CN108676090A - A kind of plasmin inhibitor and its application - Google Patents

A kind of plasmin inhibitor and its application Download PDF

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Publication number
CN108676090A
CN108676090A CN201810505110.1A CN201810505110A CN108676090A CN 108676090 A CN108676090 A CN 108676090A CN 201810505110 A CN201810505110 A CN 201810505110A CN 108676090 A CN108676090 A CN 108676090A
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nki10
gly
lys
sequence
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CN108676090B (en
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彭礼飞
邵正
罗辉
何庆丰
邓莉
彭济达
廖淑莉
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Guangdong Medical University
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Guangdong Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Abstract

The invention discloses a kind of application of plasmin inhibitor in preventing or treating the relevant diseases such as hyperfibrinolysis, bleeding, belong to biomedicine field.The amino acid sequence of the plasmin inhibitor is：SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 or SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.8 or SEQ ID NO.9 or SEQ ID NO.10.Plasmin inhibitor in the present invention has strong fibrinolytic enzyme inhibition activity and has preferable inhibition specificity, the solution fibrin effect of fibrinolysin can be significantly inhibited, hand intraoperative blood loss is reduced, the drug of prevention or treatment hyperfibrinolysis, bleeding is can be used as, there is clinical value.

Description

A kind of plasmin inhibitor and its application
Technical field
The present invention relates to biomedicine fields, and specifically the present invention relates to a kind of plasmin inhibitors to prevent or treat Application in the relevant diseases such as hyperfibrinolysis, bleeding.
Background technology
Fibrinolysin (plasmin) is human body fibrinolytic system important component, mainly fibrin degradation and fiber Proteinogen.Hyperfibrinolysis, lose blood to reduce transfusion requirement etc. have extensively preventing or treat by surgical operation for antifibrinolytic agent Application.Using antifibrinolytic agent be complicated openheart surgery and be related to cardiopulmonary by extracorporal circulatory system (CBP, Cardiopulmonary bypass) operation standard operation；It can be reduced in perioperative application antifibrinolytic agent and lose blood and reduce Transfusion requirement, in wound, postpartum haemorrhage and each surgery (liver and gall, orthopaedics, neurology department, obstetrics/gynaecology, urological department, blood vessel, youngster Section etc.) in have application (referring to following document, document 1：“Antifibrinolytic Agents in Cardiac and Noncardiac Surgery:A Comprehensive Overview and Update " (Chinese translations：Antifibrinolytic agent exists Application in heart and non-cardiac surgery：General overview and latest developments), Gerstein NS etc.,《J Cardiothorac Vasc Anesth》, the 6th phase page 2183~2205 of volume 31 in 2017；Document 2：“Antifibrinolytic Therapy And Perioperative Considerations " (Chinese translations：Anti- fibrinolytic treatment and perioperative points for attention), Levy JH etc.,《Anesthesiology》, 657-670 pages of 3 phase of volume 128 in 2018).Current antifibrinolytic agent mainly has：Ammonia first Naphthenic acid, aminocaproic acid, aminomethylbenzoic acid and Aprotinin (aprotinin).Wherein, tranexamic acid, aminocaproic acid and aminomethylbenzoic acid are By combining the lysine-binding site on plasminogen, to inhibit plasminogen to be changed into fibrinolysin, but they have been to The active unrestraint of the fibrinolysin of generation acts on.Aprotinin is then by directly inhibiting the activity of fibrinolysin there is anti-fibrinolytic to make With the fibrinolysin generated can be inhibited.
Tranexamic acid, aminocaproic acid, aminomethylbenzoic acid and Aprotinin cut both ways in clinical application.Wherein, Aprotinin is answered It can be substantially reduced in extracorporal circulatory system (CBP) art and lose blood and reduce blood transfusion needs, but Aprotinin is a kind of serine of wide spectrum Protease not only has strong inhibitory activity to plasmin activity, and to trypsase (trypsin), kallikrein (kallikrein) and the multiple proteins enzyme such as chymotrypsin (chymotrypsin) has stronger or certain inhibiting effect.Press down peptide Enzyme was suspended Clinical practice in 2007 due to being deemed likely to cause the side effects such as myocardial ischemia, renal dysfunction, but with The clinical application for the antifibrinolytic agents such as the tranexamic acid substituted increases discovery：The lysine analogues such as tranexamic acid can penetrate blood Brain barrier, influences central nervous system, in fact it could happen that the side effects such as spasm, and also the reduction of Aprotinin during surgery is lost blood and is subtracted Few blood transfusion effect is substantially better than the drugs such as tranexamic acid, meanwhile, the death rate and kidney function energy loss of openheart surgery after Aprotinin is quit listing There is no improvement for harmful incidence, and the use of allosome blood product (blood transfusion) dramatically increases (referring to following document, document 3： " rear Aprotinin epoch antifibrinolytic puzzlement and prospect ", trick is big virtuous,《Guangdong medicine》, 19 phases 2909~2913 of volume 34 in 2013 Page；Document 4：" Clinical advances of antifibrinolytic agent in openheart surgery ", Elaeocarpus decipiens outstanding person etc.,《Chinese molecular cardiology is miscellaneous Will》, the 4th 1018-1021 pages of the phase in 2014；Document 5：" progress of tranexamic acid relationship type epilepsy ", Hao Maolin etc.,《Doctor Learn summary》, 332-335 pages of 2 phase of volume 23 in 2017；Document 6：“Increased perioperative mortality following aprotinin withdrawal：a real-world analysis of blood management Strategies in adult cardiac surgery " (Chinese translations：Peri operative mortality increases after Aprotinin deactivates： The Realistic Analysis of Adult cardiac surgery blood administration strategy), Walkden GJ etc.,《Intensive Care Med》, 2013 1808-1817 pages of 10 phase of volume 39).
Also in view of Aprotinin in clinical application also with income be more than risk advantage, Canada and Europe respectively at Allow within 2011,2012 to restore Clinical practice Aprotinin.Plasmin inhibitor currently study but still unlisted Textilinin-1 may be a kind of anti-fibrinolytic medicine than Aprotinin safety, inhibit with broad spectrum serine protease with Aprotinin The feature of activity compares, and textilinin-1 has strong suppression to fibrinolysin, elastoser (elastase) and trypsase It makes and uses, but is weaker than Aprotinin to the inhibiting effect of kallikrein (kallikrein), fibrin ferment (thrombin), i.e., Textilinin-1 has certain inhibition specificity (referring to following document, document 7：“Textilinin-1,an alternative anti-bleeding agent to aprotinin:Importance of plasmin inhibition In controlling blood loss " (Chinese translations：Textilinin-1, a kind of haemostatic medicament substituting Aprotinin：Suppression Importance of the fibrinolysin processed in control is lost blood), Flight SM, etc.,《Br J Haematol》, 2 phases of volume 145 in 2009 Page 207-211；Document 8：" expression and application of the plasmin inhibitor textilinin-1 in pichia yeast ", Wang Hongying etc.,《Snake Will》, 273-277 pages of 3 phase of volume 26 in 2014).
In general, although the antifibrinolytic agent of clinical application at present is effective, also have apparent side effect or a deficiency, because This researches and develops more effective, safer anti-fibrinolytic new drug and is of great significance.
Invention content
The problem to be solved in the present invention is to provide a kind of fibre for having strong inhibitory activity to fibrinolysin and inhibiting specificity good Lyase inhibitor, for preventing or treating hyperfibrinolysis, bleeding relevant disease.
To prevent or treating hyperfibrinolysis, transfusion requirement is lost blood or reduced to hand intraoperative hemorrhage to reduce, the present invention provides A kind of application of polypeptide-NKI10 or its mutant in preventing or treating hyperfibrinolysis, bleeding relevant disease.The ammonia of NKI10 Base acid sequence is：SEQ ID NO.1, mutant are to be blocked, replaced to the partial amino-acid residue in SEQ ID NO.1 sequences The polypeptide with the amino acid sequence different from SEQ ID NO.1 amino acid sequences for changing or being formed after lacking, such as SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、 SEQ ID NO.9、SEQ ID NO.10。
Polypeptide in the present invention is a kind of very strong to fibrinolytic enzyme inhibition activity and with the fibrinolysin for preferably inhibiting specificity Inhibitor.The NKI10 of equimolar concentration can inhibit to be more than 90% human plasmin activity, and the NKI10 of 2 times of mole specific concentrations can be completely Inhibit human plasmin activity, but the NKI10 of equimolar and 2 times of mole specific concentrations is only capable of inhibiting respectively about 22%, 10% people's pancreas Proteinase activity is increased to the NKI10 of 5 times of mole specific concentrations to chymotrypsin, pancreatic elastase, fibrin ferment and plasmakinin Enzyme is discharged without obvious inhibiting effect.
SEQ ID NO.1 mutant in the present invention such as has SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10 The polypeptide of shown amino acid sequence has strong inhibitory activity to fibrinolysin, and has and preferably inhibit specificity, is that inhibitory activity is strong And inhibit the preferable plasmin inhibitor of specificity.
Polypeptide in the present invention can inhibit fibrinolysin to fibrinous dissolution, have notable anti-fibrinolysis activity, because This, it is diseases related in preventing or treating hyperfibrinolysis that polypeptide of the invention can be used as medicinal application.In addition, more in the present invention Peptide, which has, to be significantly reduced mouse docking and loses blood effect, and therefore, polypeptide of the invention can be used as medicinal application in bleeding relevant disease In to reduce transfusion requirement, be particularly applicable to reduce surgical operation lose blood.
Description of the drawings
The following drawings are only intended to schematically illustrate and explain the present invention, not delimit the scope of the invention.
Shown in FIG. 1 is the anti-cellulase solution fibrin tablet effect of NKI10.In fibrin plate, A1、A2 And A3Corresponding aperture is that fibrinolysin (2 000nmol/L) and each 5 μ L of physiological saline (control) are added per hole；B1、B2And B3Corresponding aperture is Fibrinolysin (2 000nmol/L) and each 5 μ L of NKI10 (500nmol/L) are added per hole；C1、C2And C3Corresponding aperture is that fibre is added per hole Lyase (2 000nmol/L) and each 5 μ L of NKI10 (1 000nmol/L)；D1、D2And D3Corresponding aperture is that fibrinolysin (2 is added per hole 000nmol/L) and each 5 μ L of NKI10 (2 000nmol/L).In figure, transparent circle is fibrinolysin solution fibrin shape around every hole At fibrinolytic circle, be added NKI10 concentration increase, fibrinolytic circle is smaller, shows stronger to the inhibiting effect of fibrinolysin.
Shown in Fig. 2 is the complete encoder block nucleotide sequence of NKI10 and its amino acid sequence of coding.Lowercase is Nucleotide sequence, capitalization are to encode corresponding amino acid sequence, and * is corresponding terminator codon.
NKI10, proNKI10 shown in Fig. 3 (propetide of NKI10) and Aprotinin (aprotinin), textilinin-1 and The Clustal V comparison results of the amino acid sequence of the sequence GenBank No.XP_013306948 of prediction.NKI10 with GenBank No.XP_013306948 have 92.1% amino acid sequence similarity, with Aprotinin (aprotinin), Textilinin-1 is respectively provided with 39.6%, 38.6% amino acid sequence similarity.
Specific implementation mode
For a clearer understanding of the technical characteristics, objects and effects of the present invention, now illustrate that the present invention's is specific Embodiment.Following specific examples is only illustrative of the invention and is not intended to limit the scope of the invention.
The gene order of plasmin inhibitor in 1 present invention of embodiment
(1) cDNA sequence of coding SEQ ID NO.1 and its analysis
MRNA sequence GenBank is assembled according to the prediction of Necator americanus (Necator americanus) genome No.XM_013451494 (predictive coding polypeptide GenBank No.XP_013306948), design amplimer NI10-2:5’- TCAATCCACTTTGCAACGTTTCTT-3 ', using the ends cDNA rapid amplifying (RACE) technology, with Necator americanus cDNA For template, match with SMART-RACE (Clontech products) universal primers (5 '-AAGCAGTGGTATCAACGCAGAGTAC-3 ') To expanding, obtain one it is different from nucleotide sequence shown in GenBank No.XM_013451494, and encode different ammonia The cDNA of the complete encoder block of base acid sequence.The cDNA and predictive coding amino acid sequence are shown in Fig. 2, predict to believe by online website Number peptide (http://www.cbs.dtu.dk/services/SignalP/), prediction mature peptide is amino shown in SEQ ID NO.1 The sequence of acid sequence, the sequence and known peptide/albumen in GenBank has larger difference, and not functional research report, therefore It is named as NKI10.
The preparation method of the present embodiment Central America plate mouth nematode (Necator americanus) cDNA templates and utilization The method that the ends cDNA rapid amplifying (RACE) technology obtains the cDNA of NKI10 can refer to following document, document 9：" America Identifications and characteristic research of the hookworm protease inhibitors NaKuI1 to the inhibiting effect of protease ", thin perfume (or spice) of Sui etc., Chinese parasite It learns and parasitic disease magazine the 3rd phase of volume 35 in June, 2017；Document 10：" American hookworm broad spectrum serine protease inhibitor The identification and characteristic research of NaKuI 3 ", Zhong Fangfang etc., Chinese Pathogen Biology magazine the 6th phase of volume 12 in June, 2017.
The protein/polypeptide of the amino acid sequence similarity in GenBank is searched for through BlastP, the results showed that：With SEQ ID Amino acid sequence shown in NO.1 has the forecasting sequence that the sequence of maximum comparability is GenBank No.XP_013306948.With DNAstar softwares to NKI10, proNKI10 (propetide of NKI10) amino acid sequence and Aprotinin (aprotinin), Textilinin-1 and the forecasting sequence GenBank No.XP_013306948 that other people register in GenBank are carried out ClustalV is compared, and as a result sees Fig. 3.The result shows that NKI10 and GenBank No.XP_013306948 have 92.1% amino Acid sequence similitude is respectively provided with 39.6%, 38.6% amino acid sequence with Aprotinin (aprotinin), textilinin-1 Row similitude.
From the cDNA sequence of the coding SEQ ID NO.1 obtained in Necator americanus (Necator americanus) (with reference to figure 2) is as follows：
ATGGGAATGAAGGGAAGTGGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAGAGCTAAAATGGAAAGATG GGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAA GTGAAGAGAAATGCAAGAAACGTTGCAAAGTGGAT。
(2) the gene order amplification of coding SEQ ID NO.2
Design sense primer Nm1-1：5 '-GGTCATCTATGCAATGGGGA-3 ' and downstream primer Nm1-2：5’- ATCCACTTTGCAACGTTTCTTG-3 ', using Necator americanus cDNA as template, PCR amplification obtains encoding amino acid sequence The cDNA sequence of SEQ ID NO.2, sequence are as follows：
GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAGAGCTAAAATGGAAAGATGGGGATTGGATAAGGAATC AGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGA AACGTTGCAAAGTGGAT。
(3) the gene order amplification of coding SEQ ID NO.3
Design sense primer Nm2-1：5 '-TGCAATGGGGATTTACTACC-3 ' and downstream primer Nm2-2：5’- GCAACGTTTCTTGCATTTC-3 ', using Necator americanus cDNA as template, PCR amplification obtains encoding amino acid sequence SEQ The cDNA sequence of ID NO.3, sequence are as follows：
TGCAATGGGGATTTACTACCAGGACCTTGCAGAGCTAAAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTG CAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGC。
(4) the gene order amplification of coding SEQ ID NO.4
The gene order of coding SEQ ID NO.4 is obtained with overlapping primers extension PCR method.Wherein first round amplification upstream is drawn Object Nm4-3：5 '-ATTTACTACCAGGACCTTGCAAAGCTAAAATGGAAAGATGGG-3 ', with downstream primer Nm4-2： 5 '-ATCCACTTTGCAACGTTTCTTGCATTTC-3 ' are matched, with the aforementioned coding SEQ ID obtained from Necator americanus The cDNA sequence of NO.2 is template, carries out PCR amplification and obtains first round product, using the first round PCR product as template, with design Sense primer Nm4：5 '-GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTG-3 ', with downstream primer Nm4-2： 5 '-ATCCACTTTGCAACGTTTCTTGCATTTC-3 ' are matched, and carry out the second wheel amplification, and amplification obtains coding SEQ ID NO.4 Gene order, sequence is as follows：
GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAAAGCTAAAATGGAAAGATGGGGATTGGATAAGGAATC AGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGA AACGTTGCAAAGTGGAT。
(5) the gene order amplification of coding SEQ ID NO.5
The nucleotide sequence of coding SEQ ID NO.5 is obtained with overlapping primers extension PCR method.Wherein, downstream primer is used Nm4-2：5 '-ATCCACTTTGCAACGTTTCTTGCATTTC-3 ', the first round expand sense primer Nm5-3：5’- TTGCAAAGCTAGAATGGAAAGATGGGGATTG-3 ', the second wheel amplification sense primer Nm5-1：5’- ATTTACTACCAGGACCTTGCAAAGCTAGAATGGAAAGATGGG-3 ', third round expand sense primer Nm4：5’- GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTG-3’.The first round expands template with aforementioned from Necator americanus The cDNA sequence of the coding SEQ ID NO.2 of middle acquisition, the second wheel amplification template first round PCR product, third round expand mould The second wheel PCR product of plate, as a result obtains the gene order of coding SEQ ID NO.5, sequence is as follows：
GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAAAGCTAGAATGGAAAGATGGGGATTGGATAAGGAATC AGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGA AACGTTGCAAAGTGGAT。
(6) the gene order amplification of coding SEQ ID NO.6
The nucleotide sequence of coding SEQ ID NO.6 is obtained with overlapping primers extension PCR method.Wherein, downstream primer is used Nm4-2：5 '-ATCCACTTTGCAACGTTTCTTGCATTTC-3 ', the first round expand sense primer Nm6-3：5’- TTGCAGAGCTAGAATGGAAAGATGGGGATTG-3 ', the second wheel amplification sense primer Nm6-1：5’- ATTTACTACCAGGACCTTGCAGAGCTAGAATGGAAAGATGGG-3 ', third round expand sense primer Nm4：5’- GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTG-3’.The first round expands template with aforementioned from Necator americanus The cDNA sequence of the coding SEQ ID NO.2 of middle acquisition, the second wheel amplification template first round PCR product, third round expand mould The second wheel PCR product of plate, as a result obtains the gene order of coding SEQ ID NO.6, sequence is as follows：
GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAGAGCTAGAATGGAAAGATGGGGATTGGATAAGGAATC AGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGA AACGTTGCAAAGTGGAT。
(7) the gene order amplification of coding SEQ ID NO.7
Using the aforementioned gene order for encoding SEQ ID NO.4 that obtains as template, with sense primer Nm2-1：5’- TGCAATGGGGATTTACTACC-3 ' and downstream primer Nm2-2：5 '-GCAACGTTTCTTGCATTTC-3 ' are matched, PCR amplification The gene order of encoding amino acid sequence SEQ ID NO.7 is obtained, sequence is as follows：
TGCAATGGGGATTTACTACCAGGACCTTGCAAAGCTAAAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTG CAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGC。
(8) the gene order amplification of coding SEQ ID NO.8
It is arranged as template with the aforementioned nucleotides sequence for obtaining coding SEQ ID NO.5, with sense primer Nm2-1：5’- TGCAATGGGGATTTACTACC-3 ' and downstream primer Nm2-2：5 '-GCAACGTTTCTTGCATTTC-3 ' are matched, PCR amplification The gene order of encoding amino acid sequence SEQ ID NO.8 is obtained, sequence is as follows：
TGCAATGGGGATTTACTACCAGGACCTTGCAAAGCTAGAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTG CAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGC。
(9) the gene order amplification of coding SEQ ID NO.9
It is arranged as template with the aforementioned nucleotides sequence for obtaining coding SEQ ID NO.6, with sense primer Nm2-1：5’- TGCAATGGGGATTTACTACC-3 ' and downstream primer Nm2-2：5 '-GCAACGTTTCTTGCATTTC-3 ' are matched, PCR amplification The gene order of encoding amino acid sequence SEQ ID NO.9 is obtained, sequence is as follows：
TGCAATGGGGATTTACTACCAGGACCTTGCAGAGCTAGAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTG CAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGC。
(10) the gene order amplification of coding SEQ ID NO.10
The gene order of coding SEQ ID NO.10 is obtained with overlapping primers extension PCR method.Wherein the first round expands upstream Primer Nm10-3：5 '-ATTTACTACCAGGACCTTGCAGAGTTAAAATGGAAAGATGGGG-3 ', with downstream primer Nm4- 2：5 '-ATCCACTTTGCAACGTTTCTTGCATTTC-3 ' are matched, with the aforementioned coding SEQ obtained from Necator americanus The cDNA sequence of ID NO.2 is template, carries out PCR amplification and obtains first round product, using the first round PCR product as template, is used The sense primer Nm4 of design：5 '-GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTG-3 ', with downstream primer Nm4- 2：5 '-ATCCACTTTGCAACGTTTCTTGCATTTC-3 ' are matched, and carry out the second wheel amplification, and amplification obtains coding SEQ ID The gene order of NO.10, sequence are as follows：
GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAGAGTTAAAATGGAAAGATGGGGATTGGAT AAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAA ATGCAAGAAACGTTGCAAAGTGGAT
The protease inhibiting activity of plasmin inhibitor in 2 present invention of embodiment
The method for obtaining the recombination NKI10 of the present invention, can refer to following document, document 9：" American hookworm protease presses down Identifications and characteristic research of the preparation NaKuI1 to the inhibiting effect of protease ", thin perfume (or spice) of Sui etc., Chinese parasitology and parasitic disease Magazine the 3rd phase of volume 35 in June, 2017；Document 10：" identification of American hookworm broad spectrum serine protease inhibitor NaKuI3 and Characteristic research ", Zhong Fangfang etc., Chinese Pathogen Biology magazine the 6th phase of volume 12 in June, 2017.In brief, design expression is compiled The sense primer NI10-1e of code NKI10 (SEQ ID NO.1) gene：5’-CAGGATCCATGGGAATGAAGGGAAGTGGT-3’ (underscore is BamH I restriction enzyme sites), downstream primer NI10-2e：5’-GCAAGCTTCAATCCACTTTGCAACGTTTCTT- 3 ' (underscore is Hind III digestions site), with the cDNA sequence of the acquired coding SEQ ID NO.1 of the embodiment of the present invention 1 For template, amplification obtains the gene order of coding NKI10 (SEQ ID NO.1), the gene order of acquisition is connected into protokaryon table Up to carrier pET32a-sumo, builds correct recombinant plasmid and be transferred in Escherichia coli BL21 (DE3), with IPTG induced expressions, Host bacterial is after separation, ultrasonication, and expression product purifies through nickel affinity chromatography and obtains fusion protein, through SUMO proteolytic cleavages Fusion partner is cut, and through further removing fusion partner by affinity chromatography, obtains recombination NKI10.
Recombination NKI10 is observed to the inhibiting effect in relation to serine protease with Chromogenic assay.Protease/substrate used (being concentration in bracket) is respectively：Fibrinolysin (5nmol/L)/S2288 (400 μm of ol/L), trypsase (1nmol/L) S2302 (200 μm of ol/L), pancreas chymotrypsin (1nmol/L)/S7388 (200 μm of ol/L), elastoser (5nmol/L)/S4760 (200 μm of ol/L), fibrin ferment (1nmol/L)/S2288 (400 μm of ol/L), blood plasma bradykinin release enzyme (1nmol/L)/S2302 (200μmol/L).Above-mentioned protease is in addition to pig elastoser, other protease are human protease, wherein fibrinolysin (plasmin), fibrin ferment (thrombin) be U.S.'s Haematologic Technologies Inc products, pancreas chymotrypsin, Trypsase is Germany's CalbioChem products, and plasma kallikrein is U.S. Enzyme ResearchLaboratories Product, substrate S2302 and S2288 are Italy's Chromogenix products, and S4760, S7388 and pig elastoser are the U.S. Sigma Products.Detection method is that the recombination NKI10 of 10 μ l respective concentrations is added in 96 orifice plates (100 μ l reaction systems) After 25 DEG C of incubator is incubated 15min the corresponding substrate of 40 μ l pre-temperatures is added, with enzyme mark in (blank control PBS) and 50 μ l protease Instrument (Elx808IU) A405Scanning Detction enzyme kinetics, every 15 seconds read plates 1 time continuously read 5min, record enzyme reaction rate, Every group is repeated 3 times.Inhibiting rate=(V0-V)/V0, wherein V0For control group reaction speed, V is after plasmin inhibitor is added Reaction speed.
Experimental result is shown：The NKI10 (SEQ ID NO.1) of twice as high molar ratio concentration can completely inhibit human plasmin (plasmin) active；The NKI10 (SEQ ID NO.1) of equimolar concentration can be suppressed over 90% human plasmin activity. NKI10 there is certain inhibiting effect, the NKI10 of 2 times and equimolar specific concentration can inhibit respectively people's trypsase (trypsin) About 22% and 10% people's tryptic activity.1, the NKI10 of 2 and 5 times of mole specific concentrations is to pancreas chymotrypsin (chymotrypsin), pancreatic elastase (elastase), fibrin ferment (thrombin) and blood plasma bradykinin discharge enzyme (plasma kallikrein) is without obvious inhibiting effect.The result shows that NKI10 is one kind there is very strong fibrinolysin to inhibit to live Property and have and preferably inhibit the good plasmin inhibitor of specificity.
With reference to above-mentioned implementation, mutant NKI10-1 (SEQ ID NO.2), the NKI10-2 of NKI10 are as a result shown (SEQ ID NO.3)、NKI10-3(SEQ ID NO.4)、NKI10-4(SEQ ID NO.5)、NKI10-5(SEQ ID NO.6)、 NKI10-6 (SEQ ID NO.7), NKI10-7 (SEQ ID NO.8, NKI10-8 (SEQ ID NO.9) and NKI10-9 (SEQ ID NO.10) there is strong inhibitory activity to fibrinolysin, and inhibit specificity with preferable.
The anti-fibrinolysis activity of plasmin inhibitor in 3 present invention of embodiment
Plasmin inhibitor preparation method is observed with embodiment 2 using fibrin plate dissolution experiment in the present embodiment The anti-fibrinolysis activity of plasmin inhibitor.Experimental method is：With 1 × HBSAC buffer solutions (25mM HEPES, 100mM NaCl, 5mM CaCl2, pH 7.4) the agarose solution 15mL for preparing 1.2%, it dissolves by heating, is cooled to 50~60 DEG C, 1mL is added In the bovine fibrinogen solution 10mg/mL of 37 DEG C of heat preservations, then fallen after 100 μ L fibrin ferments (200U/mL) mixings are added immediately Enter culture dish and tablet is made.Tablet punches, and the recombination NKI10 and its mutant [concentration point of respective concentration are separately added into hole Wei 0nmol/L (isometric physiological saline), 500nmol/L, 1 000nmol/L, 2 000nmol/L] and fibrinolysin (2 000nmol/L) each 5 μ L, 3 hole of each concentration incubate 3h in 37 DEG C.It is determined by observing tablet mesoporous week fibrinolytic circle size To the inhibiting effect of fibrinolysin solution fibrin, fibrinolytic circle is smaller for NKI10 and its mutant, shows to dissolve fiber to fibrinolysin The inhibiting effect of albumen is stronger.
Such as Fig. 1, under identical plasmin concentration, with the increase that NKI10 concentration is added in every hole, hole week fibrinolytic circle contracts It is small, it is added around the NKI10 of equimolar concentration and the hole of fibrinolysin without apparent fibrinolytic circle (Fig. 1, D1、D2And D3Hole), show NKI10 has the anti-fibrinolysis activity of highly significant, and its anti-fibrinolysis activity has concentration-dependent relation.To the mutant of NKI10 NKI10-1(SEQ ID NO.2)、NKI10-2(SEQ ID NO.3)、NKI10-3(SEQ ID NO.4)、NKI10-4(SEQ ID NO.5)、NKI10-5(SEQ ID NO.6)、NKI10-6(SEQ ID NO.7)、NKI10-7(SEQ ID NO.8、NKI10-8 The anti-fibrinolytic experiment of (SEQ ID NO.9) and NKI10-9 (SEQ ID NO.10) show that they have very strong anti-fibrinolytic Effect.Therefore, result of this example indicate that the plasmin inhibitor of the present invention can be used as medicinal application in prevention or treatment fibre It is molten hyperfunction diseases related.
Influence of 4 plasmin inhibitor of embodiment to the mouse tail bleeding time
Plasmin inhibitor preparation method in the present embodiment is the same as embodiment 2.Take 40 (experimental animals of Balb/c mouse Card：SCXK (Guangdong) 2013-0008；SYXK (Guangdong) 2015-0147), half male and half female, 18~22g of weight is randomly divided into 8 groups, every group 5, respectively：Saline control group, recombination NKI10 (SEQ ID NO.1) group, recombination NKI10-1 (SEQ ID NO.2) Group, recombination NKI10-2 (SEQ ID NO.3) group, recombination NKI10-3 (SEQ ID NO.4) group, recombination NKI10-4 (SEQ ID NO.5) group, recombination NKI10-5 (SEQ ID NO.6) groups and recombination NKI10-6 (SEQ ID NO.7) group.It is injected intraperitoneally 2% penta Barbital sodium anaesthetizes Balb/c mouse, 50 μ g human tissue plasmin activators (tPA) (French HBM) of each tail vein injection After 10min, tail vein injection gives the plasmin inhibitor 1.0mgkg in the present invention respectively-1Or isometric physiological saline.It gives After medicine 2min, it will be placed in 37 DEG C of physiological saline away from being cut at Mouse Tail-tip about 0.5cm, record mouse stops from cutting to bleeding Time only is the bleeding time, is the tail bleeding time, with mean ± standard deviationIt indicates.
1 is the results are shown in Table, compared with saline control group, the tail bleeding time for recombinating NKI10 and its mutant each group is aobvious It writes and reduces (P<0.05).The result shows that plasmin inhibitor can be used as the medicinal application for reducing bleeding in bleeding correlation in the present invention Disease is used especially for reducing in operation and lose blood to reduce transfusion requirement.
In 1 present invention of table plasmin inhibitor to the fibrinolytic mouse tail bleeding time influence (N=5)
* compared with the control group, P<0.05
The foregoing is merely the schematical specific implementation modes of the present invention, are not limited to the scope of the present invention.It is any Those skilled in the art, do not depart from the design of the present invention and under the premise of principle made by equivalent variations, modification and combination, The scope of protection of the invention should all be belonged to.
Sequence table
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Claims (5)

1. a kind of plasmin inhibitor and its application, the inhibitor amino acid sequence are：SEQ ID NO.1 or SEQ ID NO.2 Or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 or SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.8 or SEQ ID NO.9 or SEQ ID NO.10.
2. application as described in claim 1, it is characterised in that be applied to prevent or treat hyperfibrinolysis relevant disease.
3. application as described in claim 1, it is characterised in that be applied to prevent or treat bleeding relevant disease.
4. application as described in claim 1, it is characterised in that the plasmin inhibitor is applied to agent for stanching as component Or hemostatic material.
5. bleeding relevant disease as claimed in claim 3 is to lose blood in surgical operation.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102850451A (en) * 2011-06-29 2013-01-02 辽宁诺康生物制药有限责任公司 Recombinant textilinin-1 with plasmin inhibition activity, and preparation method and application thereof
CN105985432A (en) * 2015-03-05 2016-10-05 莫永炎 Kunitz type serine protease inhibitor and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102850451A (en) * 2011-06-29 2013-01-02 辽宁诺康生物制药有限责任公司 Recombinant textilinin-1 with plasmin inhibition activity, and preparation method and application thereof
CN105985432A (en) * 2015-03-05 2016-10-05 莫永炎 Kunitz type serine protease inhibitor and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HU WAN等: "A Spider-Derived Kunitz-Type Serine Protease Inhibitor That Acts as a Plasmin Inhibitor and an Elastase Inhibitor", 《PLOS ONE》 *
钟芳芳等: "美洲钩虫广谱丝氨酸蛋白酶抑制剂NaKuI3的鉴定及特性研究", 《中国病原生物学杂志》 *
隋细香等: "美洲钩虫蛋白酶抑制剂NaKuI1对蛋白酶的抑制作用的鉴定和特性研究", 《中国寄生虫学与寄生虫病杂志》 *

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