CN108676090B - Plasmin inhibitor and application thereof - Google Patents
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Abstract
The invention discloses an application of a plasmin inhibitor in preventing or treating related diseases such as hyperfibrinolysis, bleeding and the like, belonging to the field of biological medicines. The amino acid sequence of the plasmin inhibitor is as follows: 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. The plasmin inhibitor has strong plasmin inhibition activity and better inhibition specificity, can obviously inhibit the fibrinolysis effect of plasmin, reduces blood loss in operation, can be used as a medicament for preventing or treating hyperfibrinolysis and hemorrhage, and has clinical application value.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to application of a plasmin inhibitor in preventing or treating related diseases such as hyperfibrinolysis, bleeding and the like.
Background
Plasmin (plasmin) is an important component of the human fibrinolytic system, mainly degrading fibrin and fibrinogen. Anti-fibrinolytic drugs have wide application in preventing or treating hyperfibrinolysis, surgical blood loss to reduce transfusion requirements, and the like. The use of antifibrinolytic drugs is a complex cardiac surgery and standard procedure involving the heart and lungs through extracorporeal Circulation (CBP) surgery; perioperative use of anti-fibrinolytic drugs to reduce blood loss and blood transfusion requirements has found application in trauma, postpartum hemorrhage and Surgery (hepatobiliary, orthopedic, neurologic, obstetric/gynecologic, urological, vascular, pediatric, etc.) (see literature 1: "anti-fibrinolytic Agents in Cardiac and Noncardiac Surgery: A Comprehensive Overview and Update" (Chinese translation: use of anti-fibrinolytic drugs in Cardiac and non-Cardiac Surgery: general Overview and recent progress), Gerstein NS et al, J Cardiorac Vasc examination, Vol.31, Vol.6, pp.2183-2205, Levy JH.2: "anti-fibrinolytic Therapy and Periopathic Considerations" (Chinese translation: anti-fibrinolytic Therapy and Perioperative attention), Levy JH.120, Vol.658, Vol.670, Vol.7, publication No. 2017, Vol.8, Vol.2018, pp.3-657). The current anti-fibrinolytic drugs mainly comprise: tranexamic acid, aminocaproic acid, aminomethylbenzoic acid and aprotinin (aprotinin). Among them, tranexamic acid, aminocaproic acid and aminomethylbenzoic acid inhibit the conversion of plasminogen to plasmin by binding to lysine binding sites on plasminogen, but they do not inhibit the activity of plasmin already produced. Aprotinin has anti-fibrinolytic effect by directly inhibiting plasmin activity, and can inhibit the produced plasmin.
Tranexamic acid, aminocaproic acid, aminomethylbenzoic acid and aprotinin have advantages and disadvantages in clinical application. The aprotinin is a broad-spectrum serine protease which has strong inhibitory activity to the activity of plasmin and has strong or certain inhibitory action to various proteases such as trypsin (trypsin), kallikrein (kallikrein) and chymotrypsin (chymotrypsin) and the like. Aprotinin has been clinically suspended in 2007 because it is thought that it may cause side effects such as myocardial ischemia and renal dysfunction, but it has been found that clinical applications of anti-fibrinolytic drugs such as tranexamic acid have increased: lysine analogs such as tranexamic acid can penetrate blood brain barriers to influence a central nervous system, side effects such as spasm can occur, the blood loss reducing and blood transfusion reducing effects of aprotinin in operation are obviously superior to medicines such as tranexamic acid, meanwhile, the death rate of heart operation and the incidence rate of renal function damage are not improved after the off-market of aprotinin, but the use of allogeneic blood products (blood transfusion) is remarkably increased (see the following literature, literature 3: behind aprotinin time fibrinolysis resisting puzzares and prospects, Wen Wei, Guangdong medicine, 34 vol.19: 2909-2913 in 2013; literature 4: clinical application progress of fibrinolytic medicines in heart operation, Duyinje et al, J.M. mol. cardiopathy, 4 th 1018-, vol 23, No. 332 and page 335 in 2017; document 6: "incorporated periodic moving imaging aprotinin with thrawal: a real-world analysis of blood management strategies in the adult cardiac surgery (Chinese translation: increase in perioperative mortality after aprotinin deactivation: realistic analysis of blood management strategies in adult cardiac surgery), Walkden GJ et al, Intensive Care Med, 2013, Vol.39, Vol.10, pp.1808 and 1817).
Also in view of the advantage that aprotinin also has a larger profit than risk in clinical applications, canada and europe have allowed to recover from clinical use of aprotinin in 2011 and 2012, respectively. The plasmin inhibitor Textilinin-1 which is currently studied but not yet marketed may be an antiplasmin which is safer than aprotinin, and compared with the characteristic that aprotinin has broad-spectrum serine protease inhibitory activity, Textilinin-1 has strong inhibitory activity on plasmin, elastase (elastase) and trypsin, but has weaker inhibitory activity on kallikrein and thrombin (thrombin) than plasmin, i.e., Textilinin-1 has certain inhibitory specificity (see the following documents, document 7: "Textilinin-1, an exogenous anti-staining agent to aprotinin: Import of plasmin inhibiting in controlling blood:" hemostatic drug for inhibiting "important in controlling blood loss", FlightBr J, et al, yeast 2009, Toyoto 2. sup. 1 and Biotin III. ",207. sup. in the publication of Biotin-1, wangmangying et al, snake Zhi, 26 vol.26, 3 rd, 273 and 277 pages).
In summary, although effective, the anti-fibrinolytic drugs applied clinically at present have obvious side effects or deficiencies, so that the development of more effective and safer new anti-fibrinolytic drugs is of great significance.
Disclosure of Invention
The invention aims to provide a plasmin inhibitor with strong inhibitory activity and good inhibitory specificity on plasmin, which is used for preventing or treating diseases related to hyperfibrinolysis and hemorrhage.
In order to prevent or treat hyperfibrino lysis, hemorrhage in operation to reduce blood loss or reduce the requirement of blood transfusion, the invention provides application of polypeptide-NKI 10 or a mutant thereof in preventing or treating diseases related to hyperfibrino lysis and hemorrhage. The amino acid sequence of NKI10 is: the mutant is polypeptide which is formed by truncating, replacing or deleting partial amino acid residues in the sequence of SEQ ID NO.1 and has an amino acid sequence different from the amino acid sequence of SEQ ID NO.1, 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 and SEQ ID NO. 10.
The polypeptide of the invention is a plasmin inhibitor with strong plasmin inhibition activity and good inhibition specificity. The equimolar NKI10 can inhibit more than 90% of human plasmin activity, the 2-fold molar concentration of NKI10 can completely inhibit the human plasmin activity, but the equimolar and 2-fold molar concentration of NKI10 can only inhibit about 22% and 10% of human trypsin activity respectively, and the NKI10 increased to the 5-fold molar concentration has no obvious inhibition effect on chymotrypsin, pancreatic elastase, thrombin and plasma kallikrein.
The SEQ ID NO.1 mutant provided by the invention has strong inhibitory activity on plasmin and better inhibitory specificity, and is a plasmin inhibitor with strong inhibitory activity and better inhibitory specificity, wherein the polypeptide has amino acid sequences shown in 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 and SEQ ID NO. 10.
The polypeptide can inhibit the fibrinolysis effect of plasmin and has obvious fibrinolysis resistance, so the polypeptide can be used as a medicament to be applied to preventing or treating diseases related to hyperfibrinolysis. In addition, the polypeptide has the effect of obviously reducing the blood loss of the broken tail of a mouse, so the polypeptide can be used as a medicine to be applied to bleeding related diseases so as to reduce the blood transfusion requirement, and particularly can be applied to reducing the blood loss in surgical operation.
Drawings
The drawings are only for purposes of illustrating and explaining the present invention and are not to be construed as limiting the scope of the present invention.
FIG. 1 shows the antiplasmin-lysing fibrin plate effect of NKI 10. In fibrin plates, A1、A2And A3Plasmin (2000 nmol/L) and physiological saline (control) were added to each well at 5. mu.L each; b is1、B2And B3Plasmin (2000 nmol/L) and NKI10(500nmol/L) were added to each well at a volume of 5. mu.L each; c1、C2And C3Plasmin (2000 nmol/L) and NKI10 (1000 nmol/L) were added to each well at a volume of 5. mu.L each; d1、D2And D3For each well, 5. mu.L of plasmin (2000 nmol/L) and NKI10 (2000 nmol/L) was added. In the figure, the transparent ring around each hole is a fibrinolytic ring formed by fibrinolysis, and the smaller the fibrinolytic ring is with the increase of the concentration of NKI10, which shows that the inhibition effect on the fibrinolysis is stronger.
FIG. 2 shows the complete nucleotide sequence of the coding cassette of NKI10 and its encoded amino acid sequence. Lower case letters are nucleotide sequences, upper case letters are sequences encoding the corresponding amino acids, and upper case letters are the corresponding stop codons.
FIG. 3 shows the Clustal V alignment of the amino acid sequences of NKI10, proNKI10 (pro peptide of NKI 10), aprotinin (aprotinin), textilinin-1 and the predicted sequence GenBank No. XP-013306948. NKI10 has 92.1% amino acid sequence similarity with GenBank No. XP _013306948, and has 39.6% and 38.6% amino acid sequence similarity with aprotinin (aprotinin) and textilinin-1, respectively.
Detailed Description
In order to more clearly understand the technical features, objects, and effects of the present invention, specific embodiments of the present invention will now be described. The following specific examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
EXAMPLE 1 Gene sequence of plasmin inhibitor in the invention
(1) cDNA sequence encoding SEQ ID NO.1 and analysis thereof
According to the prediction assembly mRNA sequence GenBank No. XM _013451494 (prediction encoding polypeptide GenBank No. XP _013306948) of the American Aphanizomenon americana (Newodor americanus) genome, an amplification primer NI10-2: 5'-TCAATCCACTTTGCAACGTTTCTT-3' is designed, cDNA terminal Rapid Amplification (RACE) technology is utilized, American Aphanizomenon americana cDNA is taken as a template, and is matched with a SMART-RACE (Clontech product) universal primer (5'-AAGCAGTGGTATCAACGCAGAGTAC-3') for amplification, so that cDNA which is different from the nucleotide sequence shown in GenBank No. XM _013451494 and encodes a complete coding frame of different amino acid sequences is obtained. The cDNA and the predicted coding amino acid sequence are shown in figure 2, a signal peptide (http:// www.cbs.dtu.dk/services/SignalP /) is predicted through an online website, the predicted mature peptide is the amino acid sequence shown in SEQ ID NO.1, and the sequence has larger difference with the sequence of known polypeptide/protein in GenBank and has no function research report, so the sequence is named as NKI 10.
In this example, the following references, document 9, can be made to the method for preparing a caenorhabditis americana (Necator americanus) cDNA template and the method for obtaining NKI10 cDNA using rapid cDNA end amplification (RACE) technology: "identification and characteristic study of inhibition effect of ancylostoma americanum protease inhibitor NaKuI1 on protease", inert fragrance, etc., China parasitology and parasite disease journal, volume 35, phase 3 in 2017, 6 months; document 10: "identification and characterization of the broad-spectrum serine protease inhibitor NaKuI3 of Necator americanus", Tufang, et al, J.Med.biol.2017, Vol.12, No. 6.
The protein/polypeptide with amino acid sequence similarity in GenBank is searched by BlastP, and the result shows that: the sequence having the greatest similarity to the amino acid sequence shown in SEQ ID NO.1 is the predicted sequence of GenBank No. XP-013306948. ClustalV alignment of the amino acid sequences of NKI10 and proNKI10 (pro peptide of NKI 10) with aprotinin (aprotinin), textilinin-1 and the predicted sequences registered in GenBank by others, GenBank No. XP-013306948, was performed by DNAstar software, and the results are shown in FIG. 3. The results show that NKI10 has 92.1% amino acid sequence similarity with GenBank No. XP _013306948 and 39.6% and 38.6% amino acid sequence similarity with aprotinin (aprotinin) and textilinin-1 respectively.
The cDNA sequence encoding SEQ ID NO.1 obtained from Necator americanus (cf. FIG. 2) is as follows:
ATGGGAATGAAGGGAAGTGGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAGAGCTAAAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGCAAAGTGGAT。
(2) amplification of the Gene sequence encoding SEQ ID NO.2
Designing an upstream primer Nm 1-1: 5'-GGTCATCTATGCAATGGGGA-3', and the downstream primer Nm 1-2: 5'-ATCCACTTTGCAACGTTTCTTG-3', using American population nematode cDNA as template, PCR amplifying to obtain the cDNA sequence of coding amino acid sequence SEQ ID NO.2, the sequence is as follows:
GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAGAGCTAAAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGCAAAGTGGAT。
(3) amplification of the Gene sequence encoding SEQ ID NO.3
Designing an upstream primer Nm 2-1: 5'-TGCAATGGGGATTTACTACC-3', and the downstream primer Nm 2-2: 5'-GCAACGTTTCTTGCATTTC-3', using American population nematode cDNA as template, PCR amplifying to obtain the cDNA sequence of coding amino acid sequence SEQ ID NO.3, the sequence is as follows:
TGCAATGGGGATTTACTACCAGGACCTTGCAGAGCTAAAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGC。
(4) amplification of the Gene sequence encoding SEQ ID NO.4
The overlapping primer extension PCR method is used to obtain the gene sequence of the code SEQ ID NO. 4. Wherein the first round of amplification forward primer is amplified by Nm 4-3: 5'-ATTTACTACCAGGACCTTGCAAAGCTAAAATGGAAAGATGGG-3', and the downstream primer Nm 4-2: 5'-ATCCACTTTGCAACGTTTCTTGCATTTC-3', and performing PCR amplification by using the cDNA sequence which is obtained from the American Isatis americana and codes for SEQ ID NO.2 as a template to obtain a first round of PCR products, wherein the first round of PCR products are used as a template and the designed upstream primer Nm 4: 5'-GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTG-3', and the downstream primer Nm 4-2: 5'-ATCCACTTTGCAACGTTTCTTGCATTTC-3', performing a second round of amplification to obtain a gene sequence encoding SEQ ID NO.4, the sequence is as follows:
GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAAAGCTAAAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGCAAAGTGGAT。
(5) amplification of the Gene sequence encoding SEQ ID No.5
The nucleotide sequence encoding SEQ ID NO.5 was obtained by overlap primer extension PCR. Wherein, the downstream primers are all expressed by Nm 4-2: 5'-ATCCACTTTGCAACGTTTCTTGCATTTC-3', first round amplification forward primer with Nm 5-3: 5'-TTGCAAAGCTAGAATGGAAAGATGGGGATTG-3', second round amplification forward primer with Nm 5-1: 5'-ATTTACTACCAGGACCTTGCAAAGCTAGAATGGAAAGATGGG-3', third round of amplification with Nm 4: 5'-GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTG-3' are provided. The first round of amplification template uses the cDNA sequence coding for SEQ ID NO.2 obtained from the American Aphanizomenon americanus, the second round of amplification template uses the first round of PCR product, and the third round of amplification template uses the second round of PCR product, so as to obtain the gene sequence coding for SEQ ID NO.5, and the sequence is as follows:
GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAAAGCTAGAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGCAAAGTGGAT。
(6) amplification of the Gene sequence encoding SEQ ID NO.6
The nucleotide sequence encoding SEQ ID NO.6 was obtained by overlap primer extension PCR. Wherein, the downstream primers are all expressed by Nm 4-2: 5'-ATCCACTTTGCAACGTTTCTTGCATTTC-3', first round amplification forward primer with Nm 6-3: 5'-TTGCAGAGCTAGAATGGAAAGATGGGGATTG-3', second round amplification forward primer with Nm 6-1: 5'-ATTTACTACCAGGACCTTGCAGAGCTAGAATGGAAAGATGGG-3', third round of amplification with Nm 4: 5'-GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTG-3' are provided. The first round of amplification template uses the cDNA sequence coding for SEQ ID NO.2 obtained from the American Aphanizomenon americanus, the second round of amplification template uses the first round of PCR product, and the third round of amplification template uses the second round of PCR product, so as to obtain the gene sequence coding for SEQ ID NO.6, and the sequence is as follows:
GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAGAGCTAGAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGCAAAGTGGAT。
(7) amplification of the Gene sequence encoding SEQ ID NO.7
Using the gene sequence encoding SEQ ID NO.4 obtained as described above as a template, the sequence was determined using the upstream primer Nm 2-1: 5'-TGCAATGGGGATTTACTACC-3', and the downstream primer Nm 2-2: 5'-GCAACGTTTCTTGCATTTC-3', and PCR amplification to obtain the gene sequence of SEQ ID NO.7 with the following sequence:
TGCAATGGGGATTTACTACCAGGACCTTGCAAAGCTAAAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGC。
(8) amplification of the Gene sequence encoding SEQ ID NO.8
Using the nucleotide sequence encoding SEQ ID NO.5 obtained as described above as a template, the sequence was determined using the upstream primer Nm 2-1: 5'-TGCAATGGGGATTTACTACC-3', and the downstream primer Nm 2-2: 5'-GCAACGTTTCTTGCATTTC-3', and PCR amplification to obtain the gene sequence of SEQ ID NO.8, the sequence is as follows:
TGCAATGGGGATTTACTACCAGGACCTTGCAAAGCTAGAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGC。
(9) amplification of the Gene sequence encoding SEQ ID NO.9
Using the nucleotide sequence encoding SEQ ID NO.6 obtained as described above as a template, the sequence was determined using the upstream primer Nm 2-1: 5'-TGCAATGGGGATTTACTACC-3', and the downstream primer Nm 2-2: 5'-GCAACGTTTCTTGCATTTC-3', and PCR amplification to obtain the gene sequence of SEQ ID NO.9 with the following sequence:
TGCAATGGGGATTTACTACCAGGACCTTGCAGAGCTAGAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGC。
(10) amplification of the Gene sequence encoding SEQ ID NO.10
The overlapping primer extension PCR method is used to obtain the gene sequence of the code SEQ ID NO. 10. Wherein the first round of amplification forward primer is amplified by Nm 10-3: 5'-ATTTACTACCAGGACCTTGCAGAGTTAAAATGGAAAGATGGGG-3', and the downstream primer Nm 4-2: 5'-ATCCACTTTGCAACGTTTCTTGCATTTC-3', and performing PCR amplification by using the cDNA sequence which is obtained from the American Isatis americana and codes for SEQ ID NO.2 as a template to obtain a first round of PCR products, wherein the first round of PCR products are used as a template and the designed upstream primer Nm 4: 5'-GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTG-3', and the downstream primer Nm 4-2: 5'-ATCCACTTTGCAACGTTTCTTGCATTTC-3', performing a second round of amplification to obtain a gene sequence encoding SEQ ID NO.10, the sequence is as follows:
GGTCATCTATGCAATGGGGATTTACTACCAGGACCTTGCAGAGTTAAAATGGAAAGATGGGGATTGGATAAGGAATCAGGAAAGTGCAAAAAATTCATCTACGGTGGTTGCGGTGGAAACAGAAACAATTTTGAAAGTGAAGAGAAATGCAAGAAACGTTGCAAAGTGGAT
EXAMPLE 2 protease inhibitory Activity of plasmin inhibitors of the invention
For methods for obtaining recombinant NKI10 of the present invention, reference is made to the following, document 9: "identification and characteristic study of inhibition effect of ancylostoma americanum protease inhibitor NaKuI1 on protease", inert fragrance, etc., China parasitology and parasite disease journal, volume 35, phase 3 in 2017, 6 months; document 10: "identification and characterization of the broad-spectrum serine protease inhibitor NaKuI3 of Necator americanus", Tufang et al, J.Med.biol.2017, Vol.12, No. 6. Briefly, upstream primer NI10-1e, expressing the gene encoding NKI10(SEQ ID NO.1), was designed: 5'-CAGGATCCATGGGAATGAAGGGAAGTGGT-3' (underlined BamH I cleavage site), downstream primer NI10-2 e: 5'-GCAAGCTTCAATCCACTTTGCAACGTTTCTT-3' (underlined is Hind III restriction enzyme cutting site), using the cDNA sequence which is obtained in the embodiment 1 of the invention and codes SEQ ID NO.1 as a template, amplifying to obtain a gene sequence which codes NKI10(SEQ ID NO.1), connecting the obtained gene sequence into a prokaryotic expression vector pET32a-SUMO, constructing a correct recombinant plasmid, transferring the recombinant plasmid into Escherichia coli BL21(DE3), inducing expression by IPTG, separating and ultrasonically crushing host thalli, purifying an expression product by nickel affinity chromatography to obtain a fusion protein, cutting the fusion partner by SUMO protease, and removing the fusion partner by affinity chromatography to obtain the recombinant NKI 10.
The inhibition of the relevant serine protease by recombinant NKI10 was observed by chromogenic substrate method. The protease/substrate used (concentration in brackets) were respectively: plasmin (5nmol/L)/S2288 (400. mu. mol/L), trypsin (1nmol/L) S2302 (200. mu. mol/L), trypsin (1nmol/L)/S7388 (200. mu. mol/L), elastase (5nmol/L)/S4760 (200. mu. mol/L), thrombin (1nmol/L)/S2288 (400. mu. mol/L), plasma bradykinin-releasing enzyme (1nmol/L)/S2302 (200. mu. mol/L). The protease is human protease except for pig elastase, wherein plasmin (plasmin) and thrombin (thrombin) are products of Haematologic Technologies Inc. of the United states, trypsin chymotrypsin and trypsin are products of CalbioChem of Germany, plasma kallikrein is products of Enzyme research laboratories of the United states, substrates S2302 and S2288 are products of Chromogenix of Italy, and S4760 and S7388 and pig elastase are products of sigma company of the United states. The detection method comprises adding 10 μ l of recombinant NKI10 (PBS for blank control) and 50 μ l of protease at corresponding concentration into 96-well plate (100 μ l reaction system), incubating at 25 deg.C for 15min, adding 40 μ l of pre-warmed corresponding substrate, and labeling with enzyme reader (Elx808IU) A405And (3) scanning and detecting enzyme reaction kinetics, reading the plate for 1 time every 15 seconds, continuously reading for 5min, recording the enzyme reaction speed, and repeating each group for 3 times. Inhibition rate (V)0-V)/V0Wherein，V0As the control group reaction rate, V is the reaction rate after adding plasmin inhibitor.
The experimental results show that: NKI10(SEQ ID NO.1) in a concentration of two times of molar ratio can completely inhibit the activity of human plasmin (plasmin); equimolar concentrations of NKI10(SEQ ID NO.1) inhibited human plasmin activity by more than 90%. NKI10 has certain inhibition effect on human trypsin (trypsin), and NKI10 with 2 times and equal molar concentration can inhibit the activity of human trypsin by about 22% and 10% respectively. 1. NKI10 at 2 and 5 fold molar concentrations had no significant inhibitory effect on trypsin (chymotrypsin), trypsin (elastase), thrombin (thrombin) and plasma kallikrein (plasma kallikrein). The result shows that NKI10 is a plasmin inhibitor with strong plasmin inhibition activity and good inhibition specificity.
Referring to the implementation method, the result shows that the NKI10 mutants, namely 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(SEQ ID NO.9) and NKI10-9(SEQ ID NO.10), have strong inhibitory activity on plasmin and have good inhibitory specificity.
EXAMPLE 3 anti-fibrinolytic Effect of plasmin inhibitor of the invention
The preparation method of the plasmin inhibitor in the embodiment is the same as that in the embodiment 2, and the fibrinolysis resistance of the plasmin inhibitor is observed by using a fibrinolysis plate dissolution experiment. The experimental method comprises the following steps: 1.2% agarose solution 15mL was prepared with 1 XHBSAC buffer (25mM HEPES, 100mM NaCl, 5mM CaCl2, pH 7.4), heated to dissolve, cooled to 50-60 deg.C, added with 1mL bovine fibrinogen solution 10mg/mL which had been incubated at 37 deg.C, immediately added with 100. mu.L thrombin (200U/mL), mixed well and poured into a petri dish to make a plate. The plate was punched and recombinant NKI10 and its mutant (concentration 0nmol/L (equal volume of physiological saline), 500nmol/L, 1000 nmol/L, 2000 nmol/L) and plasmin (2000 nmol/L) were added to the wells at 5. mu.L each, 3 wells per concentration, and incubated at 37 ℃ for 3 h. The inhibition effect of NKI10 and its mutant on fibrinolysis by plasmin is determined by observing the size of perifibrinolysis ring in the plate, and the smaller the fibrinolysis ring is, the stronger the inhibition effect on fibrinolysis by plasmin is.
As shown in FIG. 1, at the same plasmin concentration, as the concentration of NKI10 in each well was increased, the fibrinolytic ring around the well was reduced, and no fibrinolytic ring was evident around the well after addition of equimolar concentrations of NKI10 and plasmin (FIG. 1, D)1、D2And D3Well) indicating that NKI10 has a very significant anti-fibrinolytic effect and that its anti-fibrinolytic effect has a concentration-dependent relationship. The results of the embodiment show that the plasmin inhibitor can be used as a medicine for preventing or treating diseases related to hyperfibrinolysis, and the plasmin inhibitor can be applied to preventing or treating diseases related to hyperfibrinolysis by anti-fibrinolysis experiments of mutants of NKI10, namely 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(SEQ ID NO.9) and NKI10-9(SEQ ID NO. 10).
Example 4 Effect of plasmin inhibitors on mouse Tail bleeding time
The plasmin inhibitor preparation in this example is the same as in example 2. 40 Balb/c mice (experimental animal evidence: SCXK (Guangdong) 2013-: a normal saline control group, a recombinant NKI10(SEQ ID NO.1) group, a recombinant NKI10-1(SEQ ID NO.2) group, a recombinant NKI10-2(SEQ ID NO.3) group, a recombinant NKI10-3(SEQ ID NO.4) group, a recombinant NKI10-4(SEQ ID NO.5) group, a recombinant NKI10-5(SEQ ID NO.6) group and a recombinant NKI10-6(SEQ ID NO.7) group. Intraperitoneal injection of 2% pentobarbital sodium is performed to narcotize Balb/c mice, 50 mu g of human tissue plasminogen activator (tPA) (French HBM) is injected into each rat tail vein for 10min, and then 1.0 mg/kg of plasmin inhibitor is respectively injected into the tail vein-1Or an equal volume of saline. After administration for 2min, cutting the mouse at a distance of about 0.5cm from the tail tip, placing in physiological saline at 37 deg.C, recording the time from cutting to stopping bleeding as bleeding time, i.e. tail bleeding time, and averaging + -standard deviation
And (4) showing.
The results are shown in table 1, and the time of tail bleeding is significantly reduced (P <0.05) for each group of recombinant NKI10 and its mutant compared to the saline control group. The results show that the plasmin inhibitor can be applied to bleeding-related diseases as a medicine for reducing bleeding, and can be particularly used for reducing blood loss in operation so as to reduce the requirement of blood transfusion.
TABLE 1 Effect of plasmin inhibitors on tail bleeding time in fibrinolytic mice of the invention: (
n＝5)
P <0.05 compared to control group
The above description is only an exemplary embodiment of the present invention, and is not intended to limit the scope of the present invention. Any equivalent alterations, modifications and combinations can be made by those skilled in the art without departing from the spirit and principles of the invention.
Sequence listing
<110> Guangdong university of medical science
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Claims (4)
1. A plasmin inhibitor, said inhibitor having the amino acid sequence: 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. The application of a plasmin inhibitor in preparing a medicament for preventing or treating hyperfibrinolysis diseases is characterized in that the amino acid sequence of the inhibitor is as follows: 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.
3. The application of a plasmin inhibitor in preparing a medicament for preventing or treating bleeding diseases is characterized in that the amino acid sequence of the inhibitor is as follows: 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.
4. The pharmaceutical use according to claim 3, wherein the bleeding disorder is surgical blood loss.
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| 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 |
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| 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 |
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| 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;隋细香等;《中国寄生虫学与寄生虫病杂志》;20170630;第35卷(第3期);第259-264页 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023131595A1 (en) * | 2022-01-04 | 2023-07-13 | Solution Shop Ag | Ovomucoid for use in a method of managing blood coagulation |
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