CN102153640A - Anticoagulation protein of tick and gene and application thereof - Google Patents
Anticoagulation protein of tick and gene and application thereof Download PDFInfo
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Abstract
The invention discloses an anticoagulation protein of a tick and a gene and application thereof. The anticoagulation protein is an anticoagulation protein Rhipilin-2 of rhipicephalus haemaphysaloides, and has an amino acid sequence shown as SEQ ID NO.1. The invention also discloses an anticoagulation protein Rhipilin-2 gene of the rhipicephalus haemaphysaloides. The gene comprises a nucleotide sequence of an amino acid sequence shown as code SEQ ID NO.1. The anticoagulation protein Rhipilin-2 of rhipicephalus haemaphysaloides has remarkable anticoagulation action, and is suitable for preparing an anticoagulation medicament or an anticoagulation biological preparation.
Description
Technical field
The present invention relates to technical field of bioengineering, relate in particular to a kind of anticoagulant protein Rhipilin-2 and gene and application of tick.
Background technology
In the organic evolution process, the blood coagulation system that Mammals has formed a complexity stops the loss of blood, and often has the coagulation function of a lot of anticoagulant proteins with the antagonism host in the organism of much making a living with blood.Tick is the vermin that a class is made a living to suck blood.Compare with other HAEMATOPHAGOUS ARTHROPODS, the time of sucking blood of tick is long especially, as the time of sucking blood of hard tick 10-14 days (Sonenshine, 1993).The process of sucking blood for a long time of tick must cause a series of hemostasis reactions of host, as vasoconstriction, platelet aggregation and blood coagulation.For sucking blood of continuing, tick must overcome host's hemostasis reaction, since Waxman (1990) identifies the anticoagulation molecule of first tick, the researchist finds that the sialisterium of tick is secreted and produces multiple anticoagulation molecule in the process of sucking blood, as zymoplasm arrestin molecule, factor Xa (FXa) arrestin molecule and tissue factor path arrestin molecule (TFPI) etc., in multiple external advantage tick bodies such as lone star tick, Dermacentor variabilis, a prominent hard tick, Rhipicephalus appendicularis, boophilus microplus, be found (Maritz-Olivier, 2007).Although the tick anticoagulant molecule of having reported is kind more than 20 nearly, identify fully also seldom.From present interpretation of result, tick anticoagulant molecule majority belongs to Kunitz family serinase specificity and suppresses molecule, characteristic contains the cysteine residues of 1 or 2 KU structure function territories and high conservative, may be from same ancestors (Lai, 2004) on evolving.
Because human health in the popular feeling, cerebrovascular disease serious threat that thrombus causes, the anti-bolt preparation of available is limited at present, and therefore, the research of medicine for treating thrombus thing is the field of very paying close attention to (Bates and Weitz, 2006) always.Hirudin has been applied to clinical medicine (Turk, 2006 according to leech (leech) anticoagulation molecule synthetic anticoagulant peptides; Bates and Weitz, 2006).In the tick anticoagulant molecule of having identified, there are 2 molecules in animal model experiment, to have the antithrombotic therapy effect, wherein the TAP molecule identified is factor Xa (FXa) inhibitor nineteen ninety, recombinant expressed TAP has shown good anti thrombotic action on a plurality of animal models, owing to reasons such as antigenicity also fail to use (Bates andWeitz at present on the human clinical, 2006), but utilize the structure of TAP molecule and mechanism of action design littler, there is not antigenic medicine for treating thrombus thing, perhaps making up novel reorganization medicine for treating thrombus thing molecule studies and makes progress, for example, use TAP functional zone sequence and new reorganization anticoagulant protein molecule of anchorin Annexin V sequence construct, its antithrombotic acitivity improves 10 times (Chen, 2005) than the activity of single anchorin Annexin V.Another tick anticoagulant molecule I xolaris is in recent years from a molecule that prominent hard tick is identified, with people's anatomic passages supressor (TFPI) homology, anticoagulant factor VIIa/tissue factor complex body, in rat model, very strong anti thrombotic action (Monteiro, 2005 have been shown; Nazareth, 2006).These studies show that the anticoagulation molecule of tick can be developed as novel antithrombotic reagent.
Crescent fan head tick (Rhipicephalus haemaphysaloides) mainly is distributed in South Asia and southern china, is one of Chinese advantage tick kind, at multiple animals such as ox, sheep, dog, the rabbit parasitism of sucking blood.Application number is 200910053692.5, publication number is the Chinese patent application of CN101928710A, discloses a kind of gene order of anticoagulation molecule Rhipilin-1 of crescent fan head tick and recombinant expressed and use.But, still lack the tick anticoagulant albumen that can be developed as novel antithrombotic reagent at present, still openly do not report both at home and abroad about other anticoagulant proteins of crescent fan head tick.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of anticoagulant protein and gene thereof of tick, and this anticoagulant protein is the anticoagulant protein Rhipilin-2 of crescent fan head tick, can be used for preparing anticoagulation medicine or anticoagulation biotechnological formulation.
In addition, also need to provide proteic recombinant expression method of a kind of tick anticoagulant and the application in preparation anticoagulation medicine or anticoagulation biotechnological formulation thereof.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of anticoagulant protein of tick, described anticoagulant protein is the anticoagulant protein Rhipilin-2 of crescent fan head tick, has the aminoacid sequence shown in the SEQ ID NO.1.
In another aspect of this invention, provide a kind of anticoagulant protein gene of tick, described gene is a crescent fan head tick anticoagulant protein Rhipilin-2 gene, and it comprises: the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO.1.
Preferably, described anticoagulant protein gene has the nucleotide sequence shown in the SEQ ID NO.2.
In another aspect of this invention, also provide a kind of recombinant vectors that comprises above-mentioned crescent fan head tick anticoagulant protein gene.
In another aspect of this invention, also provide a kind of cell that comprises above-mentioned recombinant vectors.
In another aspect of this invention, also provide a kind of tick anticoagulant proteic recombinant expression method, may further comprise the steps:
Structure contains the eukaryotic expression recombinant vectors of crescent fan head tick anticoagulant protein Rhipilin-2 coding gene sequence;
Described recombinant vectors is converted into Bacillus coli cells, and screening obtains the recombinant baculovirus grain;
With this recombinant baculovirus grain transfection insect cell, carry out the Rhipilin-2 protein expression.
In another aspect of this invention, also provide the application of a kind of above-mentioned tick anticoagulant albumen in preparation anticoagulation medicine or anticoagulation biotechnological formulation.
In another aspect of this invention, also provide the application of a kind of above-mentioned tick anticoagulant protein gene in preparation anticoagulation medicine or anticoagulation biotechnological formulation.
Crescent fan head tick anticoagulant protein Rhipilin-2 of the present invention, the Rhipilin-2 recombinant protein that obtains after recombinant expressed, by rabbit plasma partial prothrombinase activationary time (Activated Partial Thromboplastin Time, APTT) experiment, confirmed that this albumen has anticoagulant active, is very suitable for preparing the anticoagulation biotechnological formulation.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Fig. 1 is the nucleotide sequence and the corresponding amino acid sequence analysis chart of crescent fan head tick anticoagulant protein Rhipilin-2 gene of the present invention;
Fig. 2 is that the embodiment of the invention 2 indirect immunofluorescence assay detect the expression of results figure of Rhipilin-2 in insect cell;
Fig. 3 is the Western blotting figure as a result of the Rhipilin-2 recombinant protein behind the embodiment of the invention 2 purifying;
Fig. 4 is the influence figures of the embodiment of the invention 2 reorganization anticoagulant protein Rhipilin-2 to rabbit plasma partial prothrombinase activationary time and prothrombin time.
Embodiment
In the following example, the experimental technique of unreceipted actual conditions, condition routinely usually is as " molecular cloning experiment guide " (J. Sa nurse Brooker, D.W. the Russell is outstanding, Huang Peitang, Wang Jiaxi, Zhu Houchu, Deng translating. the 3rd edition, Beijing: Science Press, 2002) described in method carry out.
Sialisterium subtractive library and sequencing analysis before and after the present invention sucks blood by the female tick of structure crescent fan head tick, be cloned into the total length encoding gene of an anticoagulation molecule Rhipilin-2 from the crescent fan head tick, this full length gene is 693bp, a complete open reading frame is arranged, from the 62nd base to 646 base, 195 amino acid of encoding, the predicted protein molecular weight is 22kDa.The albumen of prediction contains a typical Kunitz structural domain, and the signal peptide sequence of 20 an amino acid formation is arranged at the N end.
In insect cell, carry out crescent fan head tick anticoagulant protein Rhipilin-2 of the present invention recombinant expressed, and by rabbit plasma partial prothrombinase activationary time (APTT) experiment confirm, this tick anticoagulant albumen Rhipilin-2 has the anticoagulation biological activity, can be used for preparing the anticoagulation biotechnological formulation.
Gene clone and the sequential analysis of embodiment 1 crescent fan head tick anticoagulant protein Rhipilin-2
1. method steps
(1) preparation of crescent fan head tick sialisterium: the crescent fan head tick is not sucked blood into tick by 1: 1 combined inoculation rabbit ear of male and female, and the female tick of half-full blood is taken out in inoculation back 4-5 days, and anatomical lens separates sialisterium down, and-70 ℃ of preservations are standby; Same tick group does not suck blood the sialisterium of female tick by separating with quadrat method.
(2) extraction of the total RNA of tick sialisterium: the Trizol reagent that adopts Invitrogen company, press description of test operation, extract crescent fan head tick do not suck blood female tick and the total RNA of the female tick of half-full blood respectively, be dissolved in the water of no RNase, measure total rna concentration, preserve standby in-70 ℃ of refrigerators then.
(3) double-stranded cDNA's is synthetic: the SMART PCR cDNA synthetic agent box that adopts Clontech company, be template with the total RNA in (2) step respectively, under the reversed transcriptive enzyme effect, synthesize the first chain cDNA (ss cDNA) with Olig (dT) 18 primers, and amplify cDNA second chain (ds cDNA) with long range PCR (LD-PCR).
(4) inhibition subtractive hybridization: use subtractive hybridization test kit Clontech PCR Select
TMCDNA Subtraction Kit.With the female tick sialisterium of half-full blood is test (Tester), is driving (Driver) with the female tick sialisterium of not sucking blood, and carries out the inhibition subtractive hybridization.Step is as follows: respectively joint 1-test cDNA (Adaptor1-Tester cDNA) and joint 2-test cDNA (Adaptor2R-Tester cDNA) are mixed with driving cDNA (Driver cDNA), 68 ℃ of incubation 8h carry out the 1st and take turns hybridization; Above-mentioned two reaction product are mixed, and add the driving cDNA of capacity once more, 68 ℃ are spent the night, and carry out the 2nd and take turns hybridization; Product to subtractive hybridization carries out the two-wheeled pcr amplification, is contrast PCR with the cDNA that adds the female tick of half-full blood that joint not subdues respectively.The primer sequence of PCR is corresponding to the sequence (being provided by test kit) of joint 1 and joint 2.
(5) screening of the structure of subtractive library, positive colony, est sequence are measured and analyzed: the subtractive hybridization product behind the pcr amplification is through PCR purification kit purifying; be connected with the pGEM-T-easy carrier; be transformed into competent cell DH5 α and screen blue hickie; picking is the white clone all; increase with nest-type PRC primer 1 and primer 2 R; positive colony is served the order-checking of sea base health biotech company; sequencing result carries out homology analysis (http://www.ncbi.nlm.nih.gov/BLAST) on GenBank, obtain 247 effective est sequences altogether.
(6) full-length gene order of anticoagulation molecule Rhipilin-2 is measured and is analyzed: according to est sequence information, select one and be numbered 201 est sequence, carry out complete sequence determination and bioinformatic analysis, the result is complete coding anticoagulation molecule homologous genes, called after Rhipilin-2 gene.
2. result and analysis
The sialisterium subtractive library successfully made up before and after the female tick of crescent fan head tick was sucked blood, and all clones are carried out the end sequencing analysis, obtained 247 effective est sequences altogether.To the clone of one of them anticoagulation molecule homologous genes, carried out complete sequence determination and bioinformatic analysis, be found to be complete encoding gene, called after Rhipilin-2 gene.As shown in Figure 1, this Rhipilin-2 full length gene is 693bp (SEQ ID NO.2), at 3 ' end polyA is arranged, contain a complete open reading frame, from the 62nd base to 646 base, 195 amino acid (SEQ ID NO.1) of encoding, the predicted protein molecular weight is 22kDa, the ATG that square frame marks among Fig. 1 is an initiator codon, TAA is a terminator codon, the protein signal peptide analysis is held the signal peptide sequence (following stroke of solid line sequence is signal peptide sequence among Fig. 1, arrow expression signal peptide cut point) that has 20 amino acid to constitute at N-; The protein function domain analysis exists by a α spiral and two single Kunitz structural domains that antiparallel βZhe Die constitutes in the 50-87 position of its amino acid sequence coded.Blast analyzes the remarkable homology of the anatomic passages supressor (TFPI) of finding one of this anticoagulant protein Rhipilin-2 and anticoagulation molecule.
Recombinant expressed and the anticoagulant active checking of embodiment 2 crescent fan head tick anticoagulant protein Rhipilin-2 genes
1. method steps
1.1 the structure of eukaryotic expression recombinant vectors and transfection
According to the coding region of gene, design contains EcoR I and Xho I restriction enzyme site special primer.
forward：5’-GCGAATTCGAGGAAAAGCCCACATGCGAT-3’(SEQ?ID?NO.3)；
reverse：5’-GGCTCGAGACTTTACTGTTTTTAGGCTTGT-3’(SEQ?ID?NO.4)。
Through conventional round pcr, with goal gene Rhipilin-2 encoding sequence subclone in baculovirus shuttle vectors pFastHTA, obtain the Rhipilin-2-pFastHTA recombinant shuttle plasmid, after order-checking is correct recombinant shuttle plasmid is converted among the intestinal bacteria DH10Bac screening recombinant baculovirus.Extracting recombinant baculovirus grain Rhipilin-2-Bacmid, transfection is carried out protein expression to insect cell sf9.
1.2 the proteic indirect immunofluorescence of eukaryotic expression detects
Rhipilin-2-Bacmid transfection under the mediation of Cellfectin II reagent is entered in the sf9 cell, and 27 ℃ of constant incubator 120h collect supernatant as first-generation recombinant virus, obtain the viral liquid of kind that height is tired after going down to posterity for three times.Get the cell that infects reorganization poison and wild-type virus respectively, PBS washing three times, with stationary liquid (methyl alcohol: the following fixedly 10min of normal temperature acetone=1: 1), carry out indirect immunofluorescence experiment, one is anti-with mouse source anti-His label monoclonal antibody (1: 300) incubated at room 1h, and PBS washing 3 times is with the sheep anti-mouse igg two anti-incubated at room 1h of FITC mark, after the PBS washing 3 times, observations under the fluorescent microscope.
1.3 recombinant protein purification and Western blotting analyze
Infect the cell of recombinant virus and the cell of the wild poison of infection with the PBS washing, ultrasonic degradation, adopt Ni-NTA metal-chelating resin to carry out the recombinant protein that purifying contains the His label, collect eluted albumen, carry out the SDS-PAGE electrophoresis, carry out Western blotting then and analyze, one is anti-with mouse source anti-His label monoclonal antibody (1: 500), two is anti-for Dylight 680 sheep anti-mouse igg antibodies (1/10000), by the double-colored imaging system observation experiment of infrared laser result.
1.4 the mensuration of recombinant protein partial prothrombinase activationary time (APTT) and prothrombin time (PT)
By volume, 9 parts of new zealand white rabbit venous blood are added 1 part 3.8% sodium citrate anticoagulant mixing, centrifugal 10 minutes of 1000g, preparation rabbit plasma.The recombinant protein Rhipilin-2 of different concns is added in the anticoagulate plasma, makes its final concentration be respectively 1.425uM, 0.713uM, 0.356uM, 0.178uM and 0.089uM, control group adds with volume PBS.Reagent D ade Actin Reagente Cefaloplastina Attivata, CaCl with APTT
2Be put into respectively among the coaglation analyzer CA-500 (Japanese SYSMEX company) with the reagent Thromborel S (U.S. SIEMENS company) of PT, the anti-freezing sample that contains recombinant protein for preparing is put to sample rack.Enter WORK LIST menu on screen, the project of having established ID number and will detect is ready to the back and begins to measure by the START key.The APTT of each sample and PT test equal triplicate.
2. result and analysis
2.1 the structure of eukaryotic expression recombinant vectors, transfection and protein expression
Eukaryotic expression recombinant plasmid Rhipilin-2-pFastHTA is converted among the E.coli DH10Bac after EcoR I and Xho I double digestion identify that (4788bp and 533bp), order-checking are correct, the extracting Rhipilin-2-Bacmid rod granule of recombinating.Universal primer and Auele Specific Primer carry out the PCR qualification result and show: the band of a 2969bp and the band of 539bp are arranged respectively, prove that goal gene recombinates in the baculovirus precursor, checking order shows that sequence correctly inserts.Recombinant baculovirus grain Rhipilin-2-Bacmid transfection is to insect cell sf9, find cytopathy behind the 72h, collecting supernatant continues to go down to posterity as recombinant virus, the indirect immunofluorescence assay of applying label protein antibodies shows, behind the transfection sf9 cell third generation, cell sends green fluorescence (seeing Fig. 2 B) under fluorescent microscope, and control group, transfection the sf9 cell then not luminous (seeing Fig. 2 A) of wild strain.The expression that proof Rhipilin-2 fusion rotein has obtained in the sf9 cell.
2.2 the purifying of recombinant protein and Western blotting detect
Collect the cell that infects, ultrasonic degradation, use the recombinant protein that the affine resin purification of Ni-NTA contains the His label, Westernblotting analysis revealed (see figure 3), recombinant protein are about 28kDa, and be consistent with expection recombinant protein size, and the sf9 cell of transfection Bacmid in contrast, then do not have band to occur, show the correctly expression in insect cell of Rhipilin-2 albumen, recombinant protein is pressed the BCA method and is measured concentration.In Fig. 3, " 1 " refers to recombinant protein; " 2 " refer to contrast.
2.3 the recombinant protein anticoagulant active is measured
By APTT the anticoagulant active of recombinant protein Ripilin-2 is detected, find the increase along with recombinant protein concentration, anticoagulation is obvious more, when recombinant protein concentration during greater than 0.713uM, and setting time (see figure 4) that can the significant prolongation rabbit plasma.And in the PT test, along with the increase of recombinant protein concentration, the setting time of rabbit plasma does not show difference.Control group is all consistent with minimum concentration sample (0.089uM).APTT is the catalytic surface that blood coagulation is provided with kephalin (partial thromboplastin) replacement thrombocyte, at Ca
2+Participation under, observe the needed time of the clotting of plasma, its measurement result is normally judged a whether normal standard of intrinsic coagulation approach.In this test along with the increase of fusion rotein Ripilin-2 concentration, the blood clotting time also significantly increases, this illustrates that this fusion rotein has restraining effect to a certain thrombin in the endogenous coagulation pathway, and this restraining effect presents linear relationship along with the increase of fusion rotein concentration; PT adds excessive tissue factor and Ca in being examined blood plasma
2+, make thrombogen change zymoplasm into, the latter makes Fibrinogen change scleroproein into, and it normally detects an index of exogenous cruor pathway.In this test, along with the increase of fusion rotein Ripilin-2 concentration, the PT value changes little, judges that thus fusion rotein is little to the blood clotting factor influence that participates in external source blood coagulation path.
The result of comprehensive APTT and PT test infers that Ripilin-2 shows anticoagulant active by the effect to the intrinsic coagulation path.
By above-mentioned experimental result as can be known, crescent fan head tick anticoagulant protein Ripilin-2 of the present invention can be used for preparing anticoagulation biotechnological formulation or anticoagulation medicine.
The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Sequence table
＜110〉China Agriculture Academe Shanghai Veterinary Institute
Guangzhou Leader Bio-technology Co., Ltd.
＜120〉anticoagulant protein of tick and gene thereof and application
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115 120 125
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130 135 140
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115 120 125
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130 135 140
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165 170 175
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Claims (8)
1. the anticoagulant protein of a tick is characterized in that, described anticoagulant protein is the anticoagulant protein Rhipilin-2 of crescent fan head tick, has the aminoacid sequence shown in the SEQ ID NO.1.
2. the anticoagulant protein gene of a tick is characterized in that, described gene is a crescent fan head tick anticoagulant protein Rhipilin-2 gene, and it comprises: the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO.1.
3. according to the anticoagulant protein gene of the described tick of claim 2, it is characterized in that described gene has the nucleotide sequence shown in the SEQ ID NO.2.
4. a recombinant vectors is characterized in that, comprises the described tick anticoagulant protein gene of claim 2.
5. a cell is characterized in that, comprises the described recombinant vectors of claim 4.
6. the proteic recombinant expression method of tick anticoagulant is characterized in that, may further comprise the steps:
Structure contains the eukaryotic expression recombinant vectors of crescent fan head tick anticoagulant protein Rhipilin-2 coding gene sequence;
Described recombinant vectors is converted into Bacillus coli cells, and screening obtains the recombinant baculovirus grain;
With this recombinant baculovirus grain transfection insect cell, carry out the Rhipilin-2 protein expression.
7. the application of the anticoagulant protein of the described tick of claim 1 in preparation anticoagulation medicine or anticoagulation biotechnological formulation.
8. the application of the anticoagulant protein gene of the described tick of claim 2 in preparation anticoagulation medicine or anticoagulation biotechnological formulation.
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| CN 201110008384 CN102153640A (en) | 2011-01-15 | 2011-01-15 | Anticoagulation protein of tick and gene and application thereof |
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| CN 201110008384 CN102153640A (en) | 2011-01-15 | 2011-01-15 | Anticoagulation protein of tick and gene and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881155A (en) * | 2017-11-21 | 2018-04-06 | 上海海洋大学 | Express GCRV spike protein VP55 recombinant baculovirus and application |
| CN110981953A (en) * | 2019-12-13 | 2020-04-10 | 首都医科大学 | Polypeptide, application of polypeptide and composition containing polypeptide |
| CN116410303A (en) * | 2023-06-07 | 2023-07-11 | 鄂尔多斯市中心医院（内蒙古自治区超声影像研究所） | Application of tick anticoagulant protein Qinghienin and encoding gene thereof in anticoagulation |
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| CN1657617A (en) * | 2004-02-18 | 2005-08-24 | 中国农业科学院上海家畜寄生虫病研究所 | Clone, expression and anti-tick immanoprotection action of falcate rhipicephalus RhcA and RhcB genes |
| CN101928710A (en) * | 2009-06-24 | 2010-12-29 | 中国农业科学院上海兽医研究所 | Anticoagulation molecule Rhipilin-1 gene sequence of Rhipicephalus haemaphysaloides, recombinant expression and application |
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| CN1657617A (en) * | 2004-02-18 | 2005-08-24 | 中国农业科学院上海家畜寄生虫病研究所 | Clone, expression and anti-tick immanoprotection action of falcate rhipicephalus RhcA and RhcB genes |
| CN101928710A (en) * | 2009-06-24 | 2010-12-29 | 中国农业科学院上海兽医研究所 | Anticoagulation molecule Rhipilin-1 gene sequence of Rhipicephalus haemaphysaloides, recombinant expression and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881155A (en) * | 2017-11-21 | 2018-04-06 | 上海海洋大学 | Express GCRV spike protein VP55 recombinant baculovirus and application |
| CN110981953A (en) * | 2019-12-13 | 2020-04-10 | 首都医科大学 | Polypeptide, application of polypeptide and composition containing polypeptide |
| CN116410303A (en) * | 2023-06-07 | 2023-07-11 | 鄂尔多斯市中心医院（内蒙古自治区超声影像研究所） | Application of tick anticoagulant protein Qinghienin and encoding gene thereof in anticoagulation |
| CN116410303B (en) * | 2023-06-07 | 2023-09-12 | 鄂尔多斯市中心医院（内蒙古自治区超声影像研究所） | Application of tick anticoagulant protein Qinghienin and encoding gene thereof in anticoagulation |
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Application publication date: 20110817 |