CN110317264A - A kind of derivative of anti-tumor protein Endostatin - Google Patents
A kind of derivative of anti-tumor protein Endostatin Download PDFInfo
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- CN110317264A CN110317264A CN201910639670.0A CN201910639670A CN110317264A CN 110317264 A CN110317264 A CN 110317264A CN 201910639670 A CN201910639670 A CN 201910639670A CN 110317264 A CN110317264 A CN 110317264A
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Abstract
The invention discloses a kind of derivatives of anti-tumor protein Endostatin, and the amino acid sequence of the Endostatin is shown in SEQ.ID.NO4；Point mutation occurs for the one of Arg of the Arg128Arg129 of the derivative amino acid sequence.The derivative of anti-tumor protein Endostatin of the invention is stable in vivo, and long half time, anti-tumor biological are high.
Description
Technical field
The present invention relates to a kind of derivative of anti-tumor protein Endostatin and applications, belong to biological medicine anti-tumor drug
Field.
Background technique
The formation of tumour growth dependence blood vessel, and tune of the formation of blood vessel by angiogenesis factor and inhibiting factor
Section, inhibiting tumor vascular growth is the effective ways for treating tumour.Endostatin (Endostatin), which has, inhibits blood vessel raw
At function, so tumour anti-new vessels treatment in receive attention.Endostatin is the hydrolysis of collagen XV III
And the 184 amino acid segments of C-terminal generated, it can effectively inhibit a variety of variety classes tumours, and biological action is in broad spectrum activity,
It has no drug resistance, is the best Angiostatin (Exp Cell Res, 312,594-607,2006) found so far.
But find that the half-life period of Endostatin is very short, exponentially drops in the clinical test of monkey study and people
Solution, the in-vivo content of 10 hours front and back Endostatins successively decrease hundred times in addition be close to zero (Acta Pharmacol Sin, 26,
124-128,2005；J Clin Oncol, 20,3792-3803,2002), show that it is very unstable in animal body, this will be big
The big antineoplaston effect for influencing Endostatin.
Summary of the invention
The purpose of the present invention is overcoming deficiency in the prior art, provide long half time, anti-tumor biological high anti-
The derivative of oncoprotein Endostatin.
A second object of the present invention is to provide the derivatives of anti-tumor protein Endostatin to prepare answering for anticancer drug
With.
Third object of the present invention is to provide the DNA segment for the derivative for encoding above-mentioned Endostatin prepare it is antitumor
The application of drug.
A kind of derivative of anti-tumor protein Endostatin, the amino acid sequence SEQ.ID.NO4 institute of the Endostatin
Show；The derivative includes: that Endostatin is connect with IgG antibody constant region；Or Arg is previously incorporated in the N-terminal of Endostatin；Or
The amino acid short peptide of glycosylation site is introduced before the N-terminal of Endostatin or behind C-terminal or before N-terminal and behind C-terminal；Or
The one of Arg of the Arg62Arg63 of the amino acid sequence of Endostatin occur point mutation or Arg128Arg129 one of them
Point mutation occurs for Arg or Arg128Arg129 one of Arg while point mutation occurs for the one of Arg of Arg62Arg63
Point mutation occurs.Or Endostatin is connect with IgG antibody constant region, is previously incorporated Arg in the N-terminal of Endostatin, is pressed down in endothelium
The amino acid short peptide of glycosylation site is introduced before the N-terminal of element or behind C-terminal or before N-terminal and behind C-terminal, Endostatin
Point mutation occurs for the one of Arg of the Arg62Arg63 of amino acid sequence or the one of Arg of Arg128Arg129 occurs point and dashes forward
Become or the one of Arg of Arg62Arg63 occurs Arg128Arg129 one of Arg while point mutation and point mutation occurs.
IgG constant region is CH1, CH1CH2 or CH1CH2CH3, and the amino acid sequence that the Endostatin is connect with CH1 is
SEQ.ID.NO8；The amino acid sequence that the Endostatin is connect with CH1CH2 is SEQ.ID.NO10；Endostatin with
The amino acid sequence of CH1CH2CH3 connection is SEQ.ID.NO12.
It is that a small peptide is connected before the N-terminal of Endostatin that the N-terminal of Endostatin, which is previously incorporated Arg, in the small peptide
Contain more than one Arg.
Glycosylation site is Asn-X-Ser or Asn-X-Thr, and wherein X is other amino acid.
It is His that point mutation, which occurs, for Arg.
The amino acid sequence of the derivative of anti-tumor protein Endostatin is preferably shown in SEQ.ID.NO20.
The nucleic acid sequence for encoding the derivative of anti-tumor protein Endostatin as claimed in claim 6 is SEQ.ID.NO19
It is shown.
The derivative of above-mentioned anti-tumor protein Endostatin is in the application for preparing anticancer drug.
The DNA segment of the derivative of above-mentioned anti-tumor protein Endostatin is encoded in the application for preparing anticancer drug.
Advantages of the present invention
Anti-tumor protein Endostatin derivative of the invention is stable in vivo, and long half time, anti-tumor biological are high.
Detailed description of the invention
Fig. 1 be embodiment 1 preamble sequence-Endostatin be separately connected IgG antibody constant region CH1, constant region CH1CH2,
The experiment of the anti-tumor activity of fusion protein constructed by constant region CH1CH2CH3.1 in figure: physiological saline；2: preamble sequence-
Endostatin；3: preamble sequence-Endostatin-IgG antibody part constant region CH1 fusion protein；4: preamble sequence-endothelium suppression
Element-IgG antibody part constant region CH1CH2 fusion protein；5: preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3
Fusion protein.
Fig. 2 be embodiment 1 preamble sequence-Endostatin be separately connected IgG antibody constant region CH1, constant region CH1CH2,
The experiment of fusion protein constructed by constant region CH1CH2CH3 and Endostatin in mouse Half-life in vivo.Sample in figure (from
Top to bottm), ● preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein；▲ preamble sequence-endothelium suppression
Element-IgG antibody part constant region CH1CH2 fusion protein；■ preamble sequence-Endostatin-IgG antibody part constant region CH1 melts
Hop protein；◆ preamble sequence-Endostatin
Fig. 3 is the Endostatin N-terminal in preamble sequence-Endostatin-IgG antibody constant region fusion protein of embodiment 2
The experiment of the point mutation of Arg in the small peptide of front.In figure: sample 1: preamble sequence-Endostatin-IgG antibody constant region fusion
Albumen；Sample 2: preamble sequence-Endostatin-IgG antibody constant region fusion protein N-terminal point mutation body 1；Sample 3: preposition sequence
Column-Endostatin-IgG antibody constant region fusion protein N-terminal point mutation body 2；Sample 4: preamble sequence-Endostatin-antibody
The point mutation body 3 of IgG constant region fusion protein N-terminal；Sample 5: physiological saline (control).
Fig. 4 is preamble sequence-Endostatin-IgG antibody constant region fusion of the introducing glycosylation site small peptide of embodiment 3
The experiment of the anti-tumor activity of albumen.Sample 1: preamble sequence-Endostatin-IgG antibody constant region fusion protein (SEQ ID
NO.12)；Sample 2: preamble sequence-Endostatin -1 (SEQ ID of IgG antibody constant region fusion protein glycosylation site mutation body
NO.14)；Sample 3: preamble sequence-Endostatin -2 (SEQ ID of IgG antibody constant region fusion protein glycosylation site mutation body
NO.16)；Sample 4: preamble sequence-Endostatin -3 (SEQ ID of IgG antibody constant region fusion protein glycosylation site mutation body
NO.18)
Fig. 5 is preamble sequence-Endostatin-IgG antibody constant region fusion protein of the introducing glycosylation small peptide of embodiment 3
In the experiment of mouse Half-life in vivo.Sample (from top to bottom): ● preamble sequence-Endostatin-IgG antibody constant region merges egg
White glycosylation site mutant 3 (SEQ ID NO.18)；▲ preamble sequence-Endostatin-IgG antibody constant region fusion protein sugar
Base site mutant 2 (SEQ ID NO.16)；The glycosylation of ■ preamble sequence-Endostatin-IgG antibody constant region fusion protein
Site mutant 1 (SEQ ID NO.14)；◆ preamble sequence-Endostatin-IgG antibody constant region fusion protein (SEQ ID
NO.12)。
Fig. 6 is preamble sequence-Endostatin-IgG antibody constant region fusion protein glycosylation site mutation body of embodiment 4
2 (SEQ ID NO.15 and SEQ ID NO.16) are carried out again on the glycosylation site (Asn210-Ser211-Thr212) of introducing
The experiment of the anti-tumor activity of the various point mutation bodies of point mutation.Sample 1: point mutation body Asn210-Ser211-Thr212；Sample
Product 2: point mutation body Asn210-Ala211-Thr212；Sample 3: point mutation body Asn210-Tyr211-Thr212；Sample 4: point
Mutant Asn210-Ser211-Ser212；Sample 5: point mutation body Asn210-Ala211-Ser212；Sample 6: it does not introduce
Preamble sequence-Endostatin of glycosylation site-IgG antibody constant region fusion protein.
Fig. 7 is experiment of the various point mutation body fusion proteins in Fig. 6 of embodiment 4 in mouse Half-life in vivo.Sample
(from top to bottom) x point mutation body (Asn210-Tyr211-Thr212)；+ point mutation body (Asn210-Ser211-Ser212)；■
Point mutation body (Asn210-Ser211-Thr212)；▲ point mutation body (Asn210-Ala211-Thr212)；● point mutation body
(Asn210-Ala211-Ser212)；◆ do not introduce preamble sequence-Endostatin-IgG antibody constant region of glycosylation site
Fusion protein.
Fig. 8 is embodiment 5 with preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein (SEQ
ID NO.11 and SEQ ID NO.12) it is used as experimental material, the various point mutation of point mutation are carried out for the position Arg85-Arg86
The experiment of the anti-tumor activity of body.1: point mutation body 1 (Ala85-Arg86)；2: point mutation body 2 (Ser85-Arg86)；3: point is prominent
Variant 3 (His85-Arg86)；4: point mutation body 4 (Arg85-Ala86)；5: point mutation body 5 (Arg85-Ser86)；6: point mutation
Body 6 (Arg85-His86)；7: not having mutein fusion protein (SEQ ID NO.12).
Fig. 9 is experiment of the various point mutation body fusion proteins in Fig. 8 of embodiment 5 in mouse Half-life in vivo.Sample
(from top to bottom) ,+point mutation body 6 (Arg85-His86)；X point mutation body 3 (His85-Arg86)；2 (Ser85- of ▲ point mutation body
Arg86)；● point mutation body 5 (Arg85-Ser86)；■ point mutation body 1 (Ala85-Arg86)；* 2 (Ser85- of point mutation body
Arg86)；◆ do not have mutein fusion protein (SEQ ID NO.12).
Figure 10 is embodiment 5 with preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein (SEQ
ID NO.11 and SEQ ID NO.12) it is used as experimental material, the various points for carrying out point mutation for the position Arg151-Arg152 are prominent
The experiment of the anti-tumor activity of variant.1: point mutation body 7 (Ala151-Arg152)；2: point mutation body 8 (Ser151-Arg152)；
3: point mutation body 9 (His151-Arg152)；4: point mutation body 10 (Arg151-Ala152)；5: 11 (Arg151- of point mutation body
Ser152)；6: point mutation body 12 (Arg151-His152)；7: not having mutein fusion protein (SEQ ID NO.12).
Figure 11 is experiment of the various point mutation body fusion proteins in Figure 10 of embodiment 5 in mouse Half-life in vivo.Sample
Product (from top to bottom), x point mutation body 9 (His151-Arg152)；+ point mutation body 12 (Arg151-His152)；* point mutation body 10
(Arg151-Ala152)；■ point mutation body 7 (Ala151-Arg152)；▲ point mutation body 8 (Ser151-Arg152)；● point is prominent
Variant 11 (Arg151-Ser152)；◆ do not have mutein fusion protein (SEQ ID NO.12).
Figure 12 is to press down before the N-terminal of the Endostatin of embodiment 6 with the subsequent each addition glycosylation site of C-terminal and endothelium
The experiment of the anti-tumor activity of the fusion protein of the transformation of 2 groups of ArgArg in element.Sample 1: preamble sequence-Endostatin-is anti-
Body IgG constant region CH1CH2CH3 merges egg (SEQ ID NO.12)；Sample 2: preamble sequence-Endostatin-IgG antibody is constant
Area CH1CH2CH3 merges egg point mutation body 1 (His85-Arg86 and His151-Arg152)；Sample 3: preamble sequence-endothelium suppression
Element-IgG antibody constant region CH1CH2CH3 fusion egg point mutation body 2 (Arg85-His86 and Arg151-His152)；Sample 4: preceding
Sequence-Endostatin (before N-terminal and the subsequent each addition glycosylation site of C-terminal)-IgG antibody constant region CH1CH2CH3 is set to melt
Hop protein (SEQ ID NO.18)；Sample 5: (before N-terminal and the subsequent each addition of C-terminal glycosylates position to preamble sequence-Endostatin
Point)-IgG antibody constant region CH1CH2CH3 fusion protein point mutation body 1 (Arg94His95 and Arg160His161)；Sample 6:
Preamble sequence-Endostatin (before N-terminal and the subsequent each addition glycosylation site of C-terminal)-IgG antibody constant region CH1CH2CH3
Fusion protein point mutation body 2 (His94Arg95 and His160Arg161) (SEQ ID NO.20).
Figure 13 is experiment of the fusion protein in mouse Half-life in vivo of the various mutant in Figure 12 of embodiment 6.Sample
Product (from top to bottom), ● before the N-terminal of Endostatin and C-terminal it is subsequent it is each be added glycosylation site fusion protein point mutation body
2 (His94Arg95 and His160Arg161) (SEQ ID NO.20)；Before the N-terminal of+Endostatin and the subsequent each addition of C-terminal
The fusion protein point mutation body 1 (Arg94His95 and Arg160His161) of glycosylation site；■ preamble sequence-Endostatin-
IgG antibody constant region CH1CH2CH3 merges egg point mutation body 1 (His85-Arg86 and His151-Arg152)；X Endostatin
Before N-terminal and C-terminal it is subsequent it is each be added glycosylation site fusion protein (SEQ ID NO.18)；▲ preamble sequence-endothelium suppression
Element-IgG antibody constant region CH1CH2CH3 fusion egg point mutation body 2 (Arg85-His86 and Arg151-His152)；◆ preposition sequence
Column-Endostatin-IgG antibody constant region CH1CH2CH3 fusion egg (SEQ ID NO.12).
Figure 14 is the photo of the mouse dissection of tumor model, 1 in figure: the injection physiology salt as control in embodiment 1
The mouse of water；2: (before the N-terminal of Endostatin and the subsequent each addition of C-terminal is sugared for 6 fusion protein of injected sample in embodiment 6
The fusion protein point mutation body 2 in base site) (SEQ ID NO.20) mouse；The referred to tumour of arrow.
Figure 15 is Endostatin and the antitumor experiment of various Endostatin derivatives of embodiment 7.1: Endostatin；2:
Preamble sequence-endostatin protein (has 2 Arg) before the N-terminal of Endostatin；3: glycosylation site small peptide-Endostatin-sugar
Base site small peptide albumen；4: the Endostatin of point mutation (His62Arg63 and His128Arg129) occurs；5: Endostatin-
IgG antibody constant region fusion protein；6: preamble sequence-Endostatin (glycosylation site is added before N-terminal and respectively behind C-terminal)-
IgG antibody constant region fusion protein point mutation body 2 (His94Arg95 and His160Arg161) (SEQ ID NO.20)；7: physiology
Salt water.
Figure 16 is Endostatin in Figure 15 of embodiment 7 and various Endostatin derivatives in mouse Half-life in vivo
Experiment.Sample (from top to bottom), ● preamble sequence-Endostatin (glycosylation site is added before N-terminal and respectively behind C-terminal)-anti-
Body IgG constant region fusion protein point mutation body 2 (His94Arg95 and His160Arg161) (SEQ ID NO.20)；The suppression of+endothelium
Element-IgG antibody constant region fusion protein；▲ glycosylation site small peptide-Endostatin-glycosylation site small peptide albumen；Point occurs for x
It is mutated the Endostatin of (His62Arg63 and His128Arg129)；■ preamble sequence-endostatin protein (the N of Endostatin
There are 2 Arg in end front)；◆ Endostatin.
Specific embodiment
Below by specific embodiment, the present invention is further illustrated.
Remodeling method one: Endostatin derivative includes Endostatin to be connected and what is constructed merge egg with antibody constant region
White, the present invention connect Endostatin with antibody constant region, especially constant region CH1, CH1CH2 of IgG antibody or
CH1CH2CH3.A preamble sequence is connected before Endostatin simultaneously, it be by human albumin signal peptide and it is mature after
5 amino acid short peptides of the N-terminal of albumin form, and signal peptide is to allow the fusion protein of expression that can be secreted into zooblast
Outside.In this way, improving the half-life period of Endostatin derivative by the molecular weight for increasing albumen.
Remodeling method two: Endostatin derivative is with preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3
Fusion protein is experimental material, is previously incorporated arginine (Arg) in the N-terminal of Endostatin, reinforces endothelium by additional Arg
The antitumor bioactivity of chalone.
Remodeling method three: Endostatin derivative is with preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3
Fusion protein is experimental material, introduces glycosylation site in the above or below of Endostatin.Eukaryon including animal is raw
The sugar chain of the albumen of object occurs on the position Asn-X-Ser/Thr, and wherein X is the amino acid other than Pro, Asp or Glu,
Sugar chain is connected on Asn.In order to improve preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein
Half-life period, Endostatin of the present invention in preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein
Glycosylation site, the glycosyl of selection are introduced before glycosylation site or N-terminal and introduced simultaneously behind C-terminal before N-terminal or behind C-terminal
The amino acid for changing site is AsnSerThr, and additional small peptide GlyAlaSer is added before and after this site, last in this way
Structure be GlyAlaSerAsnSerThrGlyAlaSer, wherein AsnSerThr is glycosylation position.
Remodeling method four: Endostatin derivative is with preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3
Fusion protein is experimental material, transformation Endostatin (SEQ ID NO.4) in 2 groups of ArgArg, i.e. Arg62Arg63 and
Arg128Arg129.Basic amino acid is often the substrate of protease, and the present invention is only to retain one in this 2 groups of ArgArg
Arg, to improve the stability of Endostatin.Although Arg is considered critically important for Heparin-binding, in order to reduce endothelium suppression
Element still helps to improve the bioactivity of Endostatin by the quantity of protease hydrolytic, such transformation.
The nucleic acid of various albumen and the essential information of amino acid sequence
SEQ ID NO.2 is the sequence of the signal peptide and 5 amino acid of N-terminal of human albumin after maturation of human albumin,
Middle 1-18 is signal peptide, and 19-23 is 5 amino acid of N-terminal of human albumin after maturation, and SEQ ID NO.1 is SEQ ID NO.2
Nucleic acid coding sequence, the information in GenBank database are NP_000468.1 and NM_000477.5, SEQ ID NO.2 this
Sequence is known as preamble sequence.
SEQ ID NO.4 is the amino acid sequence of the Endostatin in people source, and SEQ ID NO.3 is its nucleic acid encode sequence
It arranges, the information in GenBank database is NP_569712.2 and NM_130445.3, and Endostatin here shares 183 ammonia
Base acid.
SEQ ID NO.6 is the amino acid sequence of human antibody IgG constant region, and SEQ ID NO.5 is its nucleic acid encode sequence
It arranges, the information in GenBank database is M87789.1, wherein according to amino acid sequence, 1-118 is CH1 segment, 119-225
It is CH2 segment, 226-330 is CH3 segment.
Experimental material and method
Animal expression vector uses the pHEK293 Ultra Expression Vector of precious biotech firm (Takara)
II limits restriction enzyme site SmaI-XhoI-BamHI-XbaI-SalI-PstI- containing foreign gene segment insertion position
SphI-HindIII, the insertion enzyme cutting site selected here is XhoI-BamHI-XbaI and SphI-HindIII, i.e. gene segment
Front end contain XhoI-BamHI-XbaI, SphI-HindIII is contained in rear end, in addition before SphI-HindIII increase by one
His-tag and termination signal tag, i.e. catcatcatcatcatcattag.The building of expression vector is screened in Escherichia coli
It completes, then can be transfected into the transient expression that HEK293 cell carries out various albumen.The synthesis of gene segment in these carriers
It is by completions such as precious biotech firm and Shanghai bio-engineering corporations.
The culture medium of HEK293 cell is the DMEM culture medium of 10% fetal calf serum.
About isolating and purifying for albumen, since albumen possesses signal peptide, so expression is secreting type, the separation of albumen is pure
Change is to be centrifuged culture medium using His-tag method, removes cell, then utilizes HisBind Purification
Kit kit (Novagen company) purifies supernatant, Binding Buffer is first added before upper prop, then by egg
White sample upper prop is rinsed with Wash Buffer, then is eluted with Elute Buffer.If it is constant that fusion protein contains IgG antibody
Area, so that it may use ProteinA column, that is, use HiTrap Protein the A HP or HiTrap of GE Healthcare company
RProtein A FF, rinsing liquid are PBS, and eluent is 0.1M citric acid (pH2.7).With His-tag or ProteinA column method
Obtained albumen is all identified with protein electrophoresis, and the albumen that purity is a band is used for later experiment.
About tumor model, mouse is C57BL/6, and about 20-24 grams, the number of mice tested every time is 30, tumour cell
It is mice lung cancer Lewis lung carcinoma (LLC) cell strain, LLC cell is first subjected in vitro culture, is then injected into
C57BL/6 mouse peritoneal carries out large-scale culture, intraperitoneal LLC tumour cell is finally taken out, by 2x106Tumor cell inoculation
Under the right axillary of mouse.
The bioactivity of albumen is evaluated with mice tumors grew inhibiting rate and mouse Half-life in vivo etc., albumen dosage
It is all 50pmol.
A kind of derivative of the anti-tumor protein Endostatin of embodiment 1, derivative include that Endostatin and IgG antibody are constant
The fusion protein of area's connection, anti-tumor activity and the fusion protein are in mouse intracorporal half-life period.
Protein sample
Sample 1: preamble sequence-Endostatin；SEQ ID NO.2 is connected before Endostatin shown in SEQ ID NO.4
Shown in preamble sequence, the signal peptide in preamble sequence is in order to which the Endostatin of expression can be secreted into extracellularly.
Sample 2: preamble sequence-Endostatin-IgG antibody part constant region CH1 fusion protein (SEQ ID NO.8 institute
Show, the nucleic acid sequence of coding SEQ ID NO.8 is shown in SEQ ID NO.7).
Sample 3: preamble sequence-Endostatin-IgG antibody part constant region CH1CH2 fusion protein (SEQ ID NO.10
Shown, the nucleic acid sequence of coding SEQ ID NO.10 is shown in SEQ ID NO.9).
Sample 4: preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein (SEQ ID NO.12 institute
Show, the nucleic acid sequence of coding SEQ ID NO.12 is shown in SEQ ID NO.11).
Gene chemical synthesis segment above is inserted into pHEK293 Ultra Expression Vector II plasmid,
Sample 1 and the His-tag method of sample 2 are purified, and sample 3 and the ProteinA column method of sample 4 are purified.
Control sample is injecting normal saline.
1st day by 2x106LLC tumor cell inoculation is under the right axillary of mouse, the 2nd day, the 5th day, the 8th day difference vein note
Various albumen (dosage is all 50pmol) and control sample physiological saline are penetrated, the quantity of each group mouse is 30, is killed within the 10th day
Dead mouse takes out tumour and weighs.Fig. 1 is antitumor experimental result, shows to increase with the molecular weight of fusion protein, tumour
Inhibitory effect is better.
Half-life experiments intracorporal for the mouse of albumen give the various albumen of mouse one injection (100ug/0.2ml), use
Anti-endostatin antiserum detects the content of Endostatin or Endostatin fusion protein, and detection in the 1st day is after injection 2
It is carried out in hour, continuous detection 13 days.13 days changes of contents that Fig. 2 is various albumen in Mice Body, the results show that molecular weight
More big then in-vivo content is higher.
Since preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein is more stable in vivo, so
Inhibit the effect of tumour best.
The assessment of the Arg newly introduced before 2 Endostatin derivative of embodiment, that is, Endostatin N-terminal
Preamble sequence-Endostatin of 1 sample 4 of selection example-IgG antibody constant region fusion protein (SEQ ID
NO.11 and SEQ ID NO.12) it is used as experimental material, illustrated with amino acid sequence (SEQ ID NO.12), 1-23 amino acid
From albumin, 1-18 is the signal peptide of albumin, and 19-23 is 5 amino acid of the N-terminal of the albumin after maturation, wherein
There are two Arg, and 24-206 is Endostatin, and 207-536 is IgG antibody constant region, it is made of CH1, CH2 and CH3.Various realities
Candling is white:
Sample 1: preamble sequence-Endostatin-IgG antibody constant region fusion protein, as shown in SEQ ID NO.12, wherein
19-23 is Arg-Gly-Val-Phe-Arg.
Sample 2: preamble sequence-Endostatin-IgG antibody constant region fusion protein point mutation body 1, wherein 19-23 be
Ala-Gly-Val-Phe-Arg, by the agg point mutation of the 19th Arg at gcg；
Sample 3: preamble sequence-Endostatin-IgG antibody constant region fusion protein point mutation body 2, wherein 19-23 be
Arg-Gly-Val-Phe-Ala, by the cgt point mutation of the 23rd Arg at gct；
Sample 4: preamble sequence-Endostatin-IgG antibody constant region fusion protein point mutation body 3, wherein 19-23 be
Ala-Gly-Val-Phe-Ala, by the agg point mutation of the 19th Arg at gcg and by the cgt point mutation of the 23rd Arg at gct.
Injecting normal saline is as control.
Preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein (SEQ is expressed in selection example 1
Shown in ID NO.12, encode SEQ ID NO.12 nucleic acid sequence SEQ ID NO.11) plasmid (pHEK293 Ultra
Expression Vector II) it carries out various point mutation and just obtains various Endostatins-IgG antibody constant region fusion egg above
White mutant.
Various fusion proteins ProteinA column method is purified.
1st day by 2x106LLC tumor cell inoculation is under the right axillary of mouse, the 2nd day, the 5th day, the 8th day difference vein note
It penetrates various albumen (dosage is all 50pmol), the quantity of each group mouse is 30, the 10th day kill mouse, takes out tumour and claims
Weight.Fig. 3 is antitumor experimental result, and N-terminal has preamble sequence-Endostatin-IgG antibody constant region fusion protein of 2 Arg
Inhibit the effect of tumour best.
Embodiment 3 checks glycosylation site influence active for Endostatin fusion protein
With preamble sequence-Endostatin of embodiment 1-IgG antibody constant region CH1CH2CH3 fusion protein (SEQ ID
NO.11 and SEQ ID NO.12) as experimental material building derivative, i.e., it is added before the N-terminal of Endostatin or behind C-terminal
Glycosylated amino acid short peptide Gly-Ala-Ser-Asn-Ser-Thr-Gly-Ala-Ser, wherein Asn-Ser-Thr is glycosylation
Position, sugar chain is connected on Asn.Preamble sequence-Endostatin-IgG antibody constant region is expressed in selection example 1
CH1CH2CH3 fusion protein (shown in SEQ ID NO.12, encoding the nucleic acid sequence SEQ ID NO.11 of SEQ ID NO.12)
Plasmid (pHEK293 Ultra Expression Vector II) carries out various genetic manipulations, and it is prominent can also directly to synthesize these
The gene segment of variant is inserted into plasmid (pHEK293 Ultra Expression Vector II) and carries out various albumen
Expression.
Sample 1: preamble sequence-Endostatin-IgG antibody constant region fusion protein (SEQ ID NO.12), Endostatin
N-terminal before or C-terminal behind there is no glycosylated amino acid short peptide；
Sample 2: preamble sequence-Endostatin -1 (SEQ ID of IgG antibody constant region fusion protein glycosylation site mutation body
NO.13 and SEQ ID NO.14), the amino acid short peptide of a glycosylation site is added before Endostatin N-terminal；
Sample 3: preamble sequence-Endostatin -2 (SEQ ID of IgG antibody constant region fusion protein glycosylation site mutation body
NO.15 and SEQ ID NO.16), the amino acid short peptide of a glycosylation site is added behind Endostatin C-terminal；
Sample 4: preamble sequence-Endostatin -3 (SEQ ID of IgG antibody constant region fusion protein glycosylation site mutation body
NO.17 and SEQ ID NO.18), the amino acid that a glycosylation site is respectively added before the N-terminal of Endostatin and behind C-terminal is short
Peptide.
Various fusion proteins ProteinA column method is purified.
1st day by 2x106LLC tumor cell inoculation is under the right axillary of mouse, the 2nd day, the 5th day, the 8th day difference vein note
Various albumen (dosage is all 50pmol) and physiological saline are penetrated, the quantity of each group mouse is 30, and the 10th day kill mouse takes
It tumour and weighs out.Fig. 4 is antitumor experimental result, shows that there is the amino acid short peptide of glycosylation site in Endostatin front and back
Fusion protein inhibition tumour effect it is best.
Half-life experiments intracorporal for the mouse of albumen give the various albumen of mouse one injection (100ug/0.2ml), use
Anti-endostatin antiserum detects the content of Endostatin fusion protein, detection in the 1st day be after injection in 2 hours into
Row, continuous detection 13 days.13 days changes of contents that Fig. 5 is various albumen in Mice Body, the results show that before and after Endostatin all
There is the fusion protein of glycosylation site in mouse in-vivo content highest.
Here experiment shows due to Endostatin derivative i.e. preamble sequence-Endostatin-IgG antibody constant region fusion
Protein glycosylation site mutant 3 (having glycosylation site before Endostatin N-terminal and behind C-terminal) more stable in vivo, institute
To inhibit the effect of tumour best.
Embodiment 4 carries out point mutation for the amino acid of same glycosylation site again
The sugar chain of Eukaryotic albumen including animal occurs on the position Asn-X-Ser/Thr, and wherein X is to remove
Pro, Asp or Glu with for amino acid, sugar chain is connected on Asn.Here, with preamble sequence-endothelium in embodiment 3
Chalone-IgG antibody constant region fusion protein glycosylation site mutation body 2 (SEQ ID NO.15 and SEQ ID NO.16) i.e. endothelium
The fusion protein that the amino acid short peptide of a glycosylation site is added behind chalone is that material carries out new point mutation, its glycosyl
Changing site is Asn210-Ser211-Thr212, and nucleic acid sequence is aacagcacg (628-636), and the point mutation body newly increased is made
For laboratory sample.
Sample 1: point mutation body Asn210-Ser211-Thr212 (SEQ ID NO.16)
Sample 2: point mutation body Asn210-Ala211-Thr212 (being gcc by the agc point mutation for encoding Ser211)
Sample 3: point mutation body Asn210-Tyr211-Thr212 (being tac by the agc point mutation for encoding Ser211)
Sample 4: point mutation body Asn210-Ser211-Ser212 (being tcg by the acg point mutation for encoding Thr212)
Sample 5: point mutation body Asn210-Ala211-Ser212 (for gcc and incites somebody to action the agc point mutation for encoding Ser211
The acg point mutation for encoding Thr212 is tcg)
Sample 6: preamble sequence-Endostatin-IgG antibody constant region fusion protein (SEQ of glycosylation site is not introduced
ID NO.12)
With prominent containing preamble sequence-Endostatin in embodiment 3-IgG antibody constant region fusion protein glycosylation site
Melting for a glycosylated amino acid short peptide is added behind variant 2 (SEQ ID NO.15 and SEQ ID NO.16) i.e. Endostatin
The expression plasmid (pHEK293 Ultra Expression Vector II) of hop protein carries out the gene behaviour of various mutant points
Make.Various fusion proteins ProteinA column method is purified.
1st day by 2x106LLC tumor cell inoculation is under the right axillary of mouse, the 2nd day, the 5th day, the 8th day difference vein note
Various albumen (dosage is all 50pmol) and physiological saline are penetrated, the quantity of each group mouse is 30, and the 10th day kill mouse takes
It tumour and weighs out.
Fig. 6 is antitumor experimental result, is shown on same position and carries out the fusion protein of glycosylation site transformation all
There is the effect for inhibiting tumour well.
Half-life experiments intracorporal for the mouse of albumen give the various albumen of mouse one injection (100ug/0.2ml), use
Anti-endostatin antiserum detects the content of Endostatin fusion protein, detection in the 1st day be after injection in 2 hours into
Row, continuous detection 13 days.13 days changes of contents that Fig. 7 is various albumen in Mice Body, the results show that by glycosylation site
The fusion protein of transformation is all very high in the intracorporal content of mouse, as long as this shows the glycosylation of introducing Asn-X-Ser/Thr feature
Modification can improve the stability of the inhibition tumor promotion and albumen of fusion protein in vivo.
5 Endostatin derivative of embodiment is 2 groups of ArgArg being transformed in Endostatin
With preamble sequence-Endostatin of embodiment 1-IgG antibody constant region CH1CH2CH3 fusion protein (SEQ ID
NO.11 and SEQ ID NO.12) it is used as experimental material, point mutation then is carried out to 2 groups of ArgArg inside Endostatin.
Preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein (SEQ ID NO.11 and SEQ ID
NO.12 2 groups of ArgArg inside the Endostatin in) are in the middle position respectively of amino acid sequence (SEQ ID NO.4) of Endostatin
In Arg62-Arg63 and Arg128-Arg129, egg is merged in preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3
It is located at Arg85-Arg86 and Arg151-Arg152 in white amino acid sequence (SEQ ID NO.12), in the core for encoding it
Position in acid sequence (SEQ ID NO.11) is 253-258 (cgccgt) and 451-456 (cgcagg).
Point mutation is carried out firstly for the 1st group of Arg85-Arg86, nucleic acid position 253-258 (cgccgt).
Point mutation body 1:Ala85-Arg86 (being gcc by the cgc point mutation for encoding front Arg)
Point mutation body 2:Ser85-Arg86 (being agc by the cgc point mutation for encoding front Arg)
Point mutation body 3:His85-Arg86 (being cac by the cgc point mutation for encoding front Arg)
Point mutation body 4:Arg85-Ala86 (being gct by the cgt point mutation for encoding Arg below)
Point mutation body 5:Arg85-Ser86 (being agt by the cgt point mutation for encoding Arg below)
Point mutation body 6:Arg85-His86 (being cat by the cgt point mutation for encoding Arg below)
Then point mutation is carried out for the 2nd group of Arg151-Arg152, nucleic acid position 451-456 (cgcagg).
Point mutation body 7:Ala151-Arg152 (being gcc by the cgc point mutation for encoding front Arg)
Point mutation body 8:Ser151-Arg152 (being agc by the cgc point mutation for encoding front Arg)
Point mutation body 9:His151-Arg152 (being cac by the cgc point mutation for encoding front Arg)
Point mutation body 10:Arg151-Ala152 (being gcg by the agg point mutation for encoding Arg below)
Point mutation body 11:Arg151-Ser152 (being agc by the agg point mutation for encoding Arg below)
Point mutation body 12:Arg151-His152 (being cac by the agg point mutation for encoding Arg below)
Preamble sequence-expression Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein in selection example 1
The plasmid (pHEK293 Ultra Expression Vector II) of (SEQ ID NO.11 and SEQ ID NO.12) carries out each
The genetic manipulation of kind mutant point.
Various fusion proteins ProteinA column method is purified.
1st day by 2x106LLC tumor cell inoculation is under the right axillary of mouse, the 2nd day, the 5th day, the 8th day difference vein note
Various albumen (dosage is all 50pmol) and physiological saline are penetrated, the quantity of each group mouse is 30, and the 10th day kill mouse takes
It tumour and weighs out.
1st experiment is for the 1st group of Arg85-Arg86, the gene segment (SEQ of nucleic acid position 253-258 (cgccgt)
ID NO.11) it carries out, sample has a mutant 1-6 and the fusion protein not being mutated (SEQ ID NO.12) as control sample
Product.
Fig. 8 is antitumor experimental result, shows 6 (Arg85- of point mutation body 3 (His85-Arg86) and point mutation body
His86 the effect of the inhibition tumour of fusion protein) is fine.
Half-life experiments intracorporal for the mouse of albumen give the various albumen of mouse one injection (100ug/0.2ml), use
Anti-endostatin antiserum detects the content of Endostatin fusion protein, detection in the 1st day be after injection in 2 hours into
Row, continuous detection 13 days.13 days changes of contents that Fig. 9 is various albumen in Mice Body, the results show that various point mutation bodies exist
The difference of intracorporal content is little, but all higher than the in-vivo content of the fusion protein of not point mutation.
Here experiment shows to be changed due to the 1st group of Arg85-Arg86 of Endostatin, maintains the stabilization of albumen
Property be not easy to be easily degraded by proteases, so there is higher anti-tumor activity, point mutation body 3 (His85-Arg86) and point are dashed forward
In variant 6 (Arg85-His86), His maintains the property of basic amino acid instead of Arg, so embodying very high
Inhibition tumour activity and very high protein content.
Carry out now according to the 1st experimental method above and is directed to the 2nd group of Arg151-Arg152, nucleic acid position 451-456
(cgcagg) gene segment (SEQ ID NO.11) carries out point mutation, and sample has a mutant 7-12 and what is be not mutated melt
Hop protein (SEQ ID NO.12) is used as control sample.
Figure 10 is antitumor experimental result, shows point mutation body 9 (His151-Arg152) and point mutation body 12
(Arg151-His152) effect of the inhibition tumour of fusion protein is fine.
Figure 11 is various albumen in Mice Body 13 days changes of contents, the results show that various point mutation bodies are in vivo
The difference of content is little, but all higher than the in-vivo content of the fusion protein of not point mutation.
Illustrate again: with preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion protein (SEQ ID
NO.12 it) is used as experimental material, the Arg85-Arg86 inside Endostatin and Arg151-Arg152 in fusion protein are right respectively
Ying Yu is with the Arg62-Arg63 and Arg128-Arg129 in Endostatin sequence shown in SEQ ID NO.4.
Embodiment 6 is for before the N-terminal of Endostatin and in the subsequent each addition glycosylation site of C-terminal and Endostatin
The transformation of 2 groups of ArgArg reevaluate
According to the experimental result of embodiment 5, with preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion
Albumen (SEQ ID NO.11 and SEQ ID NO.12) is used as experimental material, then constructs 2 kinds of point mutation bodies:
Point mutation body 1:His85-Arg86 and His151-Arg152
It is cac by the cgc point mutation for encoding Arg85
It is cac by the cgc point mutation for encoding Arg151
Point mutation body 2:Arg85-His86 and Arg151-His152
It is cat by the cgt point mutation for encoding Arg86
It is cac by the agg point mutation for encoding Arg152
Illustrate: using SEQ ID NO.12 sequence as inside the Endostatin in experimental material fusion protein the 85th, the 86th,
151st, the 152nd amino acid position corresponds respectively to the 62nd, the 63rd, in Endostatin sequence shown in SEQ ID NO.4
128, the 129th.
According to the experimental result of embodiment 3, with before the N-terminal of Endostatin and the subsequent each addition glycosylation site of C-terminal
Fusion protein (SEQ ID NO.17 and SEQ ID NO.18) be used as experimental material, its 2 groups of ArgArg are transformed,
The amino acid position of 1st group of ArgArg is Arg94Arg95 (SEQ ID NO.18), nucleic acid position (SEQ ID
It NO.17) is 280-285 (cgccgt)；The amino acid position of 2nd group of ArgArg is Arg160Arg161 (SEQ ID NO.18),
Nucleic acid position (SEQ ID NO.17) is 478-483 (cgcagg).
In addition 2 kinds of point mutation bodies are constructed:
Point mutation body 1:Arg94His95 and Arg160His161
It is cat by the cgt point mutation for encoding Arg95
It is cac by the agg point mutation for encoding Arg161
Point mutation body 2:His94Arg95 and His160Arg161
It is cac by the cgc point mutation for encoding Arg94
It is cac by the cgc point mutation for encoding Arg160
(nucleic acid sequence SEQ ID NO.19 and amino acid sequence SEQ ID NO.20)
Illustrate: using SEQ ID NO.18 sequence as inside the Endostatin in experimental material fusion protein the 94th, the 95th,
160th, the 161st amino acid position corresponds respectively to the 62nd, the 63rd, in Endostatin sequence shown in SEQ ID NO.4
128, the 129th.
Sample used by testing now:
Sample 1: preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 fusion egg (SEQ ID NO.12)
Sample 2: preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 merges 1 (His85- of egg point mutation body
Arg86 and His151-Arg152)
Sample 3: preamble sequence-Endostatin-IgG antibody constant region CH1CH2CH3 merges 2 (Arg85- of egg point mutation body
His86 and Arg151-His152)
Sample 4 :-IgG antibody is or not preamble sequence-Endostatin (before N-terminal and the subsequent each addition glycosylation site of C-terminal)
Become area CH1CH2CH3 fusion protein (SEQ ID NO.18)
Sample 5 :-IgG antibody is or not preamble sequence-Endostatin (before N-terminal and the subsequent each addition glycosylation site of C-terminal)
Become area CH1CH2CH3 fusion protein point mutation body 1 (Arg94His95 and Arg160His161)
Sample 6 :-IgG antibody is or not preamble sequence-Endostatin (before N-terminal and the subsequent each addition glycosylation site of C-terminal)
Become area CH1CH2CH3 fusion protein point mutation body 2 (His94Arg95 and His160Arg161) (SEQ ID NO.20)
1st day inoculation LLC tumour cell, being injected intravenously within the 2nd day, the 5th day, the 8th day various albumen respectively, (dosage is all
50pmol) and physiological saline, the quantity of each group mouse are 30, the 10th day kill mouse, take out tumour and weigh.
Figure 12 is antitumor experimental result, shows to introduce 2 glycosylation sites simultaneously and change to 2 groups of 2 groups of ArgArg
The sample 5 and sample 6 made have inhibits tumor effect well.
Half-life experiments intracorporal for the mouse of albumen give the various albumen of mouse one injection (100ug/0.2ml), use
Anti-endostatin antiserum detects the content of Endostatin fusion protein, detection in the 1st day be after injection in 2 hours into
Row, continuous detection 13 days.13 days changes of contents that Figure 13 is various albumen in Mice Body, the results show that introducing 2 sugar simultaneously
Base site and all very high to sample 5 and sample 6 content in vivo of 2 groups of ArgArg transformation.
Figure 14 is that the injected sample 6 in the mouse and embodiment 6 of the injecting normal saline as control in embodiment 1 is melted
Hop protein (before the N-terminal of Endostatin and the subsequent each fusion protein point mutation body 2 that glycosylation site is added of C-terminal) (SEQ
ID NO.20) mouse dissection photo.
Embodiment 7 is compared the bioactivity of Endostatin and various Endostatin derivatives
For the DNA synthetic fragment of all insertion plasmid pHEK293 Ultra Expression Vector II, segment
XhoI-BamHI-XbaI is contained in front end, and SphI-HindIII is contained in rear end, in addition increases by one before SphI-HindIII
His-tag and termination signal tag, i.e. catcatcatcatcatcattag.
Sample 1: Endostatin
I.e. in nucleic acid sequence shown in SEQ ID NO.1 behind the DNA segment of the 1-54 of encoded signal peptide connect SEQ
The nucleic acid sequence that Endostatin is encoded shown in ID NO.3 synthesizes such DNA segment, then this DNA segment is inserted into
Plasmid pHEK293 Ultra Expression Vector II, obtains Endostatin.
Sample 2: preamble sequence-endostatin protein (has 2 Arg) before the N-terminal of Endostatin
I.e. shown in SEQ ID NO.3 on being connected behind the nucleic acid sequence for encoding preamble sequence shown in SEQ ID NO.1
Coding Endostatin nucleic acid sequence, synthesize such DNA segment, this DNA segment be then inserted into plasmid pHEK293
UltraExpression Vector II, obtains preamble sequence-endostatin protein, in this preamble sequence-Endostatin egg
Bai Zhong has 2 Arg before the N-terminal of Endostatin.
Sample 3: glycosylation site small peptide-Endostatin-glycosylation site small peptide albumen
Following DNA segment is designed first:
The DNA segment of the 1-54 of encoded signal peptide in nucleic acid sequence shown in segment 1:SEQ ID NO.1
(coding includes the small peptide of glycosylation site to segment 2:ggggcctccaacagcacgggggcctcc
GlyAlaSerAsnSerThrGlyAlaSer)
The nucleic acid sequence of Endostatin is encoded shown in segment 3:SEQ ID NO.3
Synthesize such DNA sequence dna: then this DNA segment is inserted into plasmid by segment 1- segment 2- segment 3- segment 2
PHEK293 Ultra Expression Vector II obtains glycosylation site small peptide-Endostatin-glycosylation site small peptide
Albumen.
Sample 4: the Endostatin of point mutation (His62Arg63 and His128Arg129) occurs
It is the nucleic acid sequence for encoding Endostatin shown in SEQ ID NO.3,184-189 is cgccgt, corresponding to it
Amino acid is Arg62Arg63, and the 185th g is sported a, in this way, 184-189 becomes caccgt, to produce
His62Arg63；Equally, 382-387 is cgcagg, and the amino acid corresponding to it is Arg128Arg129, and 383g is mutated
For a, in this way, 382-387 becomes cacagg, to produce His128Arg129.This is encoded, point mutation endothelium occurs
(the 1-54 of encoded signal peptide in nucleic acid sequence shown in SEQ ID NO.1 of connection signal peptide before the DNA segment of chalone
DNA segment), the DNA segment of generation point mutation (His62Arg63 and His128Arg129) Endostatin has been obtained, this is synthesized
Segment is simultaneously inserted into plasmid pHEK293 Ultra Expression Vector II, obtains that point mutation (His62Arg63 occurs
And His128Arg129) endostatin protein.
Sample 5: Endostatin-IgG antibody constant region fusion protein
Following DNA segment is designed first:
The DNA segment of the 1-54 of encoded signal peptide in nucleic acid sequence shown in segment 1:SEQ ID NO.1
The nucleic acid sequence of Endostatin is encoded shown in segment 2:SEQ ID NO.3
The nucleic acid sequence of encoding antibody IgG constant region shown in segment 3:SEQ ID NO.5
Synthesize such DNA sequence dna: then this DNA segment is inserted into plasmid by segment 1- segment 2- segment 3
PHEK293 Ultra Expression Vector II, obtains Endostatin-IgG antibody constant region fusion protein.
Sample 6: preamble sequence-Endostatin (glycosylation site is added before N-terminal and respectively behind C-terminal)-IgG antibody is constant
Area's fusion protein point mutation body 2 (His94Arg95 and His160Arg161) (SEQ ID NO.20).
Control sample is injecting normal saline.
Sample 1-4 His-tag method is purified, and sample 5 and the ProteinA column method of sample 6 are purified.
1st day by 2x106LLC tumor cell inoculation is under the right axillary of mouse, the 2nd day, the 5th day, the 8th day difference vein note
Various albumen (dosage is all 50pmol) and physiological saline are penetrated, the quantity of each group mouse is 30, and the 10th day kill mouse takes
It tumour and weighs out.
Figure 15 is Endostatin and the antitumor experiment of various Endostatin derivatives.Endostatin and various as the result is shown
Endostatin derivative has antitumor bioactivity, and sample 6 is preamble sequence-Endostatin (before N-terminal and behind C-terminal
It is each that glycosylation site is added)-IgG antibody constant region fusion protein point mutation body 2 (His94Arg95 and His160Arg161)
(SEQ ID NO.20) antitumor effect is best.
Half-life experiments intracorporal for the mouse of albumen give the various albumen of mouse one injection (100ug/0.2ml), use
Anti-endostatin antiserum detects the content of Endostatin fusion protein, detection in the 1st day be after injection in 2 hours into
Row, continuous detection 13 days.13 days changes of contents that Figure 16 is various albumen in Mice Body, the results show that containing glycosylation position
The Endostatin (sample 3) of point and the Endostatin (sample 4) that point mutation (His62Arg63 and His128Arg129) occurs begin
Retain lower in-vivo content eventually, Endostatin-IgG antibody constant region fusion protein (sample 5) contains in vivo with quite high
Amount, and preamble sequence-Endostatin (glycosylation site is added before N-terminal and respectively behind C-terminal)-IgG antibody constant region fusion egg
The in-vivo content highest of white point mutant 2 (His94Arg95 and His160Arg161) (SEQ ID NO.20) (sample 6).
As it can be seen that possessing these features simultaneously: having before the N-terminal of 2 Arg, Endostatin before the N-terminal of Endostatin and C
End is each below to be added glycosylation site, the Endostatin that point mutation (His62Arg63 and His128Arg129) occurs, endothelium suppression
The plain fusion protein for connecting and being formed with IgG antibody constant region, such Endostatin derivative such as SEQ ID NO.20 sequence
The bioactivity of the antitumor and half-life period of albumen shown in column is best.
By a series of embodiments above, the stability of Endostatin derivative includes half-life period and antitumor effect
It is all greatly improved, these transformation means are:
Endostatin derivative one, preamble sequence-Endostatin and IgG antibody constant region connect and what is formed merge egg
It is white, increase the molecular weight of Endostatin.It is worth noting that Endostatin and albumin etc. divide greatly by case here
The connection of sub- glycoprotein and the fusion protein constructed also available similar result.
Endostatin derivative two is previously incorporated Arg in the N-terminal of Endostatin, due to Endostatin Arg for it
Bioactivity is critically important, so the anti-tumor activity of Endostatin can be improved in the Arg newly increased.
Endostatin derivative three, the amino acid that glycosylation site is respectively introduced before the N-terminal of Endostatin and behind C-terminal
Small peptide is GlyAlaSerAsnSerThrGlyAlaSer here, wherein including AsnSerThr, has Asn-X-Ser/Thr special
Sign, while point mutation has been carried out, show to have the mutant of Asn-X-Ser/Thr feature that there is very high anti-tumor activity and stabilization
Property.
Endostatin derivative four, for 2 groups of ArgArg in Endostatin shown in SEQ ID NO.4 sequence
(Arg62Arg63 and Arg128Arg129) carries out point mutation, can be to avoid the hydrolysis of protease, and experiment here shows handle
As soon as Arg point mutation in ArgArg at other amino acid, improve Endostatin fusion protein anti-tumor activity and
Stability, it is clear that melt the stability of hop protein just because of Endostatin is improved, that is, improve internal Endostatin
The content for melting hop protein results in the raising that Endostatin melts hop protein antitumous effect, while experiment is shown
The combination of ArgHis or HisArg possesses better anti-tumor activity.
Endostatin derivative five, embodiment 6 have selected an optimal combination, construct a kind of Endostatin derivative, together
Shi Yongyou:
(1) glycosylation site is introduced before Endostatin N-terminal and behind C-terminal
(2) by 2 groups of ArgArg point mutation in Endostatin at ArgHis or HisArg
Preamble sequence-Endostatin-IgG antibody constant region fusion protein with 2 kinds of features above shows quite high
Anti-tumor activity and quite high content in vivo.
According to following various change can be carried out based on fusion protein sequence (SEQ ID No.11 and SEQ ID No.12)
It makes:
A) glycosylation site is introduced before Endostatin N-terminal and behind C-terminal；
B) for the point mutation of the inside ArgArg of Endostatin
C) new Arg is previously incorporated in the N-terminal of Endostatin
Sequence shown in SEQ ID NO.19 and SEQ ID NO.20, which only describes, has the advantages that fusion eggs various above
White one kind.
There is also provided a kind of preparation methods:
A) host cell expressed is zooblast；
B) a kind of expression system can carry out glycosylation site modification for pharmaceutical protein；
C) due to being secretion type expression, fusion protein can be collected from culture medium；
D) isolating and purifying for fusion protein is carried out using Protein A column.
Endostatin fusion protein recited above and a series of mutant of its transformations can be applied to tumour
The treatment of cancer.
The DNA sequences encoding of various Endostatin fusion proteins above can be applied to antitumor gene therapy.
It can be emulsifier, liposome, dispersing agent, stabilizer etc. as drug its dosage form and various injections, mouth be made together
The form of medication of the drugs such as clothes, application and surgical procedure.Other than fused protein itself can be used as drug, coding fusion
The nucleotide fragment or carrier of protein are also used as gene therapy form to apply.
The description of above embodiments and preferred embodiment belongs to of the invention the schematically illustrating to claim restriction,
Rather than the limitation present invention.It is important to note that without departing from spirit of the invention, all obvious changes with
And the similar inventions with equivalent substitution, it is all included in the scope of protection of the present invention.
Sequence table
<110>Sun Jialin
<120>a kind of derivative of anti-tumor protein Endostatin
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 69
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 1
atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60
gtgtttcgt 69
<210> 2
<211> 23
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 2
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg
20
<210> 3
<211> 549
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 3
cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120
gggctggcgg gcaccttccg cgccttcctg tcctcgcgcc tgcaggacct gtacagcatc 180
gtgcgccgtg ccgaccgcgc agccgtgccc atcgtcaacc tcaaggacga gctgctgttt 240
cccagctggg aggctctgtt ctcaggctct gagggtccgc tgaagcccgg ggcacgcatc 300
ttctcctttg acggcaagga cgtcctgagg caccccacct ggccccagaa gagcgtgtgg 360
catggctcgg accccaacgg gcgcaggctg accgagagct actgtgagac gtggcggacg 420
gaggctccct cggccacggg ccaggcctcc tcgctgctgg ggggcaggct cctggggcag 480
agtgccgcga gctgccatca cgcctacatc gtgctctgca ttgagaacag cttcatgact 540
gcctccaag 549
<210> 4
<211> 183
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 4
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala
35 40 45
Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
50 55 60
Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe
65 70 75 80
Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro
85 90 95
Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro
100 105 110
Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg
115 120 125
Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser
130 135 140
Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln
145 150 155 160
Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn
165 170 175
Ser Phe Met Thr Ala Ser Lys
180
<210> 5
<211> 990
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 5
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa 990
<210> 6
<211> 330
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 6
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 7
<211> 972
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60
gtgtttcgtc acagccaccg cgacttccag ccggtgctcc acctggttgc gctcaacagc 120
cccctgtcag gcggcatgcg gggcatccgc ggggccgact tccagtgctt ccagcaggcg 180
cgggccgtgg ggctggcggg caccttccgc gccttcctgt cctcgcgcct gcaggacctg 240
tacagcatcg tgcgccgtgc cgaccgcgca gccgtgccca tcgtcaacct caaggacgag 300
ctgctgtttc ccagctggga ggctctgttc tcaggctctg agggtccgct gaagcccggg 360
gcacgcatct tctcctttga cggcaaggac gtcctgaggc accccacctg gccccagaag 420
agcgtgtggc atggctcgga ccccaacggg cgcaggctga ccgagagcta ctgtgagacg 480
tggcggacgg aggctccctc ggccacgggc caggcctcct cgctgctggg gggcaggctc 540
ctggggcaga gtgccgcgag ctgccatcac gcctacatcg tgctctgcat tgagaacagc 600
ttcatgactg cctccaaggc ctccaccaag ggcccatcgg tcttccccct ggcaccctcc 660
tccaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 720
gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 780
gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 840
agcttgggca cccagaccta catctgcaac gtgaatcaca agcccagcaa caccaaggtg 900
gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca 960
cctgaactcc tg 972
<210> 8
<211> 324
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg His Ser His Arg Asp Phe Gln Pro Val
20 25 30
Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly
35 40 45
Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala Val Gly
50 55 60
Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln Asp Leu
65 70 75 80
Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile Val Asn
85 90 95
Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe Ser Gly
100 105 110
Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe Asp Gly
115 120 125
Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val Trp His
130 135 140
Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys Glu Thr
145 150 155 160
Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser Leu Leu
165 170 175
Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His Ala Tyr
180 185 190
Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys Ala Ser
195 200 205
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
210 215 220
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
225 230 235 240
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
245 250 255
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
260 265 270
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
275 280 285
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
290 295 300
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
305 310 315 320
Pro Glu Leu Leu
<210> 9
<211> 1293
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60
gtgtttcgtc acagccaccg cgacttccag ccggtgctcc acctggttgc gctcaacagc 120
cccctgtcag gcggcatgcg gggcatccgc ggggccgact tccagtgctt ccagcaggcg 180
cgggccgtgg ggctggcggg caccttccgc gccttcctgt cctcgcgcct gcaggacctg 240
tacagcatcg tgcgccgtgc cgaccgcgca gccgtgccca tcgtcaacct caaggacgag 300
ctgctgtttc ccagctggga ggctctgttc tcaggctctg agggtccgct gaagcccggg 360
gcacgcatct tctcctttga cggcaaggac gtcctgaggc accccacctg gccccagaag 420
agcgtgtggc atggctcgga ccccaacggg cgcaggctga ccgagagcta ctgtgagacg 480
tggcggacgg aggctccctc ggccacgggc caggcctcct cgctgctggg gggcaggctc 540
ctggggcaga gtgccgcgag ctgccatcac gcctacatcg tgctctgcat tgagaacagc 600
ttcatgactg cctccaaggc ctccaccaag ggcccatcgg tcttccccct ggcaccctcc 660
tccaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 720
gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 780
gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 840
agcttgggca cccagaccta catctgcaac gtgaatcaca agcccagcaa caccaaggtg 900
gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca 960
cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 1020
atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 1080
gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 1140
cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1200
gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 1260
atcgagaaaa ccatctccaa agccaaaggg cag 1293
<210> 10
<211> 431
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg His Ser His Arg Asp Phe Gln Pro Val
20 25 30
Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly
35 40 45
Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala Val Gly
50 55 60
Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln Asp Leu
65 70 75 80
Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile Val Asn
85 90 95
Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe Ser Gly
100 105 110
Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe Asp Gly
115 120 125
Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val Trp His
130 135 140
Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys Glu Thr
145 150 155 160
Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser Leu Leu
165 170 175
Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His Ala Tyr
180 185 190
Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys Ala Ser
195 200 205
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
210 215 220
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
225 230 235 240
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
245 250 255
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
260 265 270
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
275 280 285
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
290 295 300
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
305 310 315 320
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
325 330 335
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
340 345 350
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
355 360 365
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
370 375 380
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
385 390 395 400
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
405 410 415
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
420 425 430
<210> 11
<211> 1608
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60
gtgtttcgtc acagccaccg cgacttccag ccggtgctcc acctggttgc gctcaacagc 120
cccctgtcag gcggcatgcg gggcatccgc ggggccgact tccagtgctt ccagcaggcg 180
cgggccgtgg ggctggcggg caccttccgc gccttcctgt cctcgcgcct gcaggacctg 240
tacagcatcg tgcgccgtgc cgaccgcgca gccgtgccca tcgtcaacct caaggacgag 300
ctgctgtttc ccagctggga ggctctgttc tcaggctctg agggtccgct gaagcccggg 360
gcacgcatct tctcctttga cggcaaggac gtcctgaggc accccacctg gccccagaag 420
agcgtgtggc atggctcgga ccccaacggg cgcaggctga ccgagagcta ctgtgagacg 480
tggcggacgg aggctccctc ggccacgggc caggcctcct cgctgctggg gggcaggctc 540
ctggggcaga gtgccgcgag ctgccatcac gcctacatcg tgctctgcat tgagaacagc 600
ttcatgactg cctccaaggc ctccaccaag ggcccatcgg tcttccccct ggcaccctcc 660
tccaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 720
gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 780
gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 840
agcttgggca cccagaccta catctgcaac gtgaatcaca agcccagcaa caccaaggtg 900
gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca 960
cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 1020
atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 1080
gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 1140
cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1200
gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 1260
atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 1320
cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 1380
ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1440
aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 1500
gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 1560
ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa 1608
<210> 12
<211> 536
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 12
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg His Ser His Arg Asp Phe Gln Pro Val
20 25 30
Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly
35 40 45
Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala Val Gly
50 55 60
Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln Asp Leu
65 70 75 80
Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile Val Asn
85 90 95
Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe Ser Gly
100 105 110
Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe Asp Gly
115 120 125
Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val Trp His
130 135 140
Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys Glu Thr
145 150 155 160
Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser Leu Leu
165 170 175
Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His Ala Tyr
180 185 190
Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys Ala Ser
195 200 205
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
210 215 220
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
225 230 235 240
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
245 250 255
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
260 265 270
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
275 280 285
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
290 295 300
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
305 310 315 320
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
325 330 335
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
340 345 350
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
355 360 365
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
370 375 380
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
385 390 395 400
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
405 410 415
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
420 425 430
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
435 440 445
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
450 455 460
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
465 470 475 480
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
485 490 495
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
500 505 510
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
515 520 525
Ser Leu Ser Leu Ser Pro Gly Lys
530 535
<210> 13
<211> 1635
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60
gtgtttcgtg gggcctccaa cagcacgggg gcctcccaca gccaccgcga cttccagccg 120
gtgctccacc tggttgcgct caacagcccc ctgtcaggcg gcatgcgggg catccgcggg 180
gccgacttcc agtgcttcca gcaggcgcgg gccgtggggc tggcgggcac cttccgcgcc 240
ttcctgtcct cgcgcctgca ggacctgtac agcatcgtgc gccgtgccga ccgcgcagcc 300
gtgcccatcg tcaacctcaa ggacgagctg ctgtttccca gctgggaggc tctgttctca 360
ggctctgagg gtccgctgaa gcccggggca cgcatcttct cctttgacgg caaggacgtc 420
ctgaggcacc ccacctggcc ccagaagagc gtgtggcatg gctcggaccc caacgggcgc 480
aggctgaccg agagctactg tgagacgtgg cggacggagg ctccctcggc cacgggccag 540
gcctcctcgc tgctgggggg caggctcctg gggcagagtg ccgcgagctg ccatcacgcc 600
tacatcgtgc tctgcattga gaacagcttc atgactgcct ccaaggcctc caccaagggc 660
ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 720
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 780
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 840
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 900
aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 960
actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 1020
ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 1080
gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 1140
gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 1200
gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1260
gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1320
ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 1380
gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1440
agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1500
tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1560
ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1620
ctgtctccgg gtaaa 1635
<210> 14
<211> 545
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 14
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg Gly Ala Ser Asn Ser Thr Gly Ala Ser
20 25 30
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
35 40 45
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
50 55 60
Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala
65 70 75 80
Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
85 90 95
Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe
100 105 110
Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro
115 120 125
Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro
130 135 140
Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg
145 150 155 160
Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser
165 170 175
Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln
180 185 190
Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn
195 200 205
Ser Phe Met Thr Ala Ser Lys Ala Ser Thr Lys Gly Pro Ser Val Phe
210 215 220
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
225 230 235 240
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
245 250 255
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
260 265 270
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
275 280 285
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
290 295 300
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
305 310 315 320
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
325 330 335
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
340 345 350
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
355 360 365
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
370 375 380
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
385 390 395 400
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
405 410 415
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
420 425 430
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
435 440 445
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
450 455 460
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
465 470 475 480
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
485 490 495
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
500 505 510
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
515 520 525
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
530 535 540
Lys
545
<210> 15
<211> 1635
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60
gtgtttcgtc acagccaccg cgacttccag ccggtgctcc acctggttgc gctcaacagc 120
cccctgtcag gcggcatgcg gggcatccgc ggggccgact tccagtgctt ccagcaggcg 180
cgggccgtgg ggctggcggg caccttccgc gccttcctgt cctcgcgcct gcaggacctg 240
tacagcatcg tgcgccgtgc cgaccgcgca gccgtgccca tcgtcaacct caaggacgag 300
ctgctgtttc ccagctggga ggctctgttc tcaggctctg agggtccgct gaagcccggg 360
gcacgcatct tctcctttga cggcaaggac gtcctgaggc accccacctg gccccagaag 420
agcgtgtggc atggctcgga ccccaacggg cgcaggctga ccgagagcta ctgtgagacg 480
tggcggacgg aggctccctc ggccacgggc caggcctcct cgctgctggg gggcaggctc 540
ctggggcaga gtgccgcgag ctgccatcac gcctacatcg tgctctgcat tgagaacagc 600
ttcatgactg cctccaaggg ggcctccaac agcacggggg cctccgcctc caccaagggc 660
ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 720
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 780
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 840
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 900
aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 960
actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 1020
ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 1080
gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 1140
gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 1200
gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1260
gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1320
ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 1380
gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1440
agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1500
tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1560
ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1620
ctgtctccgg gtaaa 1635
<210> 16
<211> 545
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 16
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg His Ser His Arg Asp Phe Gln Pro Val
20 25 30
Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly
35 40 45
Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala Val Gly
50 55 60
Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln Asp Leu
65 70 75 80
Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile Val Asn
85 90 95
Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe Ser Gly
100 105 110
Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe Asp Gly
115 120 125
Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val Trp His
130 135 140
Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys Glu Thr
145 150 155 160
Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser Leu Leu
165 170 175
Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His Ala Tyr
180 185 190
Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys Gly Ala
195 200 205
Ser Asn Ser Thr Gly Ala Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
210 215 220
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
225 230 235 240
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
245 250 255
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
260 265 270
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
275 280 285
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
290 295 300
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
305 310 315 320
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
325 330 335
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
340 345 350
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
355 360 365
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
370 375 380
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
385 390 395 400
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
405 410 415
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
420 425 430
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
435 440 445
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
450 455 460
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
465 470 475 480
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
485 490 495
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
500 505 510
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
515 520 525
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
530 535 540
Lys
545
<210> 17
<211> 1662
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60
gtgtttcgtg gggcctccaa cagcacgggg gcctcccaca gccaccgcga cttccagccg 120
gtgctccacc tggttgcgct caacagcccc ctgtcaggcg gcatgcgggg catccgcggg 180
gccgacttcc agtgcttcca gcaggcgcgg gccgtggggc tggcgggcac cttccgcgcc 240
ttcctgtcct cgcgcctgca ggacctgtac agcatcgtgc gccgtgccga ccgcgcagcc 300
gtgcccatcg tcaacctcaa ggacgagctg ctgtttccca gctgggaggc tctgttctca 360
ggctctgagg gtccgctgaa gcccggggca cgcatcttct cctttgacgg caaggacgtc 420
ctgaggcacc ccacctggcc ccagaagagc gtgtggcatg gctcggaccc caacgggcgc 480
aggctgaccg agagctactg tgagacgtgg cggacggagg ctccctcggc cacgggccag 540
gcctcctcgc tgctgggggg caggctcctg gggcagagtg ccgcgagctg ccatcacgcc 600
tacatcgtgc tctgcattga gaacagcttc atgactgcct ccaagggggc ctccaacagc 660
acgggggcct ccgcctccac caagggccca tcggtcttcc ccctggcacc ctcctccaag 720
agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 780
gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 840
ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 900
ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 960
aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 1020
ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 1080
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1140
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 1200
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1260
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1320
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1380
tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1440
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1500
acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1560
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1620
aaccactaca cgcagaagag cctctccctg tctccgggta aa 1662
<210> 18
<211> 554
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 18
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg Gly Ala Ser Asn Ser Thr Gly Ala Ser
20 25 30
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
35 40 45
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
50 55 60
Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala
65 70 75 80
Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
85 90 95
Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe
100 105 110
Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro
115 120 125
Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro
130 135 140
Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg
145 150 155 160
Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser
165 170 175
Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln
180 185 190
Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn
195 200 205
Ser Phe Met Thr Ala Ser Lys Gly Ala Ser Asn Ser Thr Gly Ala Ser
210 215 220
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
225 230 235 240
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
245 250 255
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
260 265 270
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
275 280 285
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
290 295 300
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
305 310 315 320
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
325 330 335
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
340 345 350
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
355 360 365
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
370 375 380
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
385 390 395 400
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
405 410 415
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
420 425 430
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
435 440 445
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
450 455 460
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
465 470 475 480
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
485 490 495
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
500 505 510
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
515 520 525
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
530 535 540
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
545 550
<210> 19
<211> 1662
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60
gtgtttcgtg gggcctccaa cagcacgggg gcctcccaca gccaccgcga cttccagccg 120
gtgctccacc tggttgcgct caacagcccc ctgtcaggcg gcatgcgggg catccgcggg 180
gccgacttcc agtgcttcca gcaggcgcgg gccgtggggc tggcgggcac cttccgcgcc 240
ttcctgtcct cgcgcctgca ggacctgtac agcatcgtgc accgtgccga ccgcgcagcc 300
gtgcccatcg tcaacctcaa ggacgagctg ctgtttccca gctgggaggc tctgttctca 360
ggctctgagg gtccgctgaa gcccggggca cgcatcttct cctttgacgg caaggacgtc 420
ctgaggcacc ccacctggcc ccagaagagc gtgtggcatg gctcggaccc caacgggcac 480
aggctgaccg agagctactg tgagacgtgg cggacggagg ctccctcggc cacgggccag 540
gcctcctcgc tgctgggggg caggctcctg gggcagagtg ccgcgagctg ccatcacgcc 600
tacatcgtgc tctgcattga gaacagcttc atgactgcct ccaagggggc ctccaacagc 660
acgggggcct ccgcctccac caagggccca tcggtcttcc ccctggcacc ctcctccaag 720
agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 780
gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 840
ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 900
ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 960
aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 1020
ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 1080
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1140
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 1200
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1260
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1320
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1380
tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1440
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1500
acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1560
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1620
aaccactaca cgcagaagag cctctccctg tctccgggta aa 1662
<210> 20
<211> 554
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 20
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg Gly Ala Ser Asn Ser Thr Gly Ala Ser
20 25 30
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
35 40 45
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
50 55 60
Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala
65 70 75 80
Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val His Arg Ala
85 90 95
Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe
100 105 110
Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro
115 120 125
Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro
130 135 140
Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly His
145 150 155 160
Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser
165 170 175
Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln
180 185 190
Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn
195 200 205
Ser Phe Met Thr Ala Ser Lys Gly Ala Ser Asn Ser Thr Gly Ala Ser
210 215 220
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
225 230 235 240
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
245 250 255
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
260 265 270
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
275 280 285
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
290 295 300
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
305 310 315 320
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
325 330 335
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
340 345 350
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
355 360 365
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
370 375 380
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
385 390 395 400
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
405 410 415
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
420 425 430
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
435 440 445
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
450 455 460
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
465 470 475 480
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
485 490 495
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
500 505 510
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
515 520 525
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
530 535 540
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
545 550
Claims (3)
1. a kind of derivative of anti-tumor protein Endostatin, the amino acid sequence SEQ.ID.NO4 institute of the Endostatin
Show；It is characterized in that point mutation occurs for the one of Arg of the Arg128Arg129 of the derivative amino acid sequence.
2. the derivative of anti-tumor protein Endostatin according to claim 1, it is characterized in that point mutation occurs for the Arg
For His.
3. the derivative of the anti-tumor protein Endostatin of claims 1 or 2 is in the application for preparing anticancer drug.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910639670.0A CN110317264B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510761800.XA CN106674352B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin and application thereof |
| CN201910639670.0A CN110317264B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin |
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| CN201510761800.XA Division CN106674352B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin and application thereof |
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| CN110317264A true CN110317264A (en) | 2019-10-11 |
| CN110317264B CN110317264B (en) | 2022-10-11 |
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| CN201510761800.XA Active CN106674352B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin and application thereof |
| CN201910639876.3A Active CN110452295B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin |
| CN201910639670.0A Active CN110317264B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin |
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| CN201510761800.XA Active CN106674352B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin and application thereof |
| CN201910639876.3A Active CN110452295B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin |
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|---|---|---|---|---|
| WO2000058498A1 (en) * | 1999-03-30 | 2000-10-05 | Merck & Co., Inc. | Soluble recombinant endostatin |
| US20040043924A1 (en) * | 1996-10-15 | 2004-03-04 | Narhi Linda Owens | Uses of keratinocyte growth factor-2 |
| CN1884556A (en) * | 2006-06-22 | 2006-12-27 | 江苏舜唐生物工程有限公司 | Tumour-dissolving adenovirus mutant possessing multiple specific anti-tumour mechanism |
| CN101062954A (en) * | 2006-05-16 | 2007-10-31 | 中国人民解放军军事医学科学院野战输血研究所 | Fusion protein having blood vessel formation against function and its coding gene and application |
| CA2848118A1 (en) * | 2011-09-09 | 2013-03-14 | Tsinghua University | Endostatin mutants with mutations at atp binding sites |
| US20140220016A1 (en) * | 2007-06-26 | 2014-08-07 | University Of Miami | Antibody- endostatin fusion protein and its variants |
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| JP4441112B2 (en) * | 1997-11-03 | 2010-03-31 | ヒューマン ジノーム サイエンシーズ， インコーポレイテッド | VEGI is an inhibitor of angiogenesis and tumor growth |
| CN1354186A (en) * | 2000-11-30 | 2002-06-19 | 辽宁卫星生物制品研究所(有限公司) | Preparation method of recombinant human vascular endothelial cytopoiesis suppressor factor with human LgG1Fc fragment molecular structure and application of its product |
| AU2003244516A1 (en) * | 2002-02-07 | 2003-09-02 | Novozymes Delta Limited | Hiv inhibiting proteins |
| CN102816241B (en) * | 2004-02-09 | 2015-07-22 | 人类基因科学公司 | Albumin fusion proteins |
| PT2195023T (en) * | 2007-08-29 | 2018-06-08 | Sanofi Sa | Humanized anti-cxcr5 antibodies, derivatives thereof and their uses |
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| CN101265298B (en) * | 2008-04-30 | 2011-11-09 | 中国药科大学 | Endothelium chalone mutant containing non-natural amino acid and derivatives thereof |
| TWI672151B (en) * | 2008-05-02 | 2019-09-21 | 艾西利羅製藥公司 | Methods and compositions for modulating angiogenesis and pericyte composition |
| HUE040205T2 (en) * | 2010-07-13 | 2019-02-28 | Georgia State Univ Research Foundation | Anti-angiogenic agent and method of using such agent |
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| CN102731658B (en) * | 2012-05-15 | 2014-07-30 | 山东大学 | Tat PTD-Endostatin recombination protein, preparation method and application thereof |
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- 2015-11-10 CN CN201910639692.7A patent/CN110498851B/en active Active
- 2015-11-10 CN CN201510761800.XA patent/CN106674352B/en active Active
- 2015-11-10 CN CN201910639876.3A patent/CN110452295B/en active Active
- 2015-11-10 CN CN201910639670.0A patent/CN110317264B/en active Active
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| US20040043924A1 (en) * | 1996-10-15 | 2004-03-04 | Narhi Linda Owens | Uses of keratinocyte growth factor-2 |
| WO2000058498A1 (en) * | 1999-03-30 | 2000-10-05 | Merck & Co., Inc. | Soluble recombinant endostatin |
| CN101062954A (en) * | 2006-05-16 | 2007-10-31 | 中国人民解放军军事医学科学院野战输血研究所 | Fusion protein having blood vessel formation against function and its coding gene and application |
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| US20140220016A1 (en) * | 2007-06-26 | 2014-08-07 | University Of Miami | Antibody- endostatin fusion protein and its variants |
| CA2848118A1 (en) * | 2011-09-09 | 2013-03-14 | Tsinghua University | Endostatin mutants with mutations at atp binding sites |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110452295B (en) | 2022-10-11 |
| CN110498851A (en) | 2019-11-26 |
| CN110317264B (en) | 2022-10-11 |
| CN110452295A (en) | 2019-11-15 |
| CN110498851B (en) | 2022-10-11 |
| CN106674352A (en) | 2017-05-17 |
| CN106674352B (en) | 2020-05-15 |
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Effective date of registration: 20201103 Address after: 200237 Shanghai City, Xuhui District City Road 700 Lane 66, Room 201 Applicant after: Sun Jialin Applicant after: Nanjing double bond targeted drug Technology Co.,Ltd. Address before: 200237 Shanghai City, Xuhui District City Road 700 Lane 66, Room 201 Applicant before: Sun Jialin |
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