CN1539850A - Recombined protein of anti tumour, encoded gene and application - Google Patents
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Abstract
An antineoplastic recombinant protein chosen from 3 myocardiac troponins, its coding gene, and its application are disclosed. A method for efficient expression of said protein is also disclosed.
Description
Technical field
The present invention relates to antitumor recombinant protein and encoding gene and application in the bioengineering field.
Background technology
Malignant tumour is the most serious class disease of present harm humans health.The common method of treatment cancer has at present: surgical operation therapy, chemical medicinal treatment, radiotherapy, biotherapy and Chinese medical and herbal therapy.Wherein commonly used with surgical operation therapy, chemical medicinal treatment, radiotherapy.Surgical operation therapy is as the first-selection and the major programme of most of oncotherapies, makes that countless tumour patients rehabilitate, prolongs life or improved quality of life.But aspect oncotherapy, surgical operation therapy only is applicable to the patient of certain condition and scope, and because tumour can recur and shift, excision can both not reach the purpose of radical cure.Extremely important and requisite effect is also being brought into play in chemical medicinal treatment and the radiocurable tumor treatment aspect that is used in, and also in continuous development.But at present chemotherapeutics therapy of using and radiotherapy all have infringement to all active proliferative cells in the health, as hemopoietic system, gi tract etc., so symptoms such as the patient of chemicotherapy often has blood picture reduction, gastrointestinal hemorrhage, feels sick, vomitings.And owing to being subjected to the restriction that many-sided reasons such as resistance appear in patient's body and spiritual aspect holding capacity and tumour cell, chemicotherapy can not kill all tumour cells, for the recurrence of tumour and the deterioration of transfer and disease have stayed hidden danger.Biotherapy and Chinese medical and herbal therapy also are antineoplastic reasonable method, but require further study to improve curative effect.Therefore, exploitation has the antitumor drug of obvious result of treatment, safety to become the extremely urgent task that the world of medicine faces.
Studies show that the unrestricted invasive growth of malignant entity tumor and transfer thereof depend on the continuous generation of neovascularity, can suppress growth of tumor significantly so suppress vasculogenesis.Suppress vasculogenesis, the blood supply of blocking-up knurl body is the New Policy that is different from conventional antineoplaston, is also referred to as " tumour starvation cure ", has become the focus of antineoplaston research.Under the normal physiological state, ovulating except female reproductive system, corpus luteum generates, and processes such as pregnancy have outside the of short duration new vessel generative process, and adult vascular system hyperplasia is in relative static conditions.But in tumor tissues, for adapting to the quick growth of tumor tissue, vasculogenesis is very active, in time to be tumor tissue growth supply blood.Capillary blood vessel in tumor tissues source has two kinds: a kind of is the vascular endothelial growth factor that tumour cell etc. produces, and induces the knurl body to generate capillary blood vessel; Another kind is that the host blood vessel that remains in the knurl body gradually becomes tumor vessel, both the tumourization of host blood vessel.Different with normal blood vessels, tumor vascular vascular endothelial cell is imperfect, and basilar membrane is sparse and be easy to reveal.Behind the tumor-blood-vessel growth no longer further differentiation reconstruct into artery and vein, no unstriated muscle and nerve ending belong to passive blood vessel, its blood flow body that places one's entire reliance upon circulates.Newborn capillary blood vessel is a first station tumor-infiltrated and that shift, and tumour cell enters blood circulation to distant metastasis by the tumour wall of micrangium.
Entity tumor growth needs a large amount of blood supplies.Many experimental studies confirm to suppress neovascularity generation in the animal body, can be cut to the blood supply that tumor growth provides nutrient effectively, make the atrophy of knurl body so that disappear.The application of anti-neovascularization medicaments TNP-470 and anti-bFGF, VEGF monoclonal antibody, the competitive blocking VEGF signal conduction of mutant vegf receptor FlK-1, α V β 3 overexpression inducing endothelial cell apoptosis and the formation of the special inhibition tumor neogenetic blood vessels of Angiostatin etc., all can suppress tumor growth, be a kind of novel, promising antitumor drug, can remedy the limitation of current clinical cancer therapy means and replace the part treatment means.Because this class medicine is to reach by the formation that suppresses new vessel to suppress tumor growth, and the growth of people's normal tissue cell does not rely on the generation of new vessel, so healthy tissues is not almost had toxic side effect.This class medicine does not directly act on tumour cell, so the inhibition tumour has broad spectrum and do not develop immunity to drugs, is current effective, safe, promising antitumor drug of generally acknowledging in the world.It is mainly used in original position and shifts the treatment of solid tumor and the operative treatment of auxiliary tumour, also can be used for preventing the recurrence and the transfer of tumour.Therefore, exploitation has an inhibiting antitumor drug of blood vessel hyperplasia, aspect the preventing and treating of malignant tumour, has the important clinical meaning.
Cardiac muscle troponin I and cardiac troponin C all are subunits of cardiac troponin, are often used as the mark of myocardial infarction diagnosis at present, and cardiac muscle troponin I is the SEQ ID № in the sequence table: 1, form by 210 amino-acid residues; Cardiac troponin C is made up of 160 amino-acid residues.
Intestinal bacteria and pichia yeast expression system are to be used to one of the most successful expression system of expressing foreign protein, and development in recent years is very fast, and existing at present hundreds of kind albumen is expressed in this expression system.Pichia spp is a kind of methyl alcohol nutritional type yeast, its growth rapidly, culture condition is simple, can the high-density cultured continuously, genetic manipulation is similar to yeast saccharomyces cerevisiae, technology is quite ripe, is the high expression level bacterial classification of using always in the industrial production.
Summary of the invention
The purpose of this invention is to provide antitumor recombinant protein and encoding gene thereof.
Antitumor recombinant protein provided by the invention is selected from the following protein families any one:
(1) the N-terminal amino-acid residue of cardiac muscle troponin I and the part or all of protein that merges mutually of cardiac troponin C carboxyl terminal amino acid residue sequence;
(2) the C-terminal amino-acid residue of cardiac muscle troponin I and the part or all of protein that merges mutually of cardiac troponin C aminoterminal amino acid residue sequence;
(3) the N-terminal amino-acid residue of cardiac muscle troponin I and cardiac troponin C carboxyl terminal amino acid residue sequence partly or entirely, the part or all of protein of fusion mutually of the C-terminal amino-acid residue of cardiac muscle troponin I and cardiac troponin C aminoterminal amino acid residue sequence.
Above-mentioned cardiac troponin C aminoterminal amino acid residue sequence is SEQ ID № in the sequence table: 2 amino acid residue sequence, its carboxyl terminal amino acid residue sequence are SEQ ID № in the sequence table: 3 amino acid residue sequence.
Described antitumor recombinant protein can be SEQ ID № in the sequence table: 4 amino acid residue sequence or with SEQ ID №: 4 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQID: 4 is identical active by SEQ ID №: 4 deutero-protein; Described antitumor recombinant protein can be SEQ ID № in the sequence table: 5 amino acid residue sequence or with SEQ ID №: 5 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 5 is identical active by SEQ ID №: 5 deutero-protein; Described antitumor recombinant protein can be SEQ ID № in the sequence table: 6 amino acid residue sequence or with SEQ ID №: 6 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 6 is identical active by SEQ ID №: 6 deutero-protein.
Second purpose of the present invention provides the encoding gene of antitumor recombinant protein.
The encoding gene of antitumor recombinant protein provided by the invention is one of following nucleotide sequences:
(1) the SEQ ID № in the sequence table: 7 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(2) the SEQ ID № in the sequence table: 8 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(3) the SEQ ID № in the sequence table: 9 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(4) the SEQ ID № in the sequence table: 10 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(5) the SEQ ID № in the sequence table: 11 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(6) the SEQ ID № in the sequence table: 12 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(7) SEQ ID № in the code sequence tabulation: the polynucleotide of april protein sequence;
(8) SEQ ID № in the code sequence tabulation: the polynucleotide of 5 protein sequences;
(9) SEQ ID № in the code sequence tabulation: the polynucleotide of 6 protein sequences.
The expression vector and the clone that contain said gene all belong to the present invention's scope required for protection.
The 3rd purpose of the present invention provides the high-efficiency expression method of a kind of above-mentioned antitumor recombinant protein in intestinal bacteria and Pichia yeast.
The high-efficiency expression method one of antitumor recombinant protein is that the gene with the antitumor recombinant protein of coding imports in the Pichia yeast, express to obtain antitumor recombinant protein.
Specifically, this method may further comprise the steps:
1, selects the preferred codon of Pichia yeast for use, and with computer Simulation the minimum free energy of formation of translation initiation district and antitumor recombinant protein gene 5 ' end secondary structure, design and synthesize sequence 7, or sequence 8, or sequence 11 antitumor recombinant protein gene orders;
2, the synthetic gene fragment is connected with carrier pPIC9K respectively, obtains expression plasmid, obtain to express engineering bacteria behind the conversion Pichia yeast;
3, culturing engineering bacterium, expression product is secreted in the nutrient solution, obtains target protein.
In order to obtain better effect, induce with methyl alcohol in the expression process of Pichia yeast.Induce the methyl alcohol final concentration in the process to be preferably 1%-5%.
Described Pichia yeast is preferably the GS115 Pichia yeast.
The high-efficiency expression method two of antitumor recombinant protein is that the gene with the antitumor recombinant protein of coding imports in the intestinal bacteria, express to obtain antitumor recombinant protein.
Specifically, this method may further comprise the steps:
1, select the preferred codon of intestinal bacteria for use, design and synthesize sequence 9, or sequence 10, or sequence 12 antitumor recombinant protein gene orders;
2, the synthetic gene fragment is connected with carrier pET21b respectively, obtains expression plasmid, obtain to express engineering bacteria behind the transformed into escherichia coli;
3, culturing engineering bacterium, expression product obtains target protein.
Described intestinal bacteria are preferably the BL21-DE3 intestinal bacteria.
Cardiac muscle troponin I and cardiac troponin C are merged in the present invention, the synthetic new warm body of cardiac muscle troponin I-C.This syzygy has the effect of tangible anti-mouse entity tumour, and the proteic plasma stability of rat intravenous injection syzygy obviously prolongs than the plasma stability of intravenous injection cardiac muscle troponin I.Can reduce pharmaceutical quantities clinically and improve curative effect.Aspect the treatment of malignant tumour, significant.
The antitumor recombinant protein that obtains with method of the present invention accounts for the 20-60% of nutrient solution total protein, and it is simple to express process operation, and product cost is lower, can be used for large-scale production, and is significant for the antitumor drug that exploitation is new.
Description of drawings
Fig. 1 is the collection of illustrative plates of pPIC-AAF expression plasmid.
Fig. 2 cuts gel electrophoresis spectrum for pPIC-AAF expression plasmid enzyme.
Fig. 3 is the GS115 pichia spp bacteria culture fluid electrophoretogram behind the methanol induction
Fig. 4 is the collection of illustrative plates of pET-AAF expression plasmid.
Fig. 5 cuts gel electrophoresis spectrum for pET-AAF expression plasmid enzyme.
Fig. 6 is the BL21-DE3 intestinal bacteria nutrient solution electrophoretogram of IPTG after inducing.
Embodiment
The Pichia yeast vivoexpression of embodiment 1, antitumor recombinant protein
1, antitumor recombinant protein gene is synthetic
The full length sequence of sequence 13 in the artificial synthesized sequence table (encoding gene of cardiac muscle troponin I), and its 5 ' end add NdeI, 3 ' end add the NotI restriction enzyme site, obtain sequence A FF1; The 1-285 bit base sequence of sequence 7 in the artificial synthesized sequence table, and add that at its 5 ' end NdeI restriction enzyme site, 3 ' end add and 5 ' end complementary 15 bases of gene order 13, then this sequence is connected with sequence A FF1, obtain sequence A FF2; The 631-858 base of sequence 8 in the artificial synthesized sequence table, and add that at its 3 ' end NotI restriction enzyme site, 5 ' end add and 3 ' end complementary 15 bases of gene order 13, then this sequence is connected with sequence A FF1, obtain sequence A FF3.
2, antitumor recombinant protein vivoexpression
(1) complete sequence with above-mentioned synthetic is respectively charged in the pGEM-T-EASY plasmid, make up respectively pGEM-AAF1 AAF2 the AAF3 cloning vector.
(2) structure of expression plasmid
Employing is available from the efficient expression plasmid pPIC9K of American I nvitrogen company, with NdeI and NotI respectively enzyme cut pGEM-AAF1 AAF2 AAF3 plasmid and pPIC9K plasmid, target fragment is collected in gel electrophoresis, with the connection of spending the night of 16 ℃ of T4 ligase enzymes.Through the conversion of conventional method, choose bacterium, amplification obtains expression plasmid pPIC9K-AAF1 AAF2 AAF3, and its universal architecture collection of illustrative plates is as shown in Figure 1.As shown in Figure 4, enzyme is cut evaluation, proves that the structure of expression plasmid is correct.Among the figure, swimming lane 1 is cut the gel electrophoresis result for pPIC-AAF1 expression plasmid enzyme, and swimming lane 2 is cut the gel electrophoresis result for pPIC-AAF3 expression plasmid enzyme, and swimming lane 3 is cut the gel electrophoresis result for pPIC-AAF2 expression plasmid enzyme.
(3) with plasmid pPIC9K-AAF1 AAF2 AAF3 use the SacI linearizing respectively, adopt Zhejiang Xin Zhi company electricity gene introducing apparatus, with linearizing pPIC9K-AAF1 AAF2 AAF3 import respectively in the GS115 Pichia yeast (available from Invitrogen company), through cultivating, select, processes such as screening, the mono-clonal bacterium of three kinds of anti-G418mg/ml of acquisition.
(4) select the mono-clonal bacterium, be inoculated in the 5ml substratum, 30 ℃ are spent the night, and change over to then in the 250ml substratum, continue to be cultured to OD
600=10-20 collects thalline, is diluted to OD with no carbon source substratum
600=50, continue to cultivate 24 hours, adding methyl alcohol to final concentration is 1% abduction delivering, adds methyl alcohol once every 24 hours, to 96 hours.The centrifuging and taking supernatant liquor carries out gel electrophoresis analysis and functional examination.Get the 10ml supernatant liquor, use the 12%SDS-PAGE electrophoresis, and dye through Coomassie brilliant blue.The result as shown in Figure 3, the AAF1 sequence has the inductive protein expression in the position of 32KD, and according to BSA contrast, expression amount is 30mg/L; The AAF2 sequence has the inductive protein expression in the position of 38KD, and according to the BSA contrast, expression amount is 50mg/L; The AAF3 sequence has the inductive protein expression in the position of 43KD, and according to the BSA contrast, expression amount is 80mg/L.Among the figure, swimming lane 1 is the transformed bacteria nutrient solution electrophoresis result of pPIC-AAF1 expression plasmid, and swimming lane 2 is the transformed bacteria nutrient solution electrophoresis result of pPIC-AAF3 expression matter, and swimming lane 3 is the transformed bacteria nutrient solution electrophoresis result of pPIC-AAF2 expression plasmid.
In the present embodiment, induce meaning very big with methyl alcohol when cultivating the mono-clonal thalline, before not inducing, the content of target protein is very low in the nutrient solution, as shown in Figure 3, significant target protein band is arranged in the nutrient solution after inducing, and do not have significant target protein band in the nutrient solution before inducing.
3, the functional examination of the antitumor recombinant protein of the present invention
Get 24 kunming mices, be divided into and go up behind the cleer and peaceful methanol induction two groups of supernatants before the methanol induction, neck subcutaneous vaccination S180 ascites cells knurl strain respectively, go up cleer and peaceful methanol induction before the subcutaneous injection methanol induction respectively after three days after supernatant 0.5ml/ only, every injection in 12 hours once, injected continuously 7 days.Observe also and cut tumor tissue, weigh, go up before the methanol induction as a result that supernatant sequence A AF1, AAF2 and AAF3 group knurl body weight are respectively 0.82+0.22 gram and 0.11+0.05 gram behind the cleer and peaceful methanol induction, 0.13+0.08 gram, 0.09+0.05 gram, tumour inhibiting rate is respectively 86.6%, 84.1% and 89.0%.
The intestinal bacteria vivoexpression of embodiment 2, antitumor recombinant protein
1, artificial gene is synthetic: the full length sequence of sequence 14 in the artificial synthesized sequence table (encoding gene of cardiac muscle troponin I), and add that at its 5 ' end NdeI, 3 ' end add the NotI restriction enzyme site, obtain sequence A FF4; The 1-285 bit base sequence of sequence 9 in the artificial synthesized sequence table, and add that at its 5 ' end NdeI restriction enzyme site, 3 ' end add and 5 ' end complementary 15 bases of gene order 14, then this sequence is connected with sequence A FF4, obtain sequence A FF5; The 631-858 base of sequence 10 in the artificial synthesized sequence table, and add that at its 3 ' end NotI restriction enzyme site, 5 ' end add and 3 ' end complementary 15 bases of gene order 14, then this sequence is connected with sequence A FF4, obtain sequence A FF6.
2, antitumor recombinant protein vivoexpression
(1) complete sequence with above-mentioned synthetic is respectively charged in the pGEM-T-EASY plasmid, make up pGEM-AAF4 AAF5 the AAF6 cloning vector.
(2) structure of expression plasmid
Employing is available from the efficient expression plasmid pET21b of U.S. Novagen company, with SnabI and NotI respectively enzyme cut pGEM-AAF4 AAF5 AAF6 plasmid and pET21b plasmid, target fragment is collected in gel electrophoresis, with the connection of spending the night of 16 ℃ of T4 ligase enzymes.Through the conversion of conventional method, choose bacterium, amplification obtains expression plasmid pET-AAF4 AAF5 AAF6, and its universal architecture collection of illustrative plates is as shown in Figure 4.As shown in Figure 5, enzyme is cut evaluation, proves that the structure of expression plasmid is correct.Among the figure, swimming lane 1 is cut the gel electrophoresis result for pET-AAF4 expression plasmid enzyme, and swimming lane 2 is cut the gel electrophoresis result for pET-AAF6 expression plasmid enzyme, and swimming lane 3 is cut the gel electrophoresis result for pET-AAF5 expression plasmid enzyme.
(3) respectively with plasmid pET-AAF4 AAF5 AAF6 in transform importing the BL21-DE3 intestinal bacteria,, select processes such as screening, acquisition mono-clonal bacterium through cultivating.
(4) select the mono-clonal bacterium, be inoculated in the 5ml substratum, 37 ℃ are spent the night, and change over to then in the 250ml substratum, continue to be cultured to OD
600=0.8, add IPTG to final concentration be 1%37 ℃ of abduction deliverings 4 hours.The centrifuging and taking precipitation is used the lysate suspendible, ultrasonication, and centrifugal 30 minutes of 10000g abandons supernatant, and washing of precipitate twice is at last with the dissolving of 8M urea, through 50mM phosphate buffered saline buffer dialyzed overnight.Get the protein soln 10ul after the dialysis, use the 12%SDS-PAGE electrophoresis, and dye through Coomassie brilliant blue.The result as shown in Figure 6, the AAF4 sequence has the inductive protein expression in the position of 29KD, and according to BSA contrast, expression amount is 30mg/L; The AAF5 sequence has the inductive protein expression in the position of 37KD, and according to the BSA contrast, expression amount is 45mg/L; The AAF6 sequence has the inductive protein expression in the position of 42KD, and according to the BSA contrast, expression amount is 50mg/L.Among the figure, swimming lane 1 is the transformed bacteria nutrient solution electrophoresis result of pET-AAF4 expression plasmid, and swimming lane 2 is the transformed bacteria nutrient solution electrophoresis result of pET-AAF5 expression matter, and swimming lane 3 is the transformed bacteria nutrient solution electrophoresis result of pET-AAF6 expression plasmid.
3, the functional examination of the antitumor recombinant protein of the present invention
Get 24 kunming mices, be divided into phosphate buffered saline buffer and express purification liquid group, neck subcutaneous vaccination S180 ascites cells knurl strain respectively, difference subcutaneous injection phosphate buffered saline buffer and expression purification liquid 1.0mg albumen/kg after three days, every injection in 12 hours once, injected continuously 7 days.Observation also cuts tumor tissue, weighs, as a result phosphate buffered saline buffer and expression purification liquid sequence A AF4, AAF5 and AAF6 group knurl body weight are respectively 1.02+0.28 gram and 0.15+0.06 gram, 0.12+0.06 gram, 0.11+0.04 gram, tumour inhibiting rate is respectively 85.3%, 88.1% and 89.2%.
The body internal stability experiment of the intestinal bacteria vivoexpression purification liquid of embodiment 3, antitumor recombinant protein:
Get 24 kunming mices, be divided into phosphate buffered saline buffer and express purification liquid group, the expression purification liquid 2.0mg albumen/kg that obtains among subcutaneous injection phosphate buffered saline buffer and the embodiment 2 respectively, injected 8 hours, eyeground vein is got blood, separation of serum adopts conventional euzymelinked immunosorbent assay (ELISA) to measure the antitumor recombinant protein concentration of serum.Phosphate buffered saline buffer and express purification liquid sequence A AF4 as a result, AAF5 and the antitumor recombinant protein concentration of AAF6 group serum are respectively 0.52+0.08ng/ml, 3.55+0.12ng/ml, 8.22+1.08ng/ml and 9.31+1.62ng/ml, showing with the TnC fusion to increase its body internal stability, delays degraded.
Sequence table
<160>14
<210>1
<211>210
<2?12>PRT
＜213〉Genus Homo people (Homo sapiens)
<220>
<223>
<400>1
Met?Ala?Asp?Gly?Ser?Ser?Asp?Ala?Ala?Arg?Glu?Pro?Arg?Pro?Ala
1 5 10 15
Pro?Ala?Pro?Ile?Arg?Arg?Arg?Ser?Ser?Asn?Tyr?Arg?Ala?Tyr?Ala
20 25 30
Thr?Glu?Pro?His?Ala?Lys?Lys?Lys?Ser?Lys?Ile?Ser?Ala?Ser?Arg
35 40 45
Lys?Leu?Gln?Leu?Lys?Thr?Leu?Leu?Leu?Gln?Ile?Ala?Lys?Gln?Glu
50 55 60
Leu?Glu?Arg?Glu?Ala?Glu?Glu?Arg?Arg?Gly?Glu?Lys?Gly?Arg?Ala
65 70 75
Leu?Ser?Thr?Arg?Cys?Gln?Pro?Leu?Glu?Leu?Ala?Gly?Leu?Gly?Phe
80 85 90
Ala?Glu?Leu?Gln?Asp?Leu?Cys?Arg?Gln?Leu?His?Ala?Arg?Val?Asp
95 100 105
Lys?Val?Asp?Glu?Glu?Arg?Tyr?Asp?Ile?Glu?Ala?Lys?Val?Thr?Lys
110 115 120
Asn?Ile?Thr?Glu?Ile?Ala?Asp?Leu?Thr?Gln?Lys?Ile?Phe?Asp?Leu
125 130 135
Arg?Gly?Lys?Phe?Lys?Arg?Pro?Thr?Leu?Arg?Arg?Val?Arg?Ile?Ser
140 145 150
Ala?Asp?Ala?Met?Met?Gln?Ala?Leu?Leu?Gly?Ala?Arg?Ala?Lys?Glu
155 160 165
Ser?Leu?Asp?Leu?Arg?Ala?His?Leu?Lys?Gln?Val?Lys?Lys?Glu?Asp
170 175 180
Thr?Glu?Lys?Glu?Asn?Arg?Glu?Val?Gly?Asp?Trp?Arg?Lys?Asn?Ile
185 190 195
Asp?Ala?Leu?Ser?Gly?Met?Glu?Gly?Arg?Lys?Lys?Lys?Phe?Glu?Ser
200 205 210
<210>2
<211>95
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>2
Met?Asp?Asp?Ile?Tyr?Lys?Ala?Ala?Val?Glu?Gln?Leu?Thr?Glu?Glu
1 5 10 15
Gln?Lys?Asn?Glu?Phe?Lys?Ala?Ala?Phe?Asp?Ile?Phe?Val?Leu?Gly
20 25 30
Ala?Glu?Asp?Gly?Cys?Ile?Ser?Thr?Lys?Glu?Leu?Gly?Lys?Val?Met
35 40 45
Arg?Met?Leu?Gly?Gln?Asn?Pro?Thr?Pro?Glu?Glu?Leu?Gln?Glu?Met
50 55 60
Ile?Asp?Glu?Val?Asp?Glu?Asp?Gly?Ser?Gly?Thr?Val?Asp?Phe?Asp
65 70 75
Glu?Phe?Leu?Val?Met?Met?Val?Arg?Cys?Met?Lys?Asp?Asp?Ser?Lys
80 85 90
Gly?Lys?Ser?Glu?Glu
95
<210>3
<211>75
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>3
Lys?Asp?Asp?Ser?Lys?Gly?Lys?Ser?Glu?Glu?Leu?Ser?Asp?Leu?Phe
1 5 10 15
Arg?Met?Phe?Asp?Lys?Asn?Ala?Asp?Gly?Tyr?Ile?Asp?Leu?Asp?Glu
20 25 30
Leu?Lys?Ile?Met?Leu?Gln?Ala?Thr?Gly?Glu?Thr?Ile?Thr?Glu?Asp
35 40 45
Asp?Ile?Glu?Glu?Leu?Met?Lys?Asp?Gly?Asp?Lys?Asn?Asn?Asp?Gly
50 55 60
Arg?Ile?Asp?Tyr?Asp?Glu?Phe?Leu?Glu?Phe?Met?Lys?Gly?Val?Glu
65 70 75
<210>4
<211>305
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>4
Met?Asp?Asp?Ile?Tyr?Lys?Ala?Ala?Val?Glu?Gln?Leu?Thr?Glu?Glu
1 5 10 15
Gln?Lys?Asn?Glu?Phe?Lys?Ala?Ala?Phe?Asp?Ile?Phe?Val?Leu?Gly
20 25 30
Ala?Glu?Asp?Gly?Cys?Ile?Ser?Thr?Lys?Glu?Leu?Gly?Lys?Val?Met
35 40 45
Arg?Met?Leu?Gly?Gln?Asn?Pro?Thr?Pro?Glu?Glu?Leu?Gln?Glu?Met
50 55 60
Ile?Asp?Glu?Val?Asp?Glu?Asp?Gly?Ser?Gly?Thr?Val?Asp?Phe?Asp
65 70 75
Glu?Phe?Leu?Val?Met?Met?Val?Arg?Cys?Met?Lys?Asp?Asp?Ser?Lys
80 85 90
Gly?Lys?Ser?Glu?Glu?Met?Ala?Asp?Gly?Ser?Ser?Asp?Ala?Ala?Arg
95 100 105
Glu?Pro?Arg?Pro?Ala?Pro?Ala?Pro?Ile?Arg?Arg?Arg?Ser?Ser?Asn
110 115 120
Tyr?Arg?Ala?Tyr?Ala?Thr?Glu?Pro?His?Ala?Lys?Lys?Lys?Ser?Lys
125 130 135
Ile?Ser?Ala?Ser?Arg?Lys?Leu?Gln?Leu?Lys?Thr?Leu?Leu?Leu?Gln
140 145 150
Ile?Ala?Lys?Gln?Glu?Leu?Glu?Arg?Glu?Ala?Glu?Glu?Arg?Arg?Gly
155 160 165
Glu?Lys?Gly?Arg?Ala?Leu?Ser?Thr?Arg?Cys?Gln?Pro?Leu?Glu?Leu
170 175 180
Ala?Gly?Leu?Gly?Phe?Ala?Glu?Leu?Gln?Asp?Leu?Cys?Arg?Gln?Leu
185 190 195
His?Ala?Arg?Val?Asp?Lys?Val?Asp?Glu?Glu?Arg?Tyr?Asp?Ile?Glu
200 205 210
Ala?Lys?Val?Thr?Lys?Asn?Ile?Thr?Glu?Ile?Ala?Asp?Leu?Thr?Gln
215 220 225
Lys?Ile?Phe?Asp?Leu?Arg?Gly?Lys?Phe?Lys?Arg?Pro?Thr?Leu?Arg
230 235 240
Arg?Val?Arg?Ile?Ser?Ala?Asp?Ala?Met?Met?Gln?Ala?Leu?Leu?Gly
245 250 255
Ala?Arg?Ala?Lys?Glu?Ser?Leu?Asp?Leu?Arg?Ala?His?Leu?Lys?Gln
260 265 270
Val?Lys?Lys?Glu?Asp?Thr?Glu?Lys?Glu?Asn?Arg?Glu?Val?Gly?Asp
275 280 285
Trp?Arg?Lys?Asn?Ile?Asp?Ala?Leu?Ser?Gly?Met?Glu?Gly?Arg?Lys
290 295 300
Lys?Lys?Phe?Glu?Ser
305
<210>5
<211>285
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>5
Met?Ala?Asp?Gly?Ser?Ser?Asp?Ala?Ala?Arg?Glu?Pro?Arg?Pro?Ala
1 5 10 15
Pro?Ala?Pro?Ile?Arg?Arg?Arg?Ser?Ser?Asn?Tyr?Arg?Ala?Tyr?Ala
20 25 30
Thr?Glu?Pro?His?Ala?Lys?Lys?Lys?Ser?Lys?Ile?Ser?Ala?Ser?Arg
35 40 45
Lys?Leu?Gln?Leu?Lys?Thr?Leu?Leu?Leu?Gln?Ile?Ala?Lys?Gln?Glu
50 55 60
Leu?Glu?Arg?Glu?Ala?Glu?Glu?Arg?Arg?Gly?Glu?Lys?Gly?Arg?Ala
65 70 75
Leu?Ser?Thr?Arg?Cys?Gln?Pro?Leu?Glu?Leu?Ala?Gly?Leu?Gly?Phe
80 85 90
Ala?Glu?Leu?Gln?Asp?Leu?Cys?Arg?Gln?Leu?His?Ala?Arg?Val?Asp
95 100 105
Lys?Val?Asp?Glu?Glu?Arg?Tyr?Asp?Ile?Glu?Ala?Lys?Val?Thr?Lys
110 115 120
Asn?Ile?Thr?Glu?Ile?Ala?Asp?Leu?Thr?Gln?Lys?Ile?Phe?Asp?Leu
125 130 135
Arg?Gly?Lys?Phe?Lys?Arg?Pro?Thr?Leu?Arg?Arg?Val?Arg?Ile?Ser
140 145 150
Ala?Asp?Ala?Met?Met?Gln?Ala?Leu?Leu?Gly?Ala?Arg?Ala?Lys?Glu
155 160 165
Ser?Leu?Asp?Leu?Arg?Ala?His?Leu?Lys?Gln?Val?Lys?Lys?Glu?Asp
170 175 180
Thr?Glu?Lys?Glu?Asn?Arg?Glu?Val?Gly?Asp?Trp?Arg?Lys?Asn?Ile
185 190 195
Asp?Ala?Leu?Ser?Gly?Met?Glu?Gly?Arg?Lys?Lys?Lys?Phe?Glu?Ser
200 205 210
Lys?Asp?Asp?Ser?Lys?Gly?Lys?Ser?Glu?Glu?Leu?Ser?Asp?Leu?Phe
215 220 225
Arg?Met?Phe?Asp?Lys?Asn?Ala?Asp?Gly?Tyr?Ile?Asp?Leu?Asp?Glu
230 235 240
Leu?Lys?Ile?Met?Leu?Gln?Ala?Thr?Gly?Glu?Thr?Ile?Thr?Glu?Asp
245 250 255
Asp?Ile?Glu?Glu?Leu?Met?Lys?Asp?Gly?Asp?Lys?Asn?Asn?Asp?Gly
260 265 270
Arg?Ile?Asp?Tyr?Asp?Glu?Phe?Leu?Glu?Phe?Met?Lys?Gly?Val?Glu
275 280 285
<210>6
<211>380
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>6
Met?Asp?Asp?Ile?Tyr?Lys?Ala?Ala?Val?Glu?Gln?Leu?Thr?Glu?Glu
1 5 10 15
Gln?Lys?Asn?Glu?Phe?Lys?Ala?Ala?Phe?Asp?Ile?Phe?Val?Leu?Gly
20 25 30
Ala?Glu?Asp?Gly?Cys?Ile?Ser?Thr?Lys?Glu?Leu?Gly?Lys?Val?Met
35 40 45
Arg?Met?Leu?Gly?Gln?Asn?Pro?Thr?Pro?Glu?Glu?Leu?Gln?Glu?Met
50 55 60
Ile?Asp?Glu?Val?Asp?Glu?Asp?Gly?Ser?Gly?Thr?Val?Asp?Phe?Asp
65 70 75
Glu?Phe?Leu?Val?Met?Met?Val?Arg?Cys?Met?Lys?Asp?Asp?Ser?Lys
80 85 90
Gly?Lys?Ser?Glu?Glu?Met?Ala?Asp?Gly?Ser?Ser?Asp?Ala?Ala?Arg
95 100 105
Glu?Pro?Arg?Pro?Ala?Pro?Ala?Pro?Ile?Arg?Arg?Arg?Ser?Ser?Asn
110 115 120
Tyr?Arg?Ala?Tyr?Ala?Thr?Glu?Pro?His?Ala?Lys?Lys?Lys?Ser?Lys
125 130 135
Ile?Ser?Ala?Ser?Arg?Lys?Leu?Gln?Leu?Lys?Thr?Leu?Leu?Leu?Gln
140 145 150
Ile?Ala?Lys?Gln?Glu?Leu?Glu?Arg?Glu?Ala?Glu?Glu?Arg?Arg?Gly
155 160 165
Glu?Lys?Gly?Arg?Ala?Leu?Ser?Thr?Arg?Cys?Gln?Pro?Leu?Glu?Leu
170 175 180
Ala?Gly?Leu?Gly?Phe?Ala?Glu?Leu?Gln?Asp?Leu?Cys?Arg?Gln?Leu
185 190 195
His?Ala?Arg?Val?Asp?Lys?Val?Asp?Glu?Glu?Arg?Tyr?Asp?Ile?Glu
200 205 210
Ala?Lys?Val?Thr?Lys?Asn?Ile?Thr?Glu?Ile?Ala?Asp?Leu?Thr?Gln
215 220 225
Lys?Ile?Phe?Asp?Leu?Arg?Gly?Lys?Phe?Lys?Arg?Pro?Thr?Leu?Arg
230 235 240
Arg?Val?Arg?Ile?Ser?Ala?Asp?Ala?Met?Met?Gln?Ala?Leu?Leu?Gly
245 250 255
Ala?Arg?Ala?Lys?Glu?Ser?Leu?Asp?Leu?Arg?Ala?His?Leu?Lys?Gln
260 265 270
Val?Lys?Lys?Glu?Asp?Thr?Glu?Lys?Glu?Asn?Arg?Glu?Val?Gly?Asp
275 280 285
Trp?Arg?Lys?Asn?Ile?Asp?Ala?Leu?Ser?Gly?Met?Glu?Gly?Arg?Lys
290 295 300
Lys?Lys?Phe?Glu?Ser?Lys?Asp?Asp?Ser?Lys?Gly?Lys?Ser?Glu?Glu
305 310 315
Leu?Ser?Asp?Leu?Phe?Arg?Met?Phe?Asp?Lys?Asn?Ala?Asp?Gly?Tyr
320 325 330
Ile?Asp?Leu?Asp?Glu?Leu?Lys?Ile?Met?Leu?Gln?Ala?Thr?Gly?Glu
335 340 345
Thr?Ile?Thr?Glu?Asp?Asp?Ile?Glu?Glu?Leu?Met?Lys?Asp?Gly?Asp
350 355 360
Lys?Asn?Asn?Asp?Gly?Arg?Ile?Asp?Tyr?Asp?Glu?Phe?Leu?Glu?Phe
365 370 375
Met?Lys?Gly?Val?Glu
380
<210>7
<211>918
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>7
atggacgaca?tctacaaggc?tgctgtcgag?caattgaccg?aggagcaaaa?gaacgagttc 60
aaggctgctt?tcgacatctt?cgtcttgggt?gctgaggacg?gttgtatctc?caccaaggag 120
ttgggtaagg?tcatgagaat?gttgggtcaa?aacccaaccc?cagaggagtt?gcaagagatg 180
atcgacgagg?tcgacgagga?cggttccggt?accgtcgact?tcgacgagtt?cttggtcatg 240
atggtcagat?gtatgaagga?cgactccaag?ggtaagtccg?aggagatggc?tgacggttcc 300
tccgacgctg?ctagagagcc?aagaccagct?ccagctccaa?tcagaagaag?atcctccaac 360
tacagagctt?acgctaccga?gccacacgct?aagaagaagt?ccaagatctc?cgcttccaga 420
aagttgcaat?tgaagacctt?gttgttgcaa?atcgctaagc?aagagttgga?gagagaggct 480
gaggagagaa?gaggtgagaa?gggtagagct?ttgtccacca?gatgtcaacc?attggagttg 540
gctggtttgg?gtttcgctga?gttgcaagac?ttgtgtagac?aattgcacgc?tagagtcgac 600
aaggtcgacg?aggagagata?cgacatcgag?gctaaggtca?ccaagaacat?caccgagatc 660
gctgacttga?cccaaaagat?cttcgacttg?agaggtaagt?tcaagagacc?aaccttgaga 720
agagtcagaa?tctccgctga?cgctatgatg?caagctttgt?tgggtgctag?agctaaggag 780
tccttggact?tgagagctca?cttgaagcaa?gtcaagaagg?aggacaccga?gaaggagaac 840
agagaggtcg?gtgactggag?aaagaacatc?gacgctttgt?ccggtatgga?gggtagaaag 900
aagaagttcg?agtcctaa 918
<210>8
<211>858
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>8
atggctgacg?gttcctccga?cgctgctaga?gagccaagac?cagctccagc?tccaatcaga 60
agaagatcct?ccaactacag?agcttacgct?accgagccac?acgctaagaa?gaagtccaag 120
atctccgctt?ccagaaagtt?gcaattgaag?accttgttgt?tgcaaatcgc?taagcaagag 180
ttggagagag?aggctgagga?gagaagaggt?gagaagggta?gagctttgtc?caccagatgt 240
caaccattgg?agttggctgg?tttgggtttc?gctgagttgc?aagacttgtg?tagacaattg 300
cacgctagag?tcgacaaggt?cgacgaggag?agatacgaca?tcgaggctaa?ggtcaccaag 360
aacatcaccg?agatcgctga?cttgacccaa?aagatcttcg?acttgagagg?taagttcaag 420
agaccaacct?tgagaagagt?cagaatctcc?gctgacgcta?tgatgcaagc?tttgttgggt 480
gctagagcta?aggagtcctt?ggacttgaga?gctcacttga?agcaagtcaa?gaaggaggac 540
accgagaagg?agaacagaga?ggtcggtgac?tggagaaaga?acatcgacgc?tttgtccggt 600
atggagggta?gaaagaagaa?gttcgagtcc?aaggacgact?ccaagggtaa?gtccgaggag 660
ttgtccgact?tgttcagaat?gttcgacaag?aacgctgacg?gttacatcga?cttggacgag 720
ttgaagatca?tgttgcaagc?taccggtgag?accatcaccg?aggacgacat?cgaggagttg 780
atgaaggacg?gtgacaagaa?caacgacggt?agaatcgact?acgacgagtt?cttggagttc 840
atgaagggtg?tcgagtaa 858
<210>9
<211>918
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>9
atggacgaca?tctacaaagc?tgctgttgaa?cagctgactg?aagaacagaa?aaacgaattc 60
aaagctgctt?tcgacatctt?cgttctgggt?gctgaagacg?gttgcatctc?tactaaagaa 120
ctgggtaaag?ttatgcgtat?gctgggtcag?aacccgactc?cggaagaact?gcaggaaatg 180
atcgacgaag?ttgacgaaga?cggttctggt?actgttgact?tcgacgaatt?cctggttatg 240
atggttcgtt?gcatgaaaga?cgactctaaa?ggtaaatctg?aagaaatggc?tgacggttct 300
tctgacgctg?ctcgtgaacc?gcgtccggct?ccggctccga?tccgtcgtcg?ttcttctaac 360
taccgtgctt?acgctactga?accgcacgct?aaaaaaaaat?ctaaaatctc?tgcttctcgt 420
aaactgcagc?tgaaaactct?gctgctgcag?atcgctaaac?aggaactgga?acgtgaagct 480
gaagaacgtc?gtggtgaaaa?aggtcgtgct?ctgtctactc?gttgccagcc?gctggaactg 540
gctggtctgg?gtttcgctga?actgcaggac?ctgtgccgtc?agctgcacgc?tcgtgttgac 600
aaagttgacg?aagaacgtta?cgacatcgaa?gctaaagtta?ctaaaaacat?cactgaaatc 660
gctgacctga?ctcagaaaat?cttcgacctg?cgtggtaaat?tcaaacgtcc?gactctgcgt 720
cgtgttcgta?tctctgctga?cgctatgatg?caggctctgc?tgggtgctcg?tgctaaagaa 780
tctctggacc?tgcgtgctca?cctgaaacag?gttaaaaaag?aagacactga?aaaagaaaac 840
cgtgaagttg?gtgactggcg?taaaaacatc?gacgctctgt?ctggtatgga?aggtcgtaaa 900
aaaaaattcg?aatcttaa 918
<210>10
<211>858
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>10
atggctgacg?gttcttctga?cgctgctcgt?gaaccgcgtc?cggctccggc?tccgatccgt 60
cgtcgttctt?ctaactaccg?tgcttacgct?actgaaccgc?acgctaaaaa?aaaatctaaa 120
atctctgctt?ctcgtaaact?gcagctgaaa?actctgctgc?tgcagatcgc?taaacaggaa 180
ctggaacgtg?aagctgaaga?acgtcgtggt?gaaaaaggtc?gtgctctgtc?tactcgttgc 240
cagccgctgg?aactggctgg?tctgggtttc?gctgaactgc?aggacctgtg?ccgtcagctg 300
cacgctcgtg?ttgacaaagt?tgacgaagaa?cgttacgaca?tcgaagctaa?agttactaaa 360
aacatcactg?aaatcgctga?cctgactcag?aaaatcttcg?acctgcgtgg?taaattcaaa 420
cgtccgactc?tgcgtcgtgt?tcgtatctct?gctgacgcta?tgatgcaggc?tctgctgggt 480
gctcgtgcta?aagaatctct?ggacctgcgt?gctcacctga?aacaggttaa?aaaagaagac 540
actgaaaaag?aaaaccgtga?agttggtgac?tggcgtaaaa?acatcgacgc?tctgtctggt 600
atggaaggtc?gtaaaaaaaa?attcgaatct?aaagacgact?ctaaaggtaa?atctgaagaa 660
ctgtctgacc?tgttccgtat?gttcgacaaa?aacgctgacg?gttacatcga?cctggacgaa 720
ctgaaaatca?tgctgcaggc?tactggtgaa?actatcactg?aagacgacat?cgaagaactg 780
atgaaagacg?gtgacaaaaa?caacgacggt?cgtatcgact?acgacgaatt?cctggaattc 840
atgaaaggtg?ttgaataa 858
<210>11
<211>1143
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>11
atggacgaca?tctacaaggc?tgctgtcgag?caattgaccg?aggagcaaaa?gaacgagttc 60
aaggctgctt?tcgacatctt?cgtcttgggt?gctgaggacg?gttgtatctc?caccaaggag 120
ttgggtaagg?tcatgagaat?gttgggtcaa?aacccaaccc?cagaggagtt?gcaagagatg 180
atcgacgagg?tcgacgagga?cggttccggt?accgtcgact?tcgacgagtt?cttggtcatg 240
atggtcagat?gtatgaagga?cgactccaag?ggtaagtccg?aggagatggc?tgacggttcc 300
tccgacgctg?ctagagagcc?aagaccagct?ccagctccaa?tcagaagaag?atcctccaac 360
tacagagctt?acgctaccga?gccacacgct?aagaagaagt?ccaagatctc?cgcttccaga 420
aagttgcaat?tgaagacctt?gttgttgcaa?atcgctaagc?aagagttgga?gagagaggct 480
gaggagagaa?gaggtgagaa?gggtagagct?ttgtccacca?gatgtcaacc?attggagttg 540
gctggtttgg?gtttcgctga?gttgcaagac?ttgtgtagac?aattgcacgc?tagagtcgac 600
aaggtcgacg?aggagagata?cgacatcgag?gctaaggtca?ccaagaacat?caccgagatc 660
gctgacttga?cccaaaagat?cttcgacttg?agaggtaagt?tcaagagacc?aaccttgaga 720
agagtcagaa?tctccgctga?cgctatgatg?caagctttgt?tgggtgctag?agctaaggag 780
tccttggact?tgagagctca?cttgaagcaa?gtcaagaagg?aggacaccga?gaaggagaac 840
agagaggtcg?gtgactggag?aaagaacatc?gacgctttgt?ccggtatgga?gggtagaaag 900
aagaagttcg?agtccaagga?cgactccaag?ggtaagtccg?aggagttgtc?cgacttgttc 960
agaatgttcg?acaagaacgc?tgacggttac?atcgacttgg?acgagttgaa?gatcatgttg 1020
caagctaccg?gtgagaccat?caccgaggac?gacatcgagg?agttgatgaa?ggacggtgac 1080
aagaacaacg?acggtagaat?cgactacgac?gagttcttgg?agttcatgaa?gggtgtcgag 1140
taa 1143
<210>12
<211>1143
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>12
atggacgaca?tctacaaagc?tgctgttgaa?cagctgactg?aagaacagaa?aaacgaattc 60
aaagctgctt?tcgacatctt?cgttctgggt?gctgaagacg?gttgcatctc?tactaaagaa 120
ctgggtaaag?ttatgcgtat?gctgggtcag?aacccgactc?cggaagaact?gcaggaaatg 180
atcgacgaag?ttgacgaaga?cggttctggt?actgttgact?tcgacgaatt?cctggttatg 240
atggttcgtt?gcatgaaaga?cgactctaaa?ggtaaatctg?aagaaatggc?tgacggttct 300
tctgacgctg?ctcgtgaacc?gcgtccggct?ccggctccga?tccgtcgtcg?ttcttctaac 360
taccgtgctt?acgctactga?accgcacgct?aaaaaaaaat?ctaaaatctc?tgcttctcgt 420
aaactgcagc?tgaaaactct?gctgctgcag?atcgctaaac?aggaactgga?acgtgaagct 480
gaagaacgtc?gtggtgaaaa?aggtcgtgct?ctgtctactc?gttgccagcc?gctggaactg 540
gctggtctgg?gtttcgctga?actgcaggac?ctgtgccgtc?agctgcacgc?tcgtgttgac 600
aaagttgacg?aagaacgtta?cgacatcgaa?gctaaagtta?ctaaaaacat?cactgaaatc 660
gctgacctga?ctcagaaaat?cttcgacctg?cgtggtaaat?tcaaacgtcc?gactctgcgt 720
cgtgttcgta?tctctgctga?cgctatgatg?caggctctgc?tgggtgctcg?tgctaaagaa 780
tctctggacc?tgcgtgctca?cctgaaacag?gttaaaaaag?aagacactga?aaaagaaaac 840
cgtgaagttg?gtgactggcg?taaaaacatc?gacgctctgt?ctggtatgga?aggtcgtaaa 900
aaaaaattcg?aatctaaaga?cgactctaaa?ggtaaatctg?aagaactgtc?tgacctgttc 960
cgtatgttcg?acaaaaacgc?tgacggttac?atcgacctgg?acgaactgaa?aatcatgctg 1020
caggctactg?gtgaaactat?cactgaagac?gacatcgaag?aactgatgaa?agacggtgac 1080
aaaaacaacg?acggtcgtat?cgactacgac?gaattcctgg?aattcatgaa?aggtgttgaa 1140
taa 1143
<210>13
<211>633
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>13
atggctgacg?gttcctccga?cgctgctaga?gagccaagac?cagctccagc?tccaatcaga 60
agaagatcct?ccaactacag?agcttacgct?accgagccac?acgctaagaa?gaagtccaag 120
atctccgctt?ccagaaagtt?gcaattgaag?accttgttgt?tgcaaatcgc?taagcaagag 180
ttggagagag?aggctgagga?gagaagaggt?gagaagggta?gagctttgtc?caccagatgt 240
caaccattgg?agttggctgg?tttgggtttc?gctgagttgc?aagacttgtg?tagacaattg 300
cacgctagag?tcgacaaggt?cgacgaggag?agatacgaca?tcgaggctaa?ggtcaccaag 360
aacatcaccg?agatcgctga?cttgacccaa?aagatcttcg?acttgagagg?taagttcaag 420
agaccaacct?tgagaagagt?cagaatctcc?gctgacgcta?tgatgcaagc?tttgttgggt 480
gctagagcta?aggagtcctt?ggacttgaga?gctcacttga?agcaagtcaa?gaaggaggac 540
accgagaagg?agaacagaga?ggtcggtgac?tggagaaaga?acatcgacgc?tttgtccggt 600
atggagggta?gaaagaagaa?gttcgagtcc?taa 633
<210>14
<211>633
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>14
atggctgacg?gttcttctga?cgctgctcgt?gaaccgcgtc?cggctccggc?tccgatccgt 60
cgtcgttctt?ctaactaccg?tgcttacgct?actgaaccgc?acgctaaaaa?aaaatctaaa 120
atctctgctt?ctcgtaaact?gcagctgaaa?actctgctgc?tgcagatcgc?taaacaggaa 180
ctggaacgtg?aagctgaaga?acgtcgtggt?gaaaaaggtc?gtgctctgtc?tactcgttgc 240
cagccgctgg?aactggctgg?tctgggtttc?gctgaactgc?aggacctgtg?ccgtcagctg 300
cacgctcgtg?ttgacaaagt?tgacgaagaa?cgttacgaca?tcgaagctaa?agttactaaa 360
aacatcactg?aaatcgctga?cctgactcag?aaaatcttcg?acctgcgtgg?taaattcaaa 420
cgtccgactc?tgcgtcgtgt?tcgtatctct?gctgacgcta?tgatgcaggc?tctgctgggt 480
gctcgtgcta?aagaatctct?ggacctgcgt?gctcacctga?aacaggttaa?aaaagaagac 540
actgaaaaag?aaaaccgtga?agttggtgac?tggcgtaaaa?acatcgacgc?tctgtctggt 600
atggaaggtc?gtaaaaaaaa?attcgaatct?taa 633
Claims (10)
1, antitumor recombinant protein is selected from the following protein families any one:
(1) the N-terminal amino-acid residue of cardiac muscle troponin I and the part or all of protein that merges mutually of cardiac troponin C carboxyl terminal amino acid residue sequence;
(2) the C-terminal amino-acid residue of cardiac muscle troponin I and the part or all of protein that merges mutually of cardiac troponin C aminoterminal amino acid residue sequence;
(3) the N-terminal amino-acid residue of cardiac muscle troponin I and cardiac troponin C carboxyl terminal amino acid residue sequence partly or entirely, the part or all of protein of fusion mutually of the C-terminal amino-acid residue of cardiac muscle troponin I and cardiac troponin C aminoterminal amino acid residue sequence.
2, antitumor recombinant protein according to claim 1 is characterized in that: described antitumor recombinant protein is SEQ ID № in the sequence table: 4 amino acid residue sequence or with SEQ ID №: 4 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 4 is identical active by SEQ ID №: 4 deutero-protein.
3, antitumor recombinant protein according to claim 1 is characterized in that: described antitumor recombinant protein is SEQ ID № in the sequence table: 5 amino acid residue sequence or with SEQ ID №: 5 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 5 is identical active by SEQ ID №: 5 deutero-protein.
4, antitumor recombinant protein according to claim 1 is characterized in that: described antitumor recombinant protein is SEQ ID № in the sequence table: 6 amino acid residue sequence or with SEQ ID №: 6 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 6 is identical active by SEQ ID №: 6 deutero-protein.
5, the encoding gene of the described antitumor recombinant protein of claim 1 is one of following nucleotide sequences:
(1) the SEQ ID № in the sequence table: 7 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(2) the SEQ ID № in the sequence table: 8 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(3) the SEQ ID № in the sequence table: 9 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(4) the SEQ ID № in the sequence table: 10 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(5) the SEQ ID № in the sequence table: 11 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(6) the SEQ ID № in the sequence table: 12 or have 90% above homology with the dna sequence dna of its qualification, and coding identical function protein DNA sequence;
(7) SEQ ID № in the code sequence tabulation: the polynucleotide of april protein sequence;
(8) SEQ ID № in the code sequence tabulation: the polynucleotide of 5 protein sequences;
(9) SEQ ID № in the code sequence tabulation: the polynucleotide of 6 protein sequences.
6, contain described expression carrier of claim 5 and clone.
7, a kind of method that efficiently expresses the described antitumor recombinant protein of claim 1 is that the gene with the antitumor recombinant protein of coding imports in the Pichia yeast, express to obtain antitumor recombinant protein.
8, method according to claim 7 is characterized in that: induce with methyl alcohol in the expression process of Pichia yeast in the described method; Induce the methyl alcohol final concentration in the process to be preferably 1%-5%; Described Pichia yeast is the GS115 Pichia yeast.
9, a kind of method that efficiently expresses the described antitumor recombinant protein of claim 1 is that the gene with the antitumor recombinant protein of coding imports in the intestinal bacteria, express to obtain antitumor recombinant protein.
10, method according to claim 9 is characterized in that: described intestinal bacteria are preferably the BL21-DE3 intestinal bacteria.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031097871A CN100334111C (en) | 2003-04-21 | 2003-04-21 | Recombined protein of anti tumour, encoded gene and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031097871A CN100334111C (en) | 2003-04-21 | 2003-04-21 | Recombined protein of anti tumour, encoded gene and application |
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| Publication Number | Publication Date |
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| CN1539850A true CN1539850A (en) | 2004-10-27 |
| CN100334111C CN100334111C (en) | 2007-08-29 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245102A (en) * | 2016-12-30 | 2017-10-13 | 广西壮族自治区药用植物园 | Antitumor recombinant protein GPTI and its encoding gene and application |
| CN107245103A (en) * | 2016-12-30 | 2017-10-13 | 广西壮族自治区药用植物园 | Antitumor recombinant protein IFTI and its encoding gene and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6475785B1 (en) * | 1997-12-18 | 2002-11-05 | Spectral Diagnostics, Inc. | Single-chain polypeptides comprising troponin I N-terminal fragments and troponin C |
| AU1693199A (en) * | 1997-12-27 | 1999-07-19 | Matsushita Electric Industrial Co., Ltd. | Troponin ci complexes |
-
2003
- 2003-04-21 CN CNB031097871A patent/CN100334111C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245102A (en) * | 2016-12-30 | 2017-10-13 | 广西壮族自治区药用植物园 | Antitumor recombinant protein GPTI and its encoding gene and application |
| CN107245103A (en) * | 2016-12-30 | 2017-10-13 | 广西壮族自治区药用植物园 | Antitumor recombinant protein IFTI and its encoding gene and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100334111C (en) | 2007-08-29 |
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