CN110498851B - Derivative of antineoplastic protein endostatin and application thereof - Google Patents
Derivative of antineoplastic protein endostatin and application thereof Download PDFInfo
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Abstract
The invention discloses a derivative of an antitumor protein endostatin and application thereof, wherein the amino acid sequence of the endostatin is shown by SEQ ID NO. 4; the derivative introduces amino acid short peptide of glycosylation sites in front of the N end of endostatin or in front of the N end and behind the C end. The derivative of the antitumor protein endostatin has the advantages of stability in vivo, long half-life period and high antitumor bioactivity.
Description
Technical Field
The invention relates to a derivative of an antitumor protein endostatin and application thereof, belonging to the field of biological medicine antitumor drugs.
Background
Tumor growth depends on the formation of blood vessels, which are regulated by angiogenic growth factors and suppressive factors, and inhibition of tumor vascular growth is an effective method for treating tumors. Endostatin (Endostatin) has the function of inhibiting angiogenesis, so it is regarded as important in the anti-angiogenesis therapy of tumors. Endostatin is a C-terminal 184-amino acid fragment generated by hydrolysis of collagen XVIII, can effectively inhibit various tumors, has broad biological effects and no drug resistance, and is the best angiogenesis inhibitor discovered so far (Exp Cell Res,312,594-607, 2006).
However, in monkey experiments and human clinical trials, it was found that endostatin has a very short half-life and exhibits exponential degradation, and the in vivo content of endostatin decreases by hundreds of times or even approaches zero before and after 10 hours (Acta Pharmacol Sin,26,124-128,2005 j Clin oncol,20,3792-3803, 2002), which indicates that it is very unstable in animals, and this will greatly affect the anti-tumor therapeutic effect of endostatin.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the derivative of the antitumor protein endostatin with long half-life and high antitumor bioactivity.
The second purpose of the invention is to provide the application of the derivative of the antitumor protein endostatin in preparing the anticancer drugs.
The third purpose of the invention is to provide the application of the DNA segment for coding the derivative of the endostatin in preparing the antineoplastic medicine.
A derivative of antitumor protein endostatin, wherein the amino acid sequence of the endostatin is shown in SEQ.ID.NO4; the derivatives include: endostatin is linked to the antibody IgG invariant region; or Arg is introduced in front of the N end of endostatin; or an amino acid short peptide for introducing glycosylation sites in front of the N end, or behind the C end, or in front of the N end and behind the C end of endostatin; or the amino acid sequence of endostatin has a point mutation at one of Arg62Arg63, or at least one of Arg128Arg129, or at the same time as a point mutation at one of Arg62Arg63 and at the same time as a point mutation at one of Arg128Arg129. Or the endostatin is connected with an IgG invariant region, arg is introduced in front of the N end of the endostatin, amino acid short peptide of glycosylation sites is introduced in front of the N end, or behind the C end, or in front of the N end and behind the C end of the endostatin, and one Arg of Arg62Arg63, or one Arg of Arg128Arg129, or one Arg of Arg62Arg63 and Arg129 of the amino acid sequence of the endostatin are subjected to point mutation while one Arg of Arg128Arg129 is subjected to point mutation.
The IgG invariant region is CH1, CH1CH2 or CH1CH2CH3, and the amino acid sequence of the endostatin connected with CH1 is SEQ.ID.NO8; the amino acid sequence of the endostatin connected with CH1CH2 is SEQ.ID.NO10; the amino acid sequence of the endostatin connected with CH1CH2CH3 is SEQ.ID.NO12.
The Arg is introduced in front of the N end of endostatin, a short peptide is connected in front of the N end of endostatin, and the short peptide contains more than one Arg.
The glycosylation site is Asn-X-Ser or Asn-X-Thr, wherein X is other amino acid.
Point mutation of Arg to His.
The amino acid sequence of the derivative of the antitumor protein endostatin is best shown in SEQ.ID.NO20.
The nucleic acid sequence of the derivative of the antitumor protein endostatin of claim 6 is shown in SEQ.ID. NO19.
The derivative of the antitumor protein endostatin is applied to the preparation of anticancer drugs.
The DNA segment of the derivative of the antitumor protein endostatin is coded and applied to the preparation of anticancer drugs.
THE ADVANTAGES OF THE PRESENT INVENTION
The anti-tumor protein endostatin derivative of the invention has the advantages of stability in vivo, long half life and high anti-tumor bioactivity.
Drawings
FIG. 1 is a graph showing the results of the experiment of the antitumor activity of the fusion protein constructed by the pro-sequence endostatin of example 1 linked to the IgG constant region CH1, the constant region CH1CH2, and the constant region CH1CH2CH3, respectively. In the figure, 1: physiological saline; 2: pro sequence-endostatin; 3: a preposed sequence-endostatin-antibody IgG part invariant region CH1 fusion protein; 4: a leader sequence-endostatin-antibody IgG part constant region CH1CH2 fusion protein; 5: the leader sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion protein.
FIG. 2 is the experiment of the half-life of the pro-sequence endostatin of example 1 in mice, which is a fusion protein constructed by respectively connecting the antibodies IgG constant CH1, CH1CH2 and CH1CH2CH3, and endostatin. The sample in the figure (top to bottom), \ 9679; leader sequence-endostatin-antibody IgG constant region CH1CH2CH3 fusion protein; tangle-solidup preposed sequence-endostatin-antibody IgG part invariant domain CH1CH2 fusion protein; ■ A leader sequence-endostatin-antibody IgG part constant region CH1 fusion protein; preposition-endostatin
FIG. 3 is an experiment of point mutation of Arg in the short N-terminal pro-endostatin peptide in the pro sequence-endostatin-antibody IgG invariant region fusion protein of example 2. In the figure: sample 1: a pre-sequence-endostatin-antibody IgG invariant region fusion protein; sample 2: point mutant 1 at the N-terminal of the presequence-endostatin-antibody IgG invariant region fusion protein; sample 3: point mutant 2 at the N-terminal of the preposed sequence-endostatin-antibody IgG invariant region fusion protein; sample 4: point mutant 3 at the N-terminal of the presequence-endostatin-antibody IgG invariant region fusion protein; sample 5: saline (control).
FIG. 4 is an experiment of the antitumor activity of the pro-sequence-endostatin-antibody IgG constant region fusion protein introduced with glycosylation site short peptide of example 3. Sample 1: a pre-sequence-endostatin-antibody IgG constant region fusion protein (SEQ ID No. 12); sample 2: pro-sequence-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 1 (SEQ ID No. 14); sample 3: leader sequence-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 2 (SEQ ID No. 16); sample 4: pro-sequence-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 3 (SEQ ID NO. 18)
FIG. 5 is an experiment of the half-life of the pro-sequence-endostatin-antibody IgG constant region fusion protein introduced with glycosylated short peptide of example 3 in mice. Sample (top to bottom): ● Pro-sequence-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 3 (SEQ ID No. 18); a tangle-solidup preposition-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 2 (SEQ ID NO. 16); ■ Leader sequence-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 1 (SEQ ID No. 14); preposition-endostatin-antibody IgG invariant region fusion protein (SEQ ID No. 12).
FIG. 6 is an experiment on the antitumor activity of various point mutants in which the pro-sequence-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 2 (SEQ ID NO.15 and SEQ ID NO. 16) of example 4 was further subjected to point mutation at the introduced glycosylation site (Asn 210-Ser211-Thr 212). Sample 1: point mutant Asn210-Ser211-Thr212; sample 2: point mutant Asn210-Ala211-Thr212; sample 3: point mutant Asn210-Tyr211-Thr212; sample 4: point mutant Asn210-Ser211-Ser212; sample 5: point mutant Asn210-Ala211-Ser212; sample 6: the fusion protein of the preposed sequence-endostatin-antibody IgG invariant region without introducing glycosylation sites.
FIG. 7 is an experiment of the half-life of various point mutant fusion proteins in FIG. 6 of example 4 in mice. Sample (top to bottom) x-point mutant (Asn 210-Tyr211-Thr 212); + point mutant (Asn 210-Ser211-Ser 212); ■ Point mutants (Asn 210-Ser211-Thr 212); a point tangle-solidup mutant (Asn 210-Ala211-Thr 212); ● Point mutant (Asn 210-Ala211-Ser 212); no glycosylation site-introduced pro-sequence-endostatin-antibody IgG invariant region fusion proteins.
FIG. 8 is an experiment of antitumor activity of various point mutants point-mutated at Arg85-Arg86 positions using the pro-sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion proteins (SEQ ID NO.11 and SEQ ID NO. 12) of example 5 as experimental material. 1: point mutant 1 (Ala 85-Arg 86); 2: point mutant 2 (Ser 85-Arg 86); 3: point mutant 3 (His 85-Arg 86); 4: point mutant 4 (Arg 85-Ala 86); 5: point mutant 5 (Arg 85-Ser 86); 6: point mutant 6 (Arg 85-His 86); 7: no mutant fusion protein (SEQ ID NO. 12) was present.
FIG. 9 is an experiment of the in vivo half-life of various point mutant fusion proteins in FIG. 8 of example 5 in mice. Sample (top to bottom), + point mutant 6 (Arg 85-His 86); point x mutant 3 (His 85-Arg 86); a point tangle-solidup mutant 2 (Ser 85-Arg 86); ● Point mutant 5 (Arg 85-Ser 86); ■ Point mutant 1 (Ala 85-Arg 86); * Point mutant 2 (Ser 85-Arg 86); no mutant fusion protein (SEQ ID NO. 12) was present.
FIG. 10 is an experiment of antitumor activity of various point mutants point-mutated at Arg151-Arg152 positions, using the pro-sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion proteins (SEQ ID NO.11 and SEQ ID NO. 12) of example 5 as experimental material. 1: point mutant 7 (Ala 151-Arg 152); 2: point mutant 8 (Ser 151-Arg 152); 3: point mutant 9 (His 151-Arg 152); 4: point mutant 10 (Arg 151-Ala 152); 5: point mutant 11 (Arg 151-Ser 152); 6: point mutant 12 (Arg 151-His 152); 7: there was no mutant fusion protein (SEQ ID NO. 12).
FIG. 11 is an experiment of the in vivo half-life of various point mutant fusion proteins in FIG. 10 of example 5 in mice. Sample (top to bottom), point x mutant 9 (His 151-Arg 152); + point mutant 12 (Arg 151-His 152); * Point mutant 10 (Arg 151-Ala 152); ■ Point mutant 7 (Ala 151-Arg 152); point tangle-solidup mutant 8 (Ser 151-Arg 152); ● Point mutant 11 (Arg 151-Ser 152); no mutant fusion protein (SEQ ID NO. 12) was present.
Fig. 12 is an experiment of the antitumor activity of the engineered fusion proteins of group 2 argargargarg within endostatin with glycosylation sites added before the N-terminus and after the C-terminus of endostatin of example 6. Sample 1: a pre-sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion protein (SEQ ID No. 12); sample 2: leader sequence-endostatin-antibody IgG invariant domain CH1CH2CH3 fusion protein mutant 1 (His 85-Arg86 and His151-Arg 152); sample 3: leader sequence-endostatin-antibody IgG invariant domain CH1CH2CH3 fusion protein mutant 2 (Arg 85-His86 and Arg151-His 152); sample 4: pro sequence-endostatin (glycosylation sites added before the N-terminus and after the C-terminus), antibody IgG invariant CH1CH2CH3 fusion protein (SEQ ID No. 18); sample 5: pre-sequence-endostatin (glycosylation sites added before the N-terminus and after the C-terminus, respectively) -antibody IgG invariant domain CH1CH2CH3 fusion protein spot mutant 1 (Arg 94His95 and Arg160His 161); sample 6: pro sequence-endostatin (glycosylation sites added before the N-terminus and after the C-terminus, respectively) -antibody IgG invariant domain CH1CH2CH3 fusion protein site mutant 2 (His 94Arg95 and His160Arg 161) (SEQ ID No. 20).
FIG. 13 is an experiment of the in vivo half-life in mice of the fusion proteins of the various mutants in FIG. 12 of example 6. Sample (top to bottom) \9679; endostatin fusion protein dot mutant 2 (His 94Arg95 and His160Arg 161) with glycosylation sites added before the N-terminus and after the C-terminus (SEQ ID NO. 20); + a fusion protein point mutant 1 (Arg 94His95 and Arg160His 161) with glycosylation sites added before the N-terminus and after the C-terminus of endostatin; ■ Presequence-endostatin-antibody IgG invariant domain CH1CH2CH3 fusion protein mutant 1 (His 85-Arg86 and His151-Arg 152); x a fusion protein (SEQ ID NO. 18) in which glycosylation sites are added before the N-terminus and after the C-terminus of endostatin; tangle-solidup preposition-endostatin-antibody IgG invariant region CH1CH2CH3 fusion egg spot mutant 2 (Arg 85-His86 and Arg151-His 152); preposition-endostatin-antibody IgG invariant CH1CH2CH3 fusion protein (SEQ ID No. 12).
Fig. 14 is a photograph of mouse anatomy of a tumor model, in which 1: saline-injected mice as controls in example 1; 2: mice injected with the fusion protein of sample 6 (fusion protein spot mutant 2 added with glycosylation sites before the N-terminus and after the C-terminus of endostatin) (SEQ ID No. 20) in example 6; the arrows indicate the tumor.
FIG. 15 is an experiment of the endostatin and various endostatin derivatives of example 7 against tumors. 1: endostatin; 2: pro sequence-endostatin protein (2 args in front of the N-terminus of endostatin); 3: glycosylation site short peptide-endostatin-glycosylation site short peptide protein; 4: endostatin with point mutations (His 62Arg63 and His128Arg 129); 5: endostatin-antibody IgG invariant region fusion proteins; 6: pre-sequence-endostatin (glycosylation sites are added in front of the N end and behind the C end respectively) -antibody IgG invariant region fusion protein point mutant 2 (His 94Arg95 and His160Arg 161) (SEQ ID NO. 20); 7: physiological saline.
Figure 16 is an experiment showing the in vivo half-lives of endostatin and various endostatin derivatives of figure 15 of example 7 in mice. Sample (from top to bottom) \ 9679; preamble sequence-endostatin (glycosylation sites added before the N-terminus and after the C-terminus, respectively) -antibody IgG invariant region fusion protein dot mutant 2 (His 94Arg95 and His160Arg 161) (SEQ ID NO. 20); + an endostatin-antibody IgG invariant region fusion protein; tangle-solidup glycosylation site short peptide-endostatin-glycosylation site short peptide protein; endostatin with point mutation of x (His 62Arg63 and His128Arg 129); ■ Pro sequence-endostatin protein (2 args in front of the N-terminus of endostatin); endostatin.
Detailed Description
The present invention will be further illustrated by the following specific examples.
The first modification method comprises the following steps: the endostatin derivative comprises a fusion protein constructed by connecting endostatin and an antibody constant region, and the invention connects endostatin and the antibody constant region, in particular to the constant region CH1, CH1CH2 or CH1CH2CH3 of an IgG antibody. Meanwhile, a preposed sequence is connected in front of endostatin, and consists of a signal peptide of human albumin and a short peptide of 5 amino acids at the N end of the mature albumin, wherein the signal peptide is used for secreting the expressed fusion protein to the outside of animal cells. Thus, the half-life of endostatin derivatives is increased by increasing the molecular weight of the protein.
And a second modification method: the endostatin derivative is prepared by taking a preposed sequence-endostatin-IgG antibody constant region CH1CH2CH3 fusion protein as an experimental material, introducing arginine (Arg) in front of the N end of endostatin, and enhancing the anti-tumor biological activity of endostatin by additional Arg.
The transformation method comprises the following steps: the endostatin derivative is prepared by taking a preposed sequence-endostatin-IgG antibody invariant region CH1CH2CH3 fusion protein as an experimental material and introducing glycosylation sites in front of or behind endostatin. Sugar chains of proteins of eukaryotes including animals occur at Asn-X-Ser/Thr position, where X is an amino acid other than Pro, asp, or Glu, and the sugar chain is attached to Asn. In order to improve the half-life of the preposition sequence-endostatin-IgG antibody invariant region CH1CH2CH3 fusion protein, the invention introduces glycosylation sites in the preposition or behind the C-terminal of endostatin in the preposition sequence-endostatin-IgG antibody invariant region CH1CH2CH3 fusion protein or introduces glycosylation sites in front of the N-terminal and behind the C-terminal at the same time, the amino acid of the selected glycosylation site is AsnSerThr, and an additional short peptide GlyAlaSeaSer is added in front of and behind the site, so that the final structure is GlyAlaSerAsnSerThrGlyAlaS, wherein AsnSerThr is the glycosylation position.
The modification method comprises the following steps: the endostatin derivative is prepared by taking a preposed sequence-endostatin-IgG antibody invariant domain CH1CH2CH3 fusion protein as an experimental material to modify 2 groups of ArgArg (Arg 62Arg63 and Arg128Arg 129) in endostatin (SEQ ID NO. 4). Basic amino acids are often substrates for proteases, and the present invention retains only one Arg in group 2 ArgArg to improve endostatin stability. Although Arg is thought to be important for heparin binding, such modification helps to increase endostatin's biological activity in order to reduce the amount of endostatin hydrolyzed by proteases.
Basic information on nucleic acid and amino acid sequences of various proteins
SEQ ID NO.2 is a sequence of the signal peptide of human albumin and the N-terminal 5 amino acids of mature human albumin, wherein 1-18 are signal peptides, 19-23 are the N-terminal 5 amino acids of mature human albumin, SEQ ID NO.1 is the nucleic acid coding sequence of SEQ ID NO.2, the information in the GenBank database is NP-000468.1 and NM-000477.5, and the sequence of SEQ ID NO.2 is referred to as a preamble.
SEQ ID NO.4 is the amino acid sequence of endostatin of human origin, SEQ ID NO.3 is its nucleic acid coding sequence, and the information in the GenBank database is NP-569712.2 and NM-130445.3, where endostatin has a total of 183 amino acids.
SEQ ID NO.6 is the amino acid sequence of the IgG invariant region of a human antibody, SEQ ID NO.5 is its nucleic acid coding sequence, and the information in the GenBank database is M87789.1 in which, based on the amino acid sequence, 1 to 118 are CH1 fragments, 119 to 225 are CH2 fragments, and 226 to 330 are CH3 fragments.
Experimental materials and methods
The animal Expression Vector used was pHEK293Ultra Expression Vector II from Takara, which contains the insertion sites of foreign gene fragments, i.e., restriction endonuclease sites SmaI-XhoI-BamHI-XbaI-SalI-PstI-SphI-HindIII, wherein the insertion sites were selected from XhoI-BamHI-XbaI and SphI-HindIII, i.e., the gene fragment contained XhoI-BamHI-XbaI at the front end and SphI-HindIII at the rear end, and further, a His-tag and a termination signal tag, i.e., a cathcatcatcattag, were added to the front of SphI-HindIII. The construction of the expression vector was completed by selection in E.coli and then transfected into HEK293 cells for transient expression of various proteins. The synthesis of gene fragments in these vectors is carried out by Takara Shuzo Co., ltd., shanghai bioengineering Co., ltd.
The medium for HEK293 cells was DMEM medium with 10% fetal bovine serum.
For the isolation and Purification of proteins, since proteins possess signal peptides, the expression is secretory, and the isolation and Purification of proteins is performed by the His-tag method, i.e., centrifugation of a medium to remove cells, followed by Purification of the supernatant using the His Bind Purification Kit (Novagen Co.), and Binding Buffer is added before the column, followed by loading of the protein sample on the column, washing with Wash Buffer, and elution with Elute Buffer. If the fusion Protein contains an IgG-invariant region, a Protein A column, i.e., hiTrap Protein A HP or HiTrap rProtein A FF from GE Healthcare, may be used, and PBS may be used as a rinsing solution and 0.1M citric acid (pH 2.7) may be used as an eluent. The proteins obtained by His-tag or ProteinA column method were identified by protein electrophoresis, and the protein with one band purity was used in the following experiment.
Regarding the tumor model, the mice are C57BL/6, about 20-24 g, the number of mice in each experiment is 30, the tumor cells are mouse lung cancer Lewis Luccinoma (LLC) cell lines, LLC cells are cultured in vitro, then injected into the abdominal cavity of the C57BL/6 mice for large-scale culture, and finally, the LLC tumor cells in the abdominal cavity are taken out, and 2x10 cells are mixed 6 Tumor cells were inoculated in the right underarm of the mice.
The biological activity of the protein was evaluated by the tumor growth inhibition rate in mice and the half-life in mice, and the protein dose was 50pmol.
Example 1 a derivative of endostatin, an anti-tumor protein, which comprises a fusion protein of endostatin linked to an IgG invariant region, its anti-tumor activity and the half-life of the fusion protein in mice.
Protein sample
Sample 1: pro sequence-endostatin; the front of endostatin shown in SEQ ID NO.4 is connected with a preposed sequence shown in SEQ ID NO.2, and a signal peptide in the preposed sequence is used for ensuring that the expressed endostatin can be secreted out of cells.
Sample 2: the fusion protein of a preposed sequence-endostatin-IgG part constant region CH1 (shown in SEQ ID NO.8, and the nucleic acid sequence for coding the SEQ ID NO.8 is shown in SEQ ID NO. 7).
Sample 3: the leader sequence-endostatin-IgG part constant region CH1CH2 fusion protein (shown in SEQ ID NO.10, and the nucleic acid sequence for coding the SEQ ID NO.10 is shown in SEQ ID NO. 9).
Sample 4: the fusion protein of a preposed sequence-endostatin-IgG invariant region CH1CH2CH3 (shown in SEQ ID NO.12, and the nucleic acid sequence for coding the SEQ ID NO.12 is shown in SEQ ID NO. 11).
The above gene synthesis fragment was inserted into pHEK293Ultra Expression Vector II plasmid,
The control sample was saline injection.
For the half-life experiment of the protein in mice, the mice were injected with various proteins (100 ug/0.2 ml) at one time, and the content of endostatin or endostatin fusion protein was detected by using anti-endostatin antiserum, and the detection on day 1 was performed within 2 hours after the injection for 13 days continuously. FIG. 2 shows the change in the content of each protein in mice over 13 days, and the results show that the larger the molecular weight, the higher the content in vivo.
The effect of tumor inhibition is best because the pro-sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion protein is more stable in vivo.
Example 2 evaluation of endostatin derivatives, i.e., a newly introduced Arg ahead of the N-terminus of endostatin
The presequence-endostatin-antibody IgG invariant region fusion proteins (SEQ ID No.11 and SEQ ID No. 12) of sample 4 of example 1 were selected as experimental materials and are illustrated by the amino acid sequence (SEQ ID No. 12), with 1-23 amino acids derived from albumin, 1-18 being the signal peptide of albumin, 19-23 being the 5 amino acids at the N-terminus of mature albumin, two of which are Arg,24-206 being endostatin, and 207-536 being the antibody IgG invariant region, which consists of CH1, CH2, and CH3. Various experimental proteins:
sample 1: the fusion protein of the preposed sequence-endostatin-antibody IgG invariant region is shown by SEQ ID NO.12, wherein 19-23 is Arg-Gly-Val-Phe-Arg.
Sample 2: a leader sequence-endostatin-antibody IgG invariant region fusion protein point mutant 1, wherein 19-23 is Ala-Gly-Val-Phe-Arg, and the agg point of Arg at 19 is mutated into gcg;
sample 3: a pre-sequence-endostatin-antibody IgG invariant region fusion protein point mutant 2, wherein 19-23 is Arg-Gly-Val-Phe-Ala, and the cgt point of Arg of 23 is mutated into gct;
sample 4: leader sequence-endostatin-antibody IgG invariant region fusion protein point mutant 3, in which 19-23 are Ala-Gly-Val-Phe-Ala, the agg point of Arg at 19 was mutated to gcg and the cgt point of Arg at 23 was mutated to gct.
Saline was injected as a control.
The plasmid (pHEK 293Ultra Expression Vector II) expressing the leader sequence-endostatin-antibody IgG constant region CH1CH2CH3 fusion protein (SEQ ID NO.12, SEQ ID NO.11 encoding the nucleic acid sequence of SEQ ID NO. 12) of example 1 was selected for various point mutations to obtain the above various endostatin-antibody IgG constant region fusion protein mutants.
The various fusion proteins were purified using the ProteinA column method.
Example 3 examination of the Effect of glycosylation sites on endostatin fusion protein Activity
The pre-sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion protein (SEQ ID No.11 and SEQ ID No. 12) of example 1 was used as an experimental material to construct derivatives by adding a glycosylated amino acid short peptide Gly-Ala-Ser-Asn-Ser-Thr-Gly-Ala-Ser to the N-terminus or the C-terminus of endostatin, where Asn-Ser-Thr is the position of glycosylation and the sugar chain is attached to Asn. The plasmid (pHEK 293Ultra Expression Vector II) expressing the presequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion protein (SEQ ID NO.12, SEQ ID NO.11, nucleic acid sequence encoding SEQ ID NO. 12) in example 1 was selected for various genetic manipulations, or the gene fragments of these mutants were directly synthesized and inserted into the plasmid (pHEK 293Ultra Expression Vector II) for Expression of various proteins.
Sample 1: a pre-sequence-endostatin-antibody IgG invariant region fusion protein (SEQ ID NO. 12), wherein the front of the N end or the back of the C end of endostatin has no glycosylated amino acid short peptide;
sample 2: the method comprises the following steps of (1) adding an amino acid short peptide of a glycosylation site in front of the N end of endostatin to a pro-sequence-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 1 (SEQ ID NO.13 and SEQ ID NO. 14);
sample 3: a preposed sequence-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 2 (SEQ ID NO.15 and SEQ ID NO. 16), wherein an amino acid short peptide of a glycosylation site is added behind the C end of endostatin;
sample 4: the preposed sequence-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 3 (SEQ ID NO.17 and SEQ ID NO. 18) is characterized in that amino acid short peptides of a glycosylation site are added in front of the N end and behind the C end of endostatin respectively.
The various fusion proteins were purified using the ProteinA column method.
For the half-life experiment of the protein in the body of the mouse, the mouse is injected with various proteins (100 ug/0.2 ml) once, the content of the endostatin fusion protein is detected by using the anti-endostatin antiserum, the detection on the 1 st day is carried out within 2 hours after the injection, and the continuous detection is carried out for 13 days. FIG. 5 shows the variation of the contents of various proteins in the mouse body for 13 days, and the results show that the fusion protein with glycosylation sites in front and back of endostatin has the highest content in the mouse body.
The experiment shows that the endostatin derivative, namely the preposed sequence-endostatin-antibody IgG invariant region fusion protein glycosylation site mutant 3 (glycosylation sites are arranged in front of the N end and behind the C end of endostatin) is more stable in vivo, so that the effect of inhibiting tumors is best.
Example 4 additional point mutation of amino acids at the same glycosylation site
Sugar chains of proteins of eukaryotes including animals occur at Asn-X-Ser/Thr positions, where X is an amino acid other than Pro, asp or Glu, and the sugar chain is attached to Asn. Here, the pro sequence-endostatin-antibody IgG invariant fusion protein glycosylation site mutant 2 (SEQ ID NO.15 and SEQ ID NO. 16) in example 3, i.e., the fusion protein in which an amino acid short peptide having a glycosylation site added to the back of endostatin was used as a material to carry out a new point mutation, its glycosylation site was Asn210-Ser211-Thr212, the nucleic acid sequence was aacagcacg (628-636), and the newly added point mutant was used as an experimental sample.
Sample 1: point mutant Asn210-Ser211-Thr212 (SEQ ID NO. 16)
Sample 2: point mutant Asn210-Ala211-Thr212 (mutation of the agc point coding for Ser211 to gcc)
Sample 3: point mutant Asn210-Tyr211-Thr212 (mutation of agc point coding for Ser211 to tac)
Sample 4: point mutant Asn210-Ser211-Ser212 (acg point mutation encoding Thr212 to tcg)
Sample 5: point mutant Asn210-Ala211-Ser212 (agc point mutation for Ser211 to gcc and acg point mutation for Thr212 to tcg)
Sample 6: pre-sequence-endostatin-antibody IgG invariant region fusion protein (SEQ ID NO. 12) without introduction of glycosylation sites
The gene manipulations of the various mutant sites were carried out using an Expression plasmid (pHEK 293Ultra Expression Vector II) containing the prepro-endostatin-antibody IgG invariant fusion protein glycosylation site mutant 2 (SEQ ID NO.15 and SEQ ID NO. 16) of example 3, i.e., a fusion protein in which a glycosylated amino acid short peptide is added to the rear of endostatin. Various fusion proteins were purified using the ProteinA column method.
FIG. 6 shows the results of anti-tumor experiments, which show that the fusion proteins with glycosylation sites modified at the same position all have good tumor-inhibiting effect.
For the half-life experiment of the protein in the body of the mouse, the mouse is injected with various proteins (100 ug/0.2 ml) once, the content of the endostatin fusion protein is detected by using the anti-endostatin antiserum, the detection on the 1 st day is carried out within 2 hours after the injection, and the continuous detection is carried out for 13 days. FIG. 7 shows the content change of various proteins in mice for 13 days, and the results show that the content of the fusion protein modified by glycosylation sites in mice is very high, which indicates that the tumor inhibiting activity of the fusion protein and the stability of the protein in vivo can be improved by only introducing glycosylation modification with the characteristics of Asn-X-Ser/Thr.
Example 5 endostatin derivatives i.e. group 2 ArgArg in modified endostatin
The pro-sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion proteins (SEQ ID NO.11 and SEQ ID NO. 12) of example 1 were used as experimental material, and then point mutation was performed on group 2 ArgArg inside endostatin.
The 2 groups of argargarg inside endostatin in the presequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion proteins (SEQ ID No.11 and SEQ ID No. 12) are located at Arg62-Arg63 and Arg128-Arg129, respectively, in the amino acid sequence of endostatin (SEQ ID No. 4), at Arg85-Arg86 and Arg151-Arg152, respectively, in the amino acid sequence of the presequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion protein (SEQ ID No. 12), and at positions 253-258 (cgccgt) and 451-456 (cgcagg) in the nucleic acid sequence encoding it (SEQ ID No. 11).
First, for Arg85-Arg86 of group 1, nucleic acid positions 253-258 (cgccgt) were point mutated.
Point mutant 1: ala85-Arg86 (cgc point mutation encoding the preceding Arg to gcc)
Point mutant 2: ser85-Arg86 (cgc point coding for the preceding Arg was mutated to agc)
Point mutant 3: his85-Arg86 (cgc point mutation coding for the preceding Arg to cac)
Point mutant 4: arg85-Ala86 (cgt point mutation to gct encoding the following Arg)
Point mutant 5: arg85-Ser86 (cgt point mutation coding for the following Arg to agt)
Point mutant 6: arg85-His86 (cgt point mutation encoding the following Arg to cat)
Then, for Arg151-Arg152 of group 2, nucleic acid positions 451-456 (cgcagg) were point mutated.
Point mutant 7: ala151-Arg152 (cgc point mutation encoding the preceding Arg to gcc)
Point mutant 8: ser151-Arg152 (cgc point mutation coding for the preceding Arg to agc)
Point mutant 9: his151-Arg152 (cgc point mutation coding for the preceding Arg to cac)
Point mutant 10: arg151-Ala152 (agg point mutation coding for the following Arg to gcg)
Point mutant 11: arg151-Ser152 (agg point mutation coding for the following Arg to agc)
Point mutant 12: arg151-His152 (mutation of agg point coding for the following Arg to cac)
Plasmids (pHEK 293Ultra Expression Vector II) of the leader sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion proteins (SEQ ID NO.11 and SEQ ID NO. 12) of example 1 were selected for gene manipulation of various mutant sites.
The various fusion proteins were purified using the ProteinA column method.
The 1 st experiment was carried out on the gene fragment (SEQ ID NO. 11) of group 1 Arg85-Arg86, nucleic acid positions 253-258 (cgccgt), with point mutants 1-6 and the fusion protein without mutation (SEQ ID NO. 12) as control samples.
FIG. 8 shows the results of an antitumor test, which indicates that the fusion protein of Point mutant 3 (His 85-Arg 86) and Point mutant 6 (Arg 85-His 86) has a good tumor-inhibiting effect.
For the half-life experiment of the protein in the body of the mouse, the mouse is injected with various proteins (100 ug/0.2 ml) once, the content of the endostatin fusion protein is detected by using the anti-endostatin antiserum, the detection on the 1 st day is carried out within 2 hours after the injection, and the continuous detection is carried out for 13 days. FIG. 9 shows the in vivo 13-day changes in the amounts of the respective proteins in mice, and the results show that the in vivo amounts of the respective point mutants are not much different but higher than the in vivo amount of the fusion protein without the point mutation.
Experiments show that because the Arg85-Arg86 of the group 1 of endostatin is changed, the endostatin keeps the stability of protein, namely is not easily degraded by protease, so that the endostatin has higher anti-tumor activity, and in point mutant 3 (His 85-Arg 86) and point mutant 6 (Arg 85-His 86), the His replaces the Arg, but still keeps the property of basic amino acid, so that the endostatin embodies high tumor inhibition activity and high protein content.
Point mutations to the gene fragment (SEQ ID NO. 11) of group 2Arg 151-Arg152, nucleic acid positions 451-456 (cgcagg) were now carried out according to the above experimental procedure 1, with the samples having point mutants 7-12 and the fusion protein without mutations (SEQ ID NO. 12) as control samples.
FIG. 10 shows the results of an antitumor test, and shows that the fusion protein of Point mutant 9 (His 151-Arg 152) and Point mutant 12 (Arg 151-His 152) has a good tumor-inhibiting effect.
FIG. 11 shows the change in the in vivo content of each protein in mice over 13 days, and shows that the in vivo content of each point mutant is not much different but higher than that of the fusion protein without the point mutation.
Again, the following description is given: the preposed sequence-endostatin-antibody IgG invariant region CH1CH2CH3 fusion protein (SEQ ID NO. 12) is used as an experimental material, and Arg85-Arg86 and Arg151-Arg152 in endostatin in the fusion protein respectively correspond to Arg62-Arg63 and Arg128-Arg129 in the endostatin sequence shown in SEQ ID NO. 4.
Example 6 reevaluation of the engineering of groups 2 argargargarg within endostatin for the addition of glycosylation sites before the N-terminus and after the C-terminus of endostatin
According to the experimental results of example 5, 2 point mutants were constructed using the pro-sequence-endostatin-antibody IgG invariant region CH1CH2CH3 fusion proteins (SEQ ID No.11 and SEQ ID No. 12) as experimental materials:
point mutant 1: his85-Arg86 and His151-Arg152
Point mutation of cgc encoding Arg85 to cac
Mutation of the cgc point encoding Arg151 to cac
Point mutant 2: arg85-His86 and Arg151-His152
Mutation of the cgt point encoding Arg86 to cat
Mutation of the agg point encoding Arg152 to cac
Description of the drawings: the SEQ ID NO.12 sequence is taken as the experimental material, and the 85 th, 86 th, 151 th and 152 th amino acid positions in the endostatin in the fusion protein respectively correspond to the 62 th, 63 th, 128 th and 129 th amino acid positions in the endostatin sequence shown in the SEQ ID NO. 4.
According to the experimental results of example 3,2 groups of ArgArg were modified using fusion proteins (SEQ ID NO.17 and SEQ ID NO. 18) containing glycosylation sites before and after the N-terminus of endostatin,
the amino acid position of ArgArg group 1 is Arg94Arg95 (SEQ ID NO. 18) and the nucleic acid position (SEQ ID NO. 17) is 280-285 (cgccgt); the amino acid position of ArgArg group 2 is Arg160Arg161 (SEQ ID NO. 18) and the nucleic acid position (SEQ ID NO. 17) is 478-483 (cgcagg).
2 point mutants were additionally constructed:
point mutant 1: arg94His95 and Arg160His161
The cgt point encoding Arg95 was mutated to cat
Mutation of the agg point encoding Arg161 to cac
Point mutant 2: his94Arg95 and His160Arg161
Point mutation of cgc encoding Arg94 to cac
Point mutation of cgc encoding Arg160 to cac
(nucleic acid sequence SEQ ID NO.19 and amino acid sequence SEQ ID NO. 20)
Description of the drawings: the sequence of SEQ ID NO.18 is taken as the 94 th, 95 th, 160 th and 161 th amino acid positions in endostatin in the experimental material fusion protein respectively correspond to the 62 th, 63 th, 128 th and 129 th amino acid positions in the endostatin sequence shown in SEQ ID NO. 4.
Samples used in the present experiment:
sample 1: pre-sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion protein (SEQ ID NO. 12)
Sample 2: pro-sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion protein site mutant 1 (His 85-Arg86 and His151-Arg 152)
Sample 3: pro-sequence-endostatin-antibody IgG invariant CH1CH2CH3 fusion protein mutant 2 (Arg 85-His86 and Arg151-His 152)
Sample 4: presequence-endostatin (glycosylation sites added before the N-terminus and after the C-terminus) antibody IgG invariant CH1CH2CH3 fusion protein (SEQ ID NO. 18)
Sample 5: pro-sequence-endostatin (glycosylation sites added before and after the N-terminus, respectively) -antibody IgG invariant CH1CH2CH3 fusion protein Point mutant 1 (Arg 94His95 and Arg160His 161)
Sample 6: pro-endostatin (glycosylation sites added before the N-terminus and after the C-terminus) IgG invariant CH1CH2CH3 fusion protein site mutant 2 (His 94Arg95 and His160Arg 161) (SEQ ID NO. 20)
LLC tumor cells were inoculated on day 1, and each of the proteins (all at 50 pmol) and saline was injected intravenously on days 2,5, and 8, respectively, with the number of mice in each group being 30, and the mice were sacrificed on day 10, tumors removed, and weighed.
Fig. 12 is an anti-tumor experiment result, which shows that 2 glycosylation sites are introduced simultaneously, and that sample 5 and sample 6 modified by 2 groups of argargargarg have good tumor inhibition effect.
For the half-life experiment of the protein in mice, the mice were injected once with various proteins (100 ug/0.2 ml), and the endostatin-resistant antiserum was used to detect the endostatin fusion protein content, and the detection on day 1 was performed within 2 hours after the injection, and the detection was continued for 13 days. Fig. 13 shows the in vivo 13-day variation of each protein, and the results show that the in vivo content of both sample 5 and sample 6, in which 2 glycosylation sites were introduced and 2 argargargarg-engineered groups were high.
FIG. 14 is a photograph of an anatomy of a saline-injected mouse as a control in example 1 and a mouse injected with the fusion protein of sample 6 (a glycosylation site-added fusion protein spot mutant 2 (SEQ ID NO. 20) before the N-terminus and after the C-terminus of endostatin) in example 6.
Example 7 comparison of biological Activity for endostatin and various endostatin derivatives
For all DNA synthesis fragments inserted into plasmid pHEK293Ultra Expression Vector II, the fragment contained XhoI-BamHI-XbaI at the front end and SphI-HindIII at the rear end, and additionally a His-tag and a termination signal tag, i.e., a cathcatcatcatcattag, were added in front of SphI-HindIII.
Sample 1: endostatin
The DNA fragment is synthesized by connecting the DNA fragment of 1-54 of the coding signal peptide in the nucleic acid sequence shown in SEQ ID NO.1 with the nucleic acid sequence of the coding endostatin shown in SEQ ID NO.3, and then the DNA fragment is inserted into the plasmid pHEK293Ultra Expression Vector II to obtain the endostatin.
Sample 2: presequence-endostatin protein (2 Arg before N-terminus of endostatin)
The method comprises the steps of connecting the back of a nucleic acid sequence of a coding preposed sequence shown in SEQ ID NO.1 with a nucleic acid sequence of a coding endostatin shown in SEQ ID NO.3, synthesizing the DNA fragment, inserting the DNA fragment into a plasmid pHEK293Ultra Expression Vector II to obtain a preposed sequence-endostatin protein, wherein the front of the N end of the endostatin is 2 Arg.
Sample 3: glycosylation site short peptide-endostatin-glycosylation site short peptide protein
The following DNA fragments were first designed:
segment 1: DNA fragment of 1-54 encoding signal peptide in nucleic acid sequence shown in SEQ ID NO.1
Segment 2: ggggcctccaacagcacgggccctcc (encoding short peptide GlyAlaSerAsnSerThrGlyAlaSer containing glycosylation sites)
Fragment 3: nucleic acid sequence shown as SEQ ID NO.3 and used for coding endostatin
Synthesis of such DNA sequences: fragment 1-fragment 2-fragment 3-fragment 2, then this DNA fragment is inserted into plasmid pHEK293Ultra Expression Vector II to obtain glycosylation site short peptide-endostatin-glycosylation site short peptide protein.
Sample 4: endostatin with point mutation (His 62Arg63 and His128Arg 129)
SEQ ID NO.3 shows the nucleic acid sequence encoding endostatin, with amino acids Arg62Arg63 for cgccgt at 184-189, and with the mutation of g at 185 to a, so that the amino acids 184-189 become cccgt, producing His62Arg63; similarly, the 382 to 387 residues were cgcagg, which corresponds to the amino acid Arg128Arg129, and 383g was mutated to a, so that the 382 to 387 residues became cagg, thereby producing His128Arg129. The front of the DNA segment coding the endostatin with point mutation is connected with a signal peptide (the DNA segment of 1-54 coding the signal peptide in the nucleic acid sequence shown in SEQ ID NO. 1) to obtain the DNA segment coding the endostatin with point mutation (His 62Arg63 and His128Arg 129), and the segment is synthesized and inserted into a plasmid pHEK293Ultra Expression Vector II to obtain the endostatin protein with point mutation (His 62Arg63 and His128Arg 129).
Sample 5: endostatin-antibody IgG invariant region fusion protein
The following DNA fragments were first designed:
segment 1: DNA fragment 1-54 of the nucleic acid sequence shown in SEQ ID NO.1, which codes for a signal peptide
Segment 2: nucleic acid sequence shown in SEQ ID NO.3 and used for coding endostatin
Fragment 3: nucleic acid sequence shown as SEQ ID NO.5 and used for encoding IgG invariant region of antibody
Synthesis of such DNA sequences: fragment 1-fragment 2-fragment 3, then this DNA fragment was inserted into plasmid pHEK293Ultra Expression Vector II to obtain endostatin-antibody IgG constant region fusion protein.
Sample 6: pro sequence-endostatin (glycosylation sites added before the N-terminus and after the C-terminus, respectively) -antibody IgG invariant region fusion protein dot mutant 2 (His 94Arg95 and His160Arg 161) (SEQ ID No. 20).
The control sample was injected with saline.
Samples 1-4 were purified by His-tag method, and samples 5 and 6 were purified by ProteinA column method.
FIG. 15 shows the results of antitumor experiments with endostatin and various endostatin derivatives. The results show that endostatin and various endostatin derivatives have anti-tumor bioactivity, and the anti-tumor effect of sample 6, i.e., the pro-sequence-endostatin (glycosylation sites are added before the N end and after the C end), antibody IgG invariant region fusion protein point mutant 2 (His 94Arg95 and His160Arg 161) (SEQ ID No. 20) is the best.
For the half-life experiment of the protein in mice, the mice were injected once with various proteins (100 ug/0.2 ml), and the endostatin-resistant antiserum was used to detect the endostatin fusion protein content, and the detection on day 1 was performed within 2 hours after the injection, and the detection was continued for 13 days. Fig. 16 is a graph showing the in vivo 13-day content changes of the various proteins in mice, showing that endostatin containing glycosylation sites (sample 3) and endostatin with point mutations (His 62Arg63 and His128Arg 129) (sample 4) always retained low in vivo content, that endostatin-antibody IgG constant region fusion protein (sample 5) had a relatively high in vivo content, and that the in vivo content of the pro sequence-endostatin (glycosylation sites added before the N-terminus and after the C-terminus, respectively) -antibody IgG constant region fusion protein point mutant 2 (His 94Arg95 and His160Arg 161) (SEQ ID No. 20) (sample 6) was the highest.
Therefore, the method has the characteristics that: the endostatin derivative such as the protein shown in SEQ ID NO.20 has the best anti-tumor and half-life bioactivity, and is characterized in that 2Arg is arranged in front of the N end of endostatin, glycosylation sites are respectively added in front of the N end and behind the C end of endostatin, the endostatin generates point mutation (His 62Arg63 and His128Arg 129), and the endostatin is connected with an antibody IgG invariant region to form a fusion protein.
Through the above series of examples, the stability, including half-life and anti-tumor effect, of endostatin derivatives are greatly improved, and these modifications are:
the first endostatin derivative and the preposed sequence-endostatin are connected with an IgG invariant region to form a fusion protein, so that the molecular weight of the endostatin is increased. It is noted that, in this case, a fusion protein constructed by linking endostatin to a macromolecular glycoprotein such as albumin can also achieve similar results.
And the Arg of the endostatin is important for the biological activity of the endostatin, so the newly added Arg can improve the anti-tumor activity of the endostatin.
And thirdly, introducing amino acid short peptides of glycosylation sites in front of the N end and behind the C end of endostatin, wherein the amino acid short peptides are GlyAlaSerAsnSerThrGlyAlaSer, comprise AsnSerThr, have Asn-X-Ser/Thr characteristics, and are subjected to point mutation, so that the mutant with the Asn-X-Ser/Thr characteristics has high antitumor activity and stability.
The experiment shows that point mutation of one Arg in ArgArg to other amino acids improves the anti-tumor activity and stability of the endostatin fusion protein, obviously, the stability of the endostatin fusion protein is improved, namely the content of the endostatin fusion protein in vivo is improved, and the experiment shows that the combination of ArgHis or HisArg has better anti-tumor activity.
Fifth, the best combination is selected in example 6, and a class of endostatin derivatives is constructed, and simultaneously:
(1) Introduction of glycosylation sites in front of N-terminal and behind C-terminal of endostatin
(2) Point mutation of group 2 ArgArg in endostatin to ArgHis or HisArg
The pro-sequence-endostatin-antibody IgG constant region fusion protein having the above 2 characteristics showed a considerably high anti-tumor activity and a considerably high content in vivo.
Based on the fusion protein sequences (SEQ ID No.11 and SEQ ID No. 12), the following modifications can be made:
a) Introducing glycosylation sites in front of the N end and behind the C end of endostatin;
b) Point mutations to internal ArgArg of endostatin
c) Introducing new Arg in front of the N-terminus of endostatin
The sequences shown in SEQ ID NO.19 and SEQ ID NO.20 are only one of the fusion proteins described which have the above advantages.
Also provided herein is a method of making:
a) The host cell for expression is an animal cell;
b) An expression system can perform glycosylation site modification on a drug protein;
c) Because of the secretory expression, the fusion protein can be collected from the culture medium;
d) The separation and purification of the fusion Protein was performed using a Protein A column.
The endostatin fusion protein and its series of modified mutants can be used for treating tumor and cancer.
The coding DNA sequences of the endostatin fusion proteins can be applied to anti-tumor gene therapy.
The dosage form of the medicine can be prepared into various administration forms of medicines such as injection, oral administration, application, surgical treatment and the like together with an emulsifier, a liposome, a dispersant, a stabilizer and the like. In addition to the fusion protein itself as a drug, the nucleotide fragment or vector encoding the fusion protein may also be used as a form of gene therapy.
The above examples and description of the preferred embodiments are intended to be illustrative of the invention defined by the claims and not to limit the invention. It is expressly intended that all such obvious modifications and equivalents of similar inventions which do not depart from the spirit and scope of the present invention are deemed to be covered by the invention.
Sequence listing
<110> Sun Jialin
<120> derivative of antitumor protein endostatin and application thereof
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 69
<212> DNA
<213> Homo sapiens species (Homo sapiens)
<400> 1
atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60
gtgtttcgt 69
<210> 2
<211> 23
<212> PRT
<213> race of intellectual people (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> race of intellectual people (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> race of intellectual people (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> Homo sapiens species (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> race of intellectual people (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 (2)
1. A derivative of antitumor protein endostatin, wherein the amino acid sequence of the endostatin is shown by SEQ ID NO. 4; the derivatives are characterized in that the derivatives introduce amino acid short peptides of glycosylation sites in front of the N end or in front of the N end and behind the C end of endostatin; the glycosylation site is Asn-Ser-Thr or Asn-Ala-Thr or Asn-Tyr-Thr or Asn-Ser-Ser or Asn-Ala-Ser; the amino acid short peptide is Gly-Ala-Ser.
2. The use of a derivative of the antitumor protein endostatin of claim 1 in the preparation of an anticancer drug.
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| CN201910639692.7A CN110498851B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin and application thereof |
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| CN201910639692.7A CN110498851B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin and application thereof |
| CN201510761800.XA CN106674352B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin and application thereof |
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| CN201510761800.XA Active CN106674352B (en) | 2015-11-10 | 2015-11-10 | Derivative of antineoplastic protein endostatin and application thereof |
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Also Published As
| Publication number | Publication date |
|---|---|
| CN110452295A (en) | 2019-11-15 |
| CN110452295B (en) | 2022-10-11 |
| CN110317264A (en) | 2019-10-11 |
| CN106674352A (en) | 2017-05-17 |
| CN110317264B (en) | 2022-10-11 |
| CN110498851A (en) | 2019-11-26 |
| CN106674352B (en) | 2020-05-15 |
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