

cctgaagtgc agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag 1680

ccccgggaag agcagttcaa cagcacctac agggtggtga gcgtgctgac cgtgctgcac 1740

caggactggc tgaacggcaa agagtacaag tgcaaggtga gcaataaggg cctgccctcc 1800

tccatcgaga agaccatttc caaggccaag ggccagccca gggaacccca ggtgtacacc 1860

ctccctccca gccaggagga gatgaccaag aaccaggtgt ccctgacctg cctggtgaaa 1920

ggcttctacc cctccgacat tgccgtcgag tgggaaagca acggccagcc cgagaacaat 1980

tacaagacca caccccccgt gctggacagc gatggcagct ttttcctgta ctccaggctg 2040

accgtcgaca agtccaggtg gcaggagggc aacgtcttct cctgctccgt gatgcatgag 2100

gccctgcaca accactacac ccagaagtcc ctgtccctga gcctgggctg attaattaa 2159

Claims (5)

1. a kind of new fusion protein, which is characterized in that it is as coded by nucleotide sequence SEQ ID NO:18.

2. a kind of method for preparing new fusion protein, which is characterized in that by the coding of new fusion protein described in claim 1 Nucleotide sequence is inserted into expression vector, and this expression vector is introduced corresponding expression system and then carries out the table of fusion protein It reaches.

3. a kind of pharmaceutical composition, which is characterized in that by new fusion protein described in claim 1 with it is pharmaceutically acceptable Carrier or excipient composition.

4. a kind of pharmaceutical composition according to claim 3, which is characterized in that the dosage form of described pharmaceutical composition is Injection.

5. a kind of purposes of new fusion protein, which is characterized in that the new fusion protein described in claim 1 is for swelling The preparation of tumor therapeutic agent；The tumour is melanoma, lung cancer, liver cancer, lymthoma, colon cancer, colorectal cancer, breast cancer, ovum One in nest cancer, cervical carcinoma, gastric cancer, cholangiocarcinoma, gallbladder cancer, the cancer of the esophagus, kidney, glioma, cancer of pancreas and prostate cancer Kind is a variety of.