CN1523036A - Anti-coagulate protein and its coding gene and high effective expression method - Google Patents
Anti-coagulate protein and its coding gene and high effective expression method Download PDFInfo
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- CN1523036A CN1523036A CNA031046614A CN03104661A CN1523036A CN 1523036 A CN1523036 A CN 1523036A CN A031046614 A CNA031046614 A CN A031046614A CN 03104661 A CN03104661 A CN 03104661A CN 1523036 A CN1523036 A CN 1523036A
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Abstract
The present invention discloses an anticoaglant protein, its coding gene and its high-effective expression method. Said invention is aimed at providing a new artificial synthetic anticoagulant protein and its coding gene. Said anticoagulant protein has the anticoagulant protein C2 sample action, at the same time can be quickly reversal under the necessary condition. Said anticoaglant protein is the protein obtained by substituting partial or all the amino acid residues in one or several cysteinyl functional construction zone in nemic anticoagulant protein C2 by thrombase cleavage site or inserting the thrombase cleavage site into one or several cysteinyl functional construction zone of nemic anticoagulant protein C2. Said invention has important action for preventing and curing angiocardiopathy and cerebrovascular disease.
Description
Technical field
The present invention relates to a kind of anticoagulant protein in the bioengineering field and the high-efficiency expression method of encoding gene and this gene thereof.
Background technology
Thrombus be the lumen of vessels inner blood solidify or blood in the solid grumeleuse that forms of the mutual aggegation of some formed elements.Influence thrombotic factor and can be divided into blood vessel and blood two big classes.Vascular factor mainly is damaged relevant with the integrity of tunica intima.The blood factor then comprises multiple thrombin and hematoblastic interaction, causes the formation of blood inner fibrin and accumulative thrombocyte to form thrombus jointly with other formed elements.Thrombin in the blood comprises that factor I is multiple to factor XI, plasma thromboplastin antecedent I etc., and wherein modal is factor II, VII, IX and X to the anti-freezing drug influence.
Disturb the medicine of blood coagulation process, be called the anti-freezing medicine, be widely used in the prevention and the treatment of blood embolization disease clinically.The present antithrombotics that uses clinically, as heparin, r-hirudin, temparin, Antithrombin IIIs etc. all show in the use to be mixed with some untoward reactions, as the thrombopenic purpura due to the heparin.Therefore, seek new effectively, and the few antithrombotics of untoward reaction is significant.
The anticoagulant protein C2 that extracts from the dog nematode has tangible anticoagulation (seeing United States Patent (USP) 5866543), and its effect is relevant with inhibition activated factor VIIa, but the activity of factor IIa (zymoplasm) is not had influence.The polypeptide that this proteic functional part is made up of 84 amino-acid residues can prolong the clotting time, and activity is better than heparin, and its protein sequence and encoding gene thereof are seen sequence 2 and the sequence 1 in the sequence table.Including 10 halfcystines in the structure of nematode anticoagulant protein C2, is respectively its functional structure district.How resisting the blood coagulation resisting function of anticoagulant effectively, reduce the incidence of bleeding episode, is the emphasis that present anti-freezing and antithrombotic class medicine are paid close attention to.Anticoagulant protein C2 has significant anticoagulating active, but its vivo degradation still act as the master with nonspecific proteolytic enzyme, influence it at the quick reversal procedures that uses the anticoagulating active under the excessive situation, therefore, develop a kind of anticoagulant protein C2 sample effect that both had, the product that its effect simultaneously can reverse rapidly under the situation of needs aspect the preventing and treating of cardiovascular and cerebrovascular diseases, has the important clinical meaning.
Pichia yeast expression system is 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 a kind of anticoagulant protein and encoding gene thereof of new synthetic, this anticoagulant protein had both had the effect of anticoagulant protein C2 sample, can reverse rapidly under the situation of needs simultaneously.
A kind of anticoagulant protein, be that the part or all of amino-acid residue in one or more cysteinyl functional structure districts among the nematode anticoagulant protein C2 is replaced with the blood coagulation restriction enzyme site, or in one or more cysteinyl functional structure districts of nematode anticoagulant protein C2, insert the protein that the blood coagulation restriction enzyme site obtains.
Described cysteinyl functional structure district is the peptide that 2-6 amino-acid residue linking to each other with the cysteine residues carboxyl terminal formed.
Described blood coagulation restriction enzyme site be include from amino be the small peptide of forming by 2-6 amino-acid residue of arginine-glycine to carboxyl terminal.This blood coagulation restriction enzyme site preferably from aminoterminal successively by peptide chain that leucine-Xie Ansuan-proline(Pro)-arginine-6 amino-acid residues of glycine-Serine are formed.
The above-mentioned proteinic gene of encoding also belongs to protection scope of the present invention.
Second purpose of the present invention provides a kind of high-efficiency expression method of above-mentioned coding anticoagulant protein gene, makes it adapt to needs of scale production.
The high-efficiency expression method of anticoagulant protein gene is that the gene with the coding anticoagulant protein imports in the Pichia yeast, express to obtain the nematode anticoagulant protein.
Specifically, this method may further comprise the steps:
1) synthetic nematode anticoagulant protein gene order;
2) the synthetic gene fragment is connected with carrier pPIC9K, 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%.
The present invention very puts into the melting pot nematode anticoagulant protein C2 dexterously, fundamentally solved the defective that nematode anticoagulant protein C2 can not reverse fast in needs, can rapidly anticoagulation be reversed in the case of necessary, avoid the generation of profuse bleeding, aspect the preventing and treating of cardiovascular and cerebrovascular diseases, significant, be that the medicine of activeconstituents is the higher anticoagulant of a kind of safety control coefficient with the present invention.
Description of drawings
Fig. 1 is a PCR method synthetic gene fragment.
Fig. 2 is complete genome assembling synoptic diagram of the present invention
Fig. 3 is the structure collection of illustrative plates of expression plasmid
Fig. 4 cuts signing for the enzyme of expression plasmid
Fig. 5 is that the SDS-PAGE of abduction delivering analyzes
Fig. 6 is the activity identification of abduction delivering product
Embodiment
Synthesizing of embodiment 1, expressing gene
1, design anticoagulant protein gene order:
SEQ ID №: 3, at blood coagulation restriction enzyme site of the interval design of first and second halfcystines;
SEQ ID №: 5, at blood coagulation restriction enzyme site of the interval design of the 4th and the 5th halfcystine;
SEQ ID №: 7, respectively design a blood coagulation restriction enzyme site in first and second halfcystines intervals and the 5th and the 6th halfcystine interval;
SEQ ID №: 9, at blood coagulation restriction enzyme site of the interval design of the 8th and the 9th halfcystine;
SEQ ID №: 11, at blood coagulation restriction enzyme site of the interval design of the 9th and the tenth halfcystine.
2, the synthetic of small segment: with SEQ ID №: 3,5,7,9,11 anticoagulant protein genes are divided into two sections respectively, and first section sequence is cDNA normal chain, and second section sequence is the cDNA complementary strand.3 ' end at first section adds 8 and second section 3 ' complementary base, 150 bases of length overall, be respectively the sequence SEQ ID № in the sequence table: 13,15,17,19,21, with its difference called after sequence A, B, C, D, E, add 12 and first section 3 ' end complementary base at second section 3 ' end, be the SEQ ID № in the sequence table: 14,16,18,20, length overall is respectively 147,147,153,147,150 bases.Add SnaBI and NotI restriction enzyme site respectively at two fragments, 5 ' end, synthetic with dna synthesizer.
With dna synthesizer synthetic fragment, the complete sequence Syncumar protein gene that connects composition sequence A, B, C, D, E with single round-robin PCR reaction respectively, the result as shown in Figure 1, wherein A, B, C, D, E swimming lane are respectively sequence A, B, C, D, E, show that the sequence that obtains is correct.
Embodiment 2, anticoagulant protein vivoexpression of the present invention
1, cut PCR fragment among the embodiment 1, in the pGEM-T-EASY plasmid of packing into, make up pGEM-ACPA, pGEM-ACPB, pGEM-ACPC, pGEM-ACPD, pGEM-ACPE cloning vector, its structure collection of illustrative plates as shown in Figure 2.
2, the structure of pGEM-ACP expression plasmid
Employing is available from the efficient expression plasmid pPIC9K of American I nvitrogen company, with SnaBI and NotI respectively enzyme cut pGEM-ACP 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 pPIC-ACPA, pPIC-ACPB, pPIC-ACPC, pPIC-ACPD, pPIC-ACPE, and its structure collection of illustrative plates is as shown in Figure 3.Enzyme is cut evaluation, proves that the structure of expression plasmid is correct, Figure 4 shows that the different clones' of pPIC-ACPA restriction enzyme digestion and electrophoresis result.
3, with plasmid pPIC-ACP SacI linearizing. adopt Zhejiang Xin Zhi company electricity gene introducing apparatus, linearizing pPIC-ACP is imported in the GS115 Pichia yeast (available from Invitrogen company),, select through cultivating, processes such as screening obtain the mono-clonal bacterium of anti-G418mg/ml.
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.As seen the result in the position of 18KD, has the inductive protein expression, and according to the BSA contrast, the estimation expression amount is 100-150mg/L.
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 5, 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.
The functional examination of embodiment 3, anticoagulant protein of the present invention
Get 6 test tubes, be denoted as supernatant A, supernatant B behind physiological saline, the methanol induction, supernatant C, supernatant D, supernatant E, add corresponding liquid 10ul/ pipe, the rabbit arterial blood 40ul/ pipe that adds fresh collection then, mixing, the record blood coagulation time, the result as shown in Figure 6, wherein the calculation formula of blood clotting inhibiting rate is mensuration group blood coagulation pipe number/physiological saline group blood coagulation pipe number when reacting 1 hour, salt solution (NS) formed blood clot in 1.82 minutes, all are induced the back supernatant still not have blood clot after 12 hours and form.Induce the back supernatant: rabbit arterial blood is 1: 4 o'clock, and the blood clotting inhibiting rate is 100%, shows that expression product all has good anticoagulating active.
The zymoplasm of embodiment 4, anticoagulant protein of the present invention is cut reacted functional examination
Get 6 test tubes, be denoted as supernatant A, supernatant B behind physiological saline, the methanol induction, supernatant C, supernatant D, supernatant E, add corresponding liquid 10ul/ pipe, the thrombin of beef that adds 0.1 unit, hatched 30 minutes for 37 ℃, the rabbit arterial blood 50ul/ pipe that adds fresh collection then, mixing, the record blood coagulation time, salt solution (NS) formed blood clot in 1.90 minutes as a result, and supernatant A, supernatant B, supernatant C, supernatant D, supernatant E are respectively at forming blood clot in 13 minutes, 14.5 minutes, 5 minutes, 8 minutes and 25 minutes, showing that expression product can be degraded by zymoplasm in time.
Sequence table
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tgcctagttc?gtgtctgtca?ccaagactgc?gtttgcgagg?agggtttcta?cagaaacaag 180
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ggtactcgaa?actga 255
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＜213〉the nematode nematode (ancylostoma caninum) of being born in the year of dog
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50 55 60
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65 70 75
Asp?Phe?Ile?Tyr?Pro?Gly?Thr?Arg?Asn
80 84
<210>3
<211>255
<212>DNA
＜213〉artificial sequence
<220>
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tgcctagttc?gtgtctgtca?ccaagactgc?gtttgcgagg?agggtttcta?cagaaacaag 180
gacgacaagt?gtgtttctgc?tgaggactgc?gagcttgaca?atatggactt?catttaccca 240
ggtactcgaa?actga 255
<210>4
<211>84
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＜213〉artificial sequence
<220>
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Lys?Ala?Thr?Met?Gln?Cys?Leu?Val?Pro?Arg?Gly?Ser?Asp?Ser?Cys
1 5 10 15
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20 25 30
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35 40 45
Cys?His?Gln?Asp?Cys?Val?Cys?Glu?Glu?Gly?Phe?Tyr?Arg?Asn?Lys
50 55 60
Asp?Asp?Lys?Cys?Val?Ser?Ala?Glu?Asp?Cys?Glu?Leu?Asp?Asn?Met
65 70 75
Asp?Phe?Ile?Tyr?Pro?Gly?Thr?Arg?Asn
80 84
<210>5
<211>255
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＜213〉artificial sequence
<220>
<223>
<400>5
aaggctacca?tgcagtgtgg?tgagaatgag?aagtacgact?cctgcggtag?caaggagtgt 60
gacaagaagt?gtttggttcc?aagaggttgc?gaggaggacg?acgaggagcc?aaatgtccca 120
tgcctagttc?gtgtctgtca?ccaagactgc?gtttgcgagg?agggtttcta?cagaaacaag 180
gacgacaagt?gtgtttctgc?tgaggactgc?gagcttgaca?atatggactt?catttaccca 240
ggtactcgaa?actga 255
<210>6
<211>84
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>6
Lys?Ala?Thr?Met?Gln?Cys?Gly?Glu?Asn?Glu?Lys?Tyr?Asp?Ser?Cys
1 5 10 15
Gly?Ser?Lys?Glu?Cys?Asp?Lys?Lys?Cys?Leu?Val?Pro?Arg?Gly?Ser
20 25 30
Glu?Glu?Asp?Asp?Glu?Glu?Pro?Asn?Val?Pro?Cys?Leu?Val?Arg?Val
35 40 45
Cys?His?Gln?Asp?Cys?Val?Cys?Glu?Glu?Gly?Phe?Tyr?Arg?Asn?Lys
50 55 60
Asp?Asp?Lys?Cys?Val?Ser?Ala?Glu?Asp?Cys?Glu?Leu?Asp?Asn?Met
65 70 75
Asp?Phe?Ile?Tyr?Pro?Gly?Thr?Arg?Asn
80 84
<210>7
<211>261
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>7
aaggctacca?tgcagtgttt?ggttccaaga?ggttgcgact?cctgcggtag?caaggagtgt 60
gacaagaagt?gtaagtacga?cggtgttgag?gaggaggacg?acgaggagcc?aaatgtccca 120
tgcttggttc?caagaggttg?ctgtcaccaa?gactgcgttt?gcgaggaggg?tttctacaga 180
aacaaggacg?acaagtgtgt?ttctgctgag?gactgcgagc?ttgacaatat?ggacttcatt 240
tacccaggta?ctcgaaactg?a 261
<210>8
<211>86
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>8
Lys?Ala?Thr?Met?Gln?Cys?Leu?Val?Pro?Arg?Gly?Ser?Asp?Ser?Cys
1 5 10 15
Gly?Ser?Lys?Glu?Cys?Asp?Lys?Lys?Cys?Lys?Tyr?Asp?Gly?Val?Glu
20 25 30
Glu?Glu?Asp?Asp?Glu?Glu?Pro?Asn?Val?Pro?Cys?Leu?Val?Pro?Arg
35 40 45
Gly?Ser?Cys?His?Gln?Asp?Cys?Val?Cys?Glu?Glu?Gly?Phe?Tyr?Arg
50 55 60
Asn?Lys?Asp?Asp?Lys?Cys?Val?Ser?Ala?Glu?Asp?Cys?Glu?Leu?Asp
65 70 75
Asn?Met?Asp?Phe?Ile?Tyr?Pro?Gly?Thr?Arg?Asn
80 86
<210>9
<211>255
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>9
aaggctacca?tgcagtgtgg?tgagaatgag?aagtacgact?cctgcggtag?caaggagtgt 60
gacaagaagt?gtaagtacga?cggtgttgag?gaggaggacg?acgaggagcc?aaatgtccca 120
tgcctagttc?gtgtctgtca?ccaagactgc?gtttgcttgg?ttccaagagg?ttgcaacaag 180
gacgacaagt?gtgtttctgc?tgaggactgc?gagcttgaca?atatggactt?catttaccca 240
ggtactcgaa?actga 255
<210>10
<211>84
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>10
Lys?Ala?Thr?Met?Gln?Cys?Gly?Glu?Asn?Glu?Lys?Tyr?Asp?Ser?Cys
1 5 10 15
Gly?Ser?Lys?Glu?Cys?Asp?Lys?Lys?Cys?Lys?Tyr?Asp?Gly?Val?Glu
20 25 30
Glu?Glu?Asp?Asp?Glu?Glu?Pro?Asn?Val?Pro?Cys?Leu?Val?Arg?Val
35 40 45
Cys?His?Gln?Asp?Cys?Val?Cys?Leu?Val?Pro?Arg?Gly?Ser?Asn?Lys
50 55 60
Asp?Asp?Lys?Cys?Val?Ser?Ala?Glu?Asp?Cys?Glu?Leu?Asp?Asn?Met
65 70 75
Asp?Phe?Ile?Tyr?Pro?Gly?Thr?Arg?Asn
80 84
<210>11
<211>85
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>11
aaggctacca?tgcagtgtgg?tgagaatgag?aagtacgact?cctgcggtag?caaggagtgt 60
gacaagaagt?gtaagtacga?cggtgttgag?gaggaggacg?acgaggagcc?aaatgtccca 120
tgcctagttc?gtgtctgtca?ccaagactgc?gtttgcgagg?agggtttcta?cagaaacaag 180
gacgacaagt?gtttggttcc?aagaggttgc?tgcgagcttg?acaatatgga?cttcatttac 240
ccaggtactc?gaaactga 258
<210>12
<211>85
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>12
Lys?Ala?Thr?Met?Gln?Cys?Gly?Glu?Asn?Glu?Lys?Tyr?Asp?Ser?Cys
1 5 10 15
Gly?Ser?Lys?Glu?Cys?Asp?Lys?Lys?Cys?Lys?Tyr?Asp?Gly?Val?Glu
20 25 30
Glu?Glu?Asp?Asp?Glu?Glu?Pro?Asn?Val?Pro?Cys?Leu?Val?Arg?Val
35 40 45
Cys?His?Gln?Asp?Cys?Val?Cys?Glu?Glu?Gly?Phe?Tyr?Arg?Asn?Lys
50 55 60
Asp?Asp?Lys?Cys?Leu?Val?Pro?Arg?Gly?Ser?Cys?Glu?Leu?Asp?Asn
65 70 75
Met?Asp?Phe?Ile?Tyr?Pro?Gly?Thr?Arg?Asn
80 85
<210>13
<211>150
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>13
gaagcttacg?taaaggctac?catgcagtgt?ttggttccaa?gaggttgcga?ctcctgcggt 60
agcaaggagt?gtgacaagaa?gtgtaagtac?gacggtgttg?aggaggagga?cgacgaggag 120
ccaaatgtcc?catgcctagt?tcgtgtctgt 150
<210>14
<211>141
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>14
attcgcggcc?gctcagtttc?gagtacctgg?gtaaatgaag?tccatattgt?caagctcgca 60
gtcctcagca?gaaacacact?tgtcgtcctt?gtttctgtag?aaaccctcct?cgcaaacgca 120
gtcttggtga?cagacacgaa?c 141
<210>15
<211>150
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>15
gaagcttacg?taaaggctac?catgcagtgt?ggtgagaatg?agaagtacga?ctcctgcggt 60
agcaaggagt?gtgacaagaa?gtgtttggtt?ccaagaggtt?gcgaggagga?cgacgaggag 120
ccaaatgtcc?catgcctagt?tcgtgtctgt 150
<210>16
<211>141
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>16
attcgcggcc?gctcagtttc?gagtacctgg?gtaaatgaag?tccatattgt?caagctcgca 60
gtcctcagca?gaaacacact?tgtcgtcctt?gtttctgtag?aaaccctcct?cgcaaacgca 120
gtcttggtga?cagacacgaa?c 141
<210>17
<211>150
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>17
gaagcttacg?taaaggctac?catgcagtgt?ttggttccaa?gaggttgcga?ctcctgcggt 60
agcaaggagt?gtgacaagaa?gtgtaagtac?gacggtgttg?aggaggagga?cgacgaggag 120
ccaaatgtcc?catgcttggt?tccaagaggt 150
<210>18
<211>147
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>18
attcgcggcc?gctcagtttc?gagtacctgg?gtaaatgaag?tccatattgt?caagctcgca 60
gtcctcagca?gaaacacact?tgtcgtcctt?gtttctgtag?aaaccctcct?cgcaaacgca 120
gtcttggtga?cagcaacctc?ttggaac 147
<210>19
<211>150
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>19
gaagcttacg?taaaggctac?catgcagtgt?ggtgagaatg?agaagtacga?ctcctgcggt 60
agcaaggagt?gtgacaagaa?gtgtaagtac?gacggtgttg?aggaggagga?cgacgaggag 120
ccaaatgtcc?catgcctagt?tcgtgtctgt 150
<210>20
<211>141
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>20
attcgcggcc?gctcagtttc?gagtacctgg?gtaaatgaag?tccatattgt?caagctcgca 60
gtcctcagca?gaaacacact?tgtcgtcctt?gttgcaacct?cttggaacca?agcaaacgca 120
gtcttggtga?cagacacgaa?c 141
<210>21
<211>150
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>21
gaagcttacg?taaaggctac?catgcagtgt?ggtgagaatg?agaagtacga?ctcctgcggt 60
agcaaggagt?gtgacaagaa?gtgtaagtac?gacggtgttg?aggaggagga?cgacgaggag 120
ccaaatgtcc?catgcctagt?tcgtgtctgt 150
<210>22
<211>144
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>22
attcgcggcc?gctcagtttc?gagtacctgg?gtaaatgaag?tccatattgt?caagctcgca 60
gcaacctctt?ggaaccaaac?acttgtcgtc?cttgtttctg?tagaaaccct?cctcgcaaac 120
gcagtcttgg?tgacagacac?gaac 144
Claims (9)
1, a kind of anticoagulant protein, be that the part or all of amino-acid residue in one or more cysteinyl functional structure districts among the nematode anticoagulant protein C2 is replaced with the blood coagulation restriction enzyme site, or in one or more cysteinyl functional structure districts of nematode anticoagulant protein C2, insert the protein that the blood coagulation restriction enzyme site obtains.
2, anticoagulant protein according to claim 1 is characterized in that: described cysteinyl functional structure district is meant the aminoacid sequence between nematode anticoagulant protein C2 two adjacent cysteine residues.
3, anticoagulant protein according to claim 1 is characterized in that: described blood coagulation restriction enzyme site is to include from the small peptide 2-6 amino-acid residue be made of of aminoterminal for arginine-glycine.
4, anticoagulant protein according to claim 1 is characterized in that: described blood coagulation restriction enzyme site for from aminoterminal successively by peptide chain that LEU-VAL-proline(Pro)-6 amino-acid residues of arginine-glycine-Serine are formed.
5, the described proteinic gene of coding claim 1-4.
6, the high-efficiency expression method of anticoagulant protein gene is that the gene with claim 5 imports in the Pichia yeast, expresses obtaining the nematode anticoagulant protein.
7, method according to claim 6 is characterized in that: this method may further comprise the steps:
1) synthetic nematode anticoagulant protein gene order;
2) the synthetic gene fragment is connected with carrier pPIC9K, 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.
8, according to claim 6 or 7 described methods, it is characterized in that: induce with methyl alcohol in the expression process of Pichia yeast.
9, method according to claim 8 is characterized in that: inducing the methyl alcohol final concentration in the process is 1%-5%.
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|---|---|---|---|
| CNB031046614A CN100355783C (en) | 2003-02-20 | 2003-02-20 | Anti-coagulate protein and its coding gene and high effective expression method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031046614A CN100355783C (en) | 2003-02-20 | 2003-02-20 | Anti-coagulate protein and its coding gene and high effective expression method |
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| Publication Number | Publication Date |
|---|---|
| CN1523036A true CN1523036A (en) | 2004-08-25 |
| CN100355783C CN100355783C (en) | 2007-12-19 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101041690B (en) * | 2006-03-22 | 2010-10-06 | 北京大学 | Recombinant dog hookworm coagulate peptide resistant 5 mutant, its encoding gene, preparation and application thereof |
| CN102617722A (en) * | 2007-03-05 | 2012-08-01 | 广东医学院 | Ancylostoma caninum anticoagulant peptide, preparation and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0788546B9 (en) * | 1994-10-18 | 2007-06-13 | Dendreon Corporation | Nematode-extracted serine protease inhibitors and anticoagulant proteins |
| CN1194009C (en) * | 2000-09-12 | 2005-03-23 | 南京大学 | Anticoagulant protein mediated by anticoagulant protein of human placenta |
-
2003
- 2003-02-20 CN CNB031046614A patent/CN100355783C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101041690B (en) * | 2006-03-22 | 2010-10-06 | 北京大学 | Recombinant dog hookworm coagulate peptide resistant 5 mutant, its encoding gene, preparation and application thereof |
| CN102617722A (en) * | 2007-03-05 | 2012-08-01 | 广东医学院 | Ancylostoma caninum anticoagulant peptide, preparation and application thereof |
| CN102617722B (en) * | 2007-03-05 | 2013-10-16 | 广东医学院 | Ancylostoma caninum anticoagulant peptide, preparation and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100355783C (en) | 2007-12-19 |
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