A new type of recombinant protein Plasminogen-CTM and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a novel recombinant protein Plasminogen-CTM and its application. The present invention first provides a novel recombinant protein Plasminogen based on natural Plasminogen protein, and after the amino acid sites 742-810 of the natural Plasminogen protein are mutated in a combined manner, multiple recombinant protein mutants are obtained. The novel recombinant protein Plasminogen provided by the present invention has enzyme activity without activation by an activator, and can therefore be an effective means to replace natural plasmin directly isolated from plasma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a novel recombinant protein Plasminogen-CTM and its application. Background Art

[0002] Plasminogen, also known as plasma trypsinogen, is a precursor of plasmin (plasmin) and does not have the ability to enzymatically hydrolyze fibrin. In normal organisms, plasmin exists in the state of zymogen and becomes active only through the action of plasminogen activator. Plasmin is a proteolytic enzyme that can specifically hydrolyze fibrin gel and is an important component in the fibrinolytic system. The coagulation and fibrinolytic systems in the body are closely interdependent. Once the body produces a coagulation reaction, it will almost simultaneously activate the fibrinolytic system to remove excess thrombus in the body and reduce the level of fibrinogen in the body through a negative feedback effect, thereby avoiding excessive coagulation of fibrin. The lack of plasminogen will lead to the accumulation of fibrin, which in turn leads to the development of lesions, thereby damaging normal tissue and organ function.

[0003] At present, the most commonly used method for obtaining plasmin is plasma separation. However, this method has problems such as high cost of plasmin separation, limited source of raw materials, and expensive price of plasmin finished products. Although the plasmin obtained by gene mutation and synthetic biology can overcome the problems of high preparation cost and limited source of raw materials of plasma plasmin to a certain extent, the currently synthesized plasmin still has problems such as low activity or no activity. For example, the patent with publication number CN118308334A and name "A recombinant human plasminogen and its preparation method and application" discloses a recombinant human plasminogen and its preparation method and application. The patent also discloses a recombinant human plasminogen protein with a length of about 248 amino acids. However, the recombinant human plasminogen prepared by this scheme is in an inactive zymogen form, and an activator (e.g., urokinase) needs to be added to activate it to obtain active plasmin.

[0004] In summary, it is necessary to provide new methods or strategies to improve the deficiencies of the existing technologies. Summary of the invention

[0005] In view of this, the object of the present invention is to provide multiple variants and applications of a novel recombinant protein Plasminogen, and the specific technical solutions are as follows.

[0006] A novel recombinant protein Plasminogen, wherein the novel recombinant protein Plasminogen is based on a natural Plasminogen protein, and any one of the mutants after the following mutations are made at the amino acid positions 742-810 of the natural Plasminogen protein:

[0007] (1) The first mutant: Y at position 772 mutates to F, Q at position 775 mutates to L, T at position 778 mutates to V, N at position 788 mutates to L, Y at position 793 mutates to F, T at position 801 mutates to V, N at position 809 mutates to L and N at position 810 mutates to L (SEQ ID NO. 7); or,

[0008] (2) The second mutant: Q at position 757 mutates to L, Y at position 772 mutates to F, Q at position 775 mutates to L, T at position 778 mutates to V, N at position 788 mutates to L, Y at position 793 mutates to F, T at position 801 mutates to V, N at position 809 mutates to L and N at position 810 mutates to L (SEQ ID NO. 8); or,

[0009] (3) a third mutant: T at position 742 mutated to V, T at position 753 mutated to V, Q at position 757 mutated to L, Y at position 772 mutated to F, Q at position 775 mutated to L, T at position 778 mutated to V, N at position 788 mutated to L, Y at position 793 mutated to F, T at position 801 mutated to V, N at position 809 mutated to L and N at position 810 mutated to L (SEQ ID NO. 9); or,

[0010] (4) The fourth mutant: T at position 753 mutated to V, Q at position 757 mutated to L, Y at position 772 mutated to F, Q at position 775 mutated to L, T at position 778 mutated to V, N at position 788 mutated to L, Y at position 793 mutated to F, T at position 801 mutated to V, N at position 809 mutated to L and N at position 810 mutated to L (SEQ ID NO. 1).

[0011] As a preferred technical means, the novel recombinant protein Plasminogen is based on the natural Plasminogen protein, and the following mutations are made at the amino acid sites 753-810 of the natural Plasminogen protein:

[0012] The 753rd T mutated to V, the 757th Q mutated to L, the 772nd Y mutated to F, the 775th Q mutated to L, the 778th T mutated to V, the 788th N mutated to L, the 793rd Y mutated to F, the 801st T mutated to V, the 809th N mutated to L and the 810th N mutated to L.

[0013] As a preferred technical means, the amino acid sequence of the novel recombinant protein Plasminogen is shown as SEQ ID NO.1.

[0014] Furthermore, the amino acid of the novel recombinant protein Plasminogen also includes a sequence as shown in any one of SEQ ID NOs. 7-9.

[0015] A recombinant expression vector comprising any one of the novel recombinant protein Plasminogens described above.

[0016] Furthermore, the types of the vector include plasmid vectors, phage vectors or animal and plant virus vectors.

[0017] A recombinant bacterium expressing any one of the novel recombinant proteins Plasminogen.

[0018] Furthermore, the recombinant bacteria include Escherichia coli, Bacillus subtilis, Pichia pastoris or Saccharomyces cerevisiae.

[0019] Use of any of the above novel recombinant proteins Plasminogen in the preparation of a medicament for dissolving fibrin clots.

[0020] Use of any of the above novel recombinant proteins Plasminogen in the preparation of a drug for preventing the formation of fibrin clots.

[0021] Furthermore, the dosage form of the drug includes tablets, injections and / or sprays.

[0022] Use of any of the above novel recombinant proteins Plasminogen in the preparation of an in vitro fibrin clot dissolving reagent.

[0023] Beneficial technical effects:

[0024] The present invention obtains multiple recombinant Plasminogen protein variants with enzymatic activity by mutating multiple sites of the C-terminal (C-terminus) of the natural Plasminogen protein in a combined manner, among which the recombinant Plasminogen protein variant with the best effect has an enzymatic activity comparable to that of natural plasmin, and can effectively promote the dissolution of fibrin clots; in addition, the crystal structure of the optimal mutant is similar to that of the natural protein, and the overall hydrophilicity and hydrophobicity are also similar to those of the natural Plasminogen protein, so it has broad clinical application value and market development value.

[0025] Although the enzyme activity of several other mutants is weaker than that of the optimal mutant and natural plasmin, they do not require additional activators to activate them to have enzyme activity. Therefore, they can also become an alternative to natural plasmin directly isolated from plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0027] Figure 1 The electrophoresis band diagrams of multiple recombinant proteins prepared by the present invention;

[0028] Figure 2 is the crystal structure of Plasminogen-CTM predicted by Alphafold 3;

[0029] Figure 3 The crystal structure of the natural Plasminogen protein predicted by Alphafold 3;

[0030] Figure 4 The hydrophilicity analysis results of the recombinant protein Plasminogen-CTM prepared by the present invention;

[0031] Figure 5 It is the electrophoresis band diagram of the recombinant protein Plasminogen without mutation;

[0032] Figure 6 These are the results of enzyme activity assays of the recombinant protein Plasminogen-CTM and other recombinant proteins or plasmin prepared by the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0035] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0036] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0037] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0038] Materials and Reagents:

[0039] Sprague Dawley rats were purchased from the Experimental Animal Center of Nantong University, male rats, 4 weeks old, order number: B241009410.

[0040] Escherichia coli BL21 (DE3) competent cells were purchased from Beijing Solebow Technology Co., Ltd.

[0041] Plasmin (derived from human plasma) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., catalog number: P920011.

[0042] The pET-22b(+) plasmid is a commercially available E. coli expression vector purchased from Beijing Qingke Biotechnology Co., Ltd. The vector tags are N-pelB and C-His, and the vector resistance is Ampicillin.

[0043] Restriction endonucleases NdeI and XhoI were purchased from NEB (Beijing) Co., Ltd. His-tag protein purification resin (nickel column) was purchased from Shanghai Lianmai Bioengineering Co., Ltd., catalog number LM-616. IPTG, ampicillin, and DMSO (dimethyl sulfoxide) were all purchased from Beijing Solebow Technology Co., Ltd. DMEM medium was purchased from GIBCO. CCK8 reagent was purchased from Chongqing Baoguang Technology Co., Ltd. and used according to the reagent instructions.

[0044] Culture medium:

[0045] Each liter of LB medium contains: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and the pH is adjusted to 7.0.

[0046] Preparation method: Dissolve 5 g yeast extract, 10 g tryptone, and 10 g sodium chloride in 950 mL double distilled water, adjust the pH to 7.0 with sodium hydroxide solution, and dilute to 1 L with double distilled water. If preparing solid culture medium, add agar at 1.5 g / 100 mL. Sterilize at 121°C with high pressure steam for 30 min.

[0047] The experimental reagents not specifically described in the present invention are all conventional reagents in the art, and can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and reference can be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions of relevant reagent manufacturers.

[0048] Example 1

[0049] 1. Sequence design, expression and purification of recombinant protein Plasminogen-CTM.

[0050] The present embodiment provides a novel recombinant protein, which is obtained by performing site-directed amino acid mutagenesis on the natural Plasminogen protein, and is named as the recombinant protein Plasminogen-CTM, whose amino acid sequence is shown in SEQ ID NO.1, with a total length of 820 amino acids; the nucleotide sequence is shown in SEQ ID NO.2, with a total length of 2460 bp. When synthesizing the gene, NdeI restriction site (CATATG) and XhoI restriction site (CTCGAG) are added to the 5' end and 3' end of the gene respectively. The gene sequence with restriction sites is shown in SEQ ID NO.3. The synthesized gene is sequenced and verified, and the gene with the correct sequence is used for subsequent vector construction. The specific sequences involved are shown in Table 1.

[0051] Table 1

[0052]

[0053] 2. Vector Construction

[0054] In this embodiment, pET-22b(+) plasmid is preferably used as an expression vector. The pET-22b(+) plasmid and the target gene (SEQ ID NO.1) are subjected to double restriction digestion reaction with restriction endonucleases NdeI and XhoI, respectively, and then the target gene is connected to the pET-22b(+) vector by ligation reaction. It is understood that this embodiment can also be implemented by using phage vectors or animal and plant virus vectors.

[0055] 3. Recombinant Bacterial Transformation

[0056] (1) Take out the competent Escherichia coli BL21(DE3) cells (Beijing Solebow) from the -80℃ freezer and place them on ice for 5 min.

[0057] (2) After the glycerol containing the BL21(DE3) competent cells has melted, add the competent cells to the ligation product, pipette and release the pipette three times to mix, and place on ice for 30 min.

[0058] (3) Quickly wipe the water off the tube wall with absorbent paper, then heat shock at 42℃ for 90 seconds, and immediately place in an ice bath for 2 minutes.

[0059] (4) Under sterile conditions, add 800 μL of LB liquid culture medium and culture at 37°C, 150 rpm for 45 min.

[0060] (5) Collect the cells by centrifugation at 8000 rpm for 5 min, discard part of the supernatant, and use the remaining 100 μL of supernatant to resuspend the E. coli. Then, spread it evenly on LB solid culture medium containing 100 μg / mL ampicillin, place it in a 37°C incubator, and invert and culture for 12 h.

[0061] (6) Pick a single clone and inoculate it into liquid LB medium containing 100 μg / mL ampicillin. After culturing at 37°C for 12-16 h, identify the positive clone.

[0062] In this embodiment, Escherichia coli is preferably used as the recombinant bacteria. It is understandable that this embodiment can also be implemented using Bacillus subtilis, Pseudomonas aeruginosa or Saccharomyces cerevisiae strains.

[0063] 4. Protein Expression and Purification

[0064] (1) Inoculation: Prepare liquid LB medium and sterilize it. Place the sterilized liquid LB medium in a clean bench to cool to room temperature. Add ampicillin to the LB medium in the clean bench and mix well to make the final concentration of ampicillin 100 μg / mL. Then inoculate Escherichia coli (positive clone) containing the target gene plasmid into the LB medium at a volume of 200 μL / L. Place the LB medium in a shaker and culture at a speed of 170 rpm and a temperature of 37°C for 8-10 h.

[0065] (2) Induction: After culturing on a shaking table for 8-10 h, take out 2 mL of the bacterial solution and measure its OD600 value using a spectrophotometer. When the OD600 value of the bacterial solution reaches 0.6-0.8, add IPTG with a final concentration of 200 μL / mL to the bacterial solution, and then culture on a shaking table at 37°C and 170 rpm for 8 h.

[0066] (3) Purification: After adding IPTG and culturing on a shaking table for 8 h, remove the bacterial solution and centrifuge it at 8000 rpm at 4°C for 5 min. After centrifugation, remove the supernatant and retain the precipitate (the precipitate is Escherichia coli).

[0067] (4) Ultrasonic disruption: Ultrasonic disruption of E. coli was followed by centrifugation. The precipitate obtained by centrifugation contained the target protein. The precipitate was first washed with washing solution I (50 mmol / L Tris-HCl, 1 mol / L urea, 10 mL / L Triton X-100), then washed with washing solution II (50 mmol / L Tris-HCl, 2 mol / L urea, 5 mL / L Triton X-100), and then dissolved with inclusion body dissolution solution (50 mmol / L Tris-HCl, 8 mol / L urea, 100 mmol / L NaCl).

[0068] (5) Finally, the inclusion body solution was subjected to gradient renaturation as follows: the inclusion body solution was placed in a dialysis bag (Beijing Solebow Technology Co., Ltd., catalog number: YA1071), and then the dialysis bag was placed in 6 M, 4 M, 2 M, 1 M, and 0.5 M urea solutions for gradient renaturation. The renaturation was performed for 2-4 h at each urea concentration, and the renaturation was performed at a low temperature of 4°C. After the renaturation, the solution was purified by His-tag protein purification resin (nickel column, Shanghai Lianmai) to obtain the recombinant protein Plasminogen-CTM (the gene sequence of the target protein was designed with a histidine tag). After purification, polyacrylamide gel electrophoresis (SDS-PAGE) was performed to identify whether the purified target protein was successfully obtained. The results showed that a target protein of approximately 91.870 kDa was obtained (see Figure 1 ).

[0069] The high-level structures of the recombinant protein Plasminogen-CTM and the natural Plasminogen protein prepared in this example were predicted. Figure 2 and Figure 3 . Figure 2 The protein crystal structure of the novel recombinant protein Plasminogen-CTM is shown; Figure 3 The crystal structure of natural Plasminogen is shown; the results show that the three-dimensional structure of Plasminogen-CTM is similar to that of natural Plasminogen, without significant changes.

[0070] Example 2

[0071] This example provides verification of the hydrophilic and hydrophobic properties of the novel recombinant protein Plasminogen-CTM.

[0072] Furthermore, the hydrophilicity and hydrophobicity of the recombinant protein Plasminogen-CTM prepared in Example 1 were predicted. Figure 4 The horizontal axis represents the amino acid number of the protein; the vertical axis represents the hydrophilicity, and the larger the vertical axis value, the more hydrophobic it is; if it is a negative value, it represents hydrophilicity.

[0073] The results showed that the overall hydrophilicity and hydrophobicity of the new recombinant protein Plasminogen-CTM were similar to those of natural Plasminogen, with no significant difference.

[0074] Example 3

[0075] This example provides a recombinant Plasminogen protein and its hydrophilicity detection.

[0076] First, a natural Plasminogen protein is synthesized by recombinant means, which is called a recombinant Plasminogen protein (without mutation of hydrophilic and hydrophobic amino acids). The prokaryotic expression vector construction, recombinant bacterial transformation, protein expression and purification of the recombinant Plasminogen protein are the same as those in Example 1. The amino acid sequence SEQ ID NO.4, the nucleotide sequence SEQ ID NO.5 and the nucleotide sequence SEQ ID NO.6 with restriction enzyme cleavage sites of the unmutated recombinant protein Plasminogen constructed in this example are shown in Table 2.

[0077] Table 2

[0078]

[0079] After purification, SDS-PAGE was performed to identify whether the purified target protein was successfully obtained. Figure 5 .

[0080] The results showed that the target protein (non-mutated recombinant protein Plasminogen) with a size of about 90 kDa was obtained, which was consistent with its theoretical prediction value (91.94 kDa). Table 3 shows the basic information (theoretical value) of Plasminogen-CTM and non-mutated recombinant protein Plasminogen, as shown below.

[0081] Table 3

[0082]

[0083] As shown in Table 3, the hydrophilicity of Plasminogen-CTM is similar to that of the unmutated recombinant protein Plasminogen.

[0084] Example 4

[0085] This example provides other mutants of recombinant Plasminogen proteins, as shown in Table 4.

[0086] Table 4

[0087]

[0088] The prokaryotic expression vector construction, transformation, protein expression and purification of other recombinant Plasminogen protein mutants provided in this example are the same as those in Example 1. After purification, SDS-PAGE was performed to identify whether the purified target protein was successfully obtained. The results are as follows: Figure 1 shown.

[0089] Depend on Figure 1 The SDS-PAGE results show that after the hydrophilic amino acid mutation modification, the recombinant Plasminogen proteins with mutations at different sites can be effectively expressed, and their isoelectric points and hydrophilicity values ​​are shown in Table 5.

[0090] Table 5

[0091]

[0092] As shown in Table 5, the molecular weights, isoelectric points and average hydrophilicity values ​​of Plasminogen-8, Plasminogen-9 and Plasminogen-11 are similar, with no significant differences.

[0093] The amino acid sequences of the mutants Plasminogen-8, Plasminogen-9 and Plasminogen-11 of the recombinant Plasminogen protein involved in this example are shown in the following table.

[0094] Table 6

[0095]

[0096] Example 5

[0097] This example provides enzyme activity assays of multiple recombinant Plasminogen protein mutants prepared in Example 4.

[0098] Preparation of protease solution: Accurately weigh 50 μg of recombinant protein mutants Plasminogen-8, Plasminogen-9, Plasminogen-CTM, Plasminogen-11 and natural cellulase in turn, dissolve them in 500 mL of 0.02 M dibasic phosphate-citrate buffer, pH 6.0 at 40°C (operate in a constant temperature water bath), and the resulting solution is set aside.

[0099] Enzyme activity determination: add 10 mL of 3% fibrin solution to a large test tube, add 3 mL of buffer solution and balance in a 60℃ water bath for about 5 min, add 0.5 mL of the recombinant protein mutant solution of each experimental group, mix thoroughly and record the time. After 2 min, take out about 0.25 mL of the reaction solution with a dropper and drop it into the color adjustment well pre-filled with dilute iodine solution (0.75 mL). When the color of the liquid in the well changes from purple to brown-red, which is the same as the standard color, the reaction endpoint is reached and the time is recorded as T (min). Three parallel experiments were carried out in each experimental group, and the average enzyme activity of the recombinant protein in each experimental group was calculated in IU / mg.

[0100] The results are as follows Figure 6 As shown, the average enzyme activity of Plasminogen-CTM is comparable to that of natural plasmin, while the average enzyme activity of Plasminogen-8, Plasminogen-9 and Plasminogen-11 is weaker. The specific results are shown in Table 7.

[0101] Table 7

[0102]

[0103] The above results show that the unmutated Plasminogen (i.e., natural Plasminogen protein) has almost no enzyme activity, and the new recombinant protein Plasminogen-CTM exhibits the highest enzyme activity, which is not significantly different from the enzyme activity of natural plasmin. This shows that the new recombinant protein Plasminogen-CTM has enzyme activity without the need for an activator, and the enzyme activity is high, and it has the potential to become an effective means to replace the natural plasmin directly isolated from plasma. On the other hand, although the enzyme activity of the recombinant protein Plasminogen-8, Plasminogen-9 or Plasminogen-11 is weaker than that of Plasminogen-CTM and natural plasmin, it does not require additional activators to activate and has enzyme activity. Among them, Plasminogen-11, which has the smallest enzyme activity, can also reach about 30% of the enzyme activity value of natural plasmin, so Plasminogen-8, Plasminogen-9 and Plasminogen-11 can also become an alternative to natural plasmin directly isolated from plasma.

[0104] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0105] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A novel recombinant protein Plasminogen, characterized in that: The novel recombinant protein Plasminogen is based on the natural Plasminogen protein, and any one of the mutants after the following mutations are made at the amino acid positions 742-810 of the natural Plasminogen protein: (1) A first mutant: Y at position 772 mutates to F, Q at position 775 mutates to L, T at position 778 mutates to V, N at position 788 mutates to L, Y at position 793 mutates to F, T at position 801 mutates to V, N at position 809 mutates to L and N at position 810 mutates to L; the amino acid sequence of the first mutant is shown in SEQ ID NO.7; or, (2) A second mutant: Q at position 757 mutates to L, Y at position 772 mutates to F, Q at position 775 mutates to L, T at position 778 mutates to V, N at position 788 mutates to L, Y at position 793 mutates to F, T at position 801 mutates to V, N at position 809 mutates to L and N at position 810 mutates to L; the amino acid sequence of the second mutant is shown in SEQ ID NO.8; or, (3) A third mutant: T at position 742 mutates to V, T at position 753 mutates to V, Q at position 757 mutates to L, Y at position 772 mutates to F, Q at position 775 mutates to L, T at position 778 mutates to V, N at position 788 mutates to L, Y at position 793 mutates to F, T at position 801 mutates to V, N at position 809 mutates to L and N at position 810 mutates to L; the amino acid sequence of the third mutant is shown in SEQ ID NO.9; or, (4) The fourth mutant: T at position 753 mutated to V, Q at position 757 mutated to L, Y at position 772 mutated to F, Q at position 775 mutated to L, T at position 778 mutated to V, N at position 788 mutated to L, Y at position 793 mutated to F, T at position 801 mutated to V, N at position 809 mutated to L and N at position 810 mutated to L; the amino acid sequence of the fourth mutant is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: The recombinant expression vector is connected to a target gene, and the target gene is a gene of a novel recombinant protein Plasminogen of any mutant described in claim 1.

3. A recombinant bacterium, characterized in that A novel recombinant protein Plasminogen expressing any one of the mutants described in claim 1.

4. Use of the novel recombinant protein Plasminogen of any mutant described in claim 1 in the preparation of a medicament for dissolving fibrin clots.

5. Use of the novel recombinant protein Plasminogen of any mutant according to claim 1 in the preparation of a drug for preventing the formation of fibrin clots.

6. The use according to claim 4 or 5, characterized in that The dosage form of the medicine is tablet, injection or spray.

7. Use of the novel recombinant protein Plasminogen of any mutant according to claim 1 in the preparation of an in vitro fibrin clot dissolving reagent.

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