A recombinant leukocyte inhibitor and leech peptide chimeric protein mutant

Abstract

This invention belongs to the field of molecular biology, specifically disclosing a recombinant leukocyte inhibitory factor and a leech peptide chimeric protein mutant. Based on TNHH, this invention mutates cysteine, which is prone to translational mismatches, and mutates some nonpolar amino acids at the thrombin binding site to other amino acids, resulting in a TNHH mutant. TNHH is a bifunctional chimeric protein. Through site-directed mutagenesis, we have simultaneously solved two problems, not only reducing its isoform content and improving protein purity, but also significantly enhancing its biological activity, which has high practical significance.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a recombinant leukocyte inhibitory factor and a leech peptide chimeric protein mutant. Background Technology

[0002] In my country, cerebrovascular diseases are the second leading cause of death after malignant tumors, with ischemic brain injury accounting for two-thirds of these deaths. Ischemic stroke mainly falls into two categories: stroke caused by cerebral arteriosclerosis or cerebral embolism, and stroke caused by low cerebral blood flow due to cardiac arrest or other reasons. Currently, treatment primarily involves thrombolytic therapy using antiplatelet drugs such as aspirin and clopidogrel. While this can improve the condition of a stroke patient, the treatment period is long and carries serious sequelae, such as hemiplegia and cognitive impairment. Furthermore, the treatment is expensive and has numerous side effects.

[0003] Studies have shown that ischemic brain injury can lead to increased thrombin activity and leukocyte adhesion and aggregation in the human body. A novel biological drug, TNHH, can effectively inhibit both of these phenomena. TNHH (Targeted Neutrophil Inhibitory Factor and Hirugen Hybrid) was synthesized and researched by Chongqing Fujin Biopharmaceutical Co., Ltd. It is a bifunctional recombinant chimeric protein formed by adding a cross-linked peptide between leukocyte inhibitory factor (NIF) and hirugen (Hirulog), possessing the functions of both. It can inhibit both leukocyte migration and aggregation and thrombin activity.

[0004] TNHH, a novel protein drug for treating acute cerebrovascular diseases, is designed based on the pathological mechanism of local prothrombin activation during cerebral hemorrhage and thrombosis, leading to thrombin formation and subsequent platelet-induced microvascular obstruction and cerebral edema, as well as brain tissue damage caused by leukocyte infiltration and activation. It is a novel bifunctional anti-stroke protein targeting leukocyte activation and thrombin activity. Administered within 24 hours of acute onset and for approximately 7 days, this drug can achieve multiple objectives: restoring damaged brain tissue, reducing cerebral edema, and improving local microcirculation. Due to its good safety and significant efficacy, the dual-target fusion protein design of TNHH reduces the risks associated with NIF and Hirulog fragments while increasing effectiveness. Therefore, TNHH has become a novel drug with significant therapeutic and preventative effects (accelerating recovery and reducing sequelae) for acute stroke (especially acute cerebral embolism). This will bring good news to the increasing number of elderly patients with acute cerebrovascular events, while saving significant medical and social costs.

[0005] TNHH consists of 282 amino acids, with an N-terminal NIF of 257 amino acids and a C-terminal 20-peptide hirudin (Hirulog). The middle region is composed of 5 glycine hinge regions, with the first amino acid at the N-terminus being methionine. The NIF region in TNHH is identical to the natural NIF except for the addition of Met at position 1. However, the hirudin (Hirulog region) differs from the Hirulog reported in foreign literature in two amino acid sequences: the first D-proline in the binding peptide FPRPGGGG is replaced with an L-proline, and the sixth glycine is replaced with serine. Its nucleotide sequence is shown in SEQ.ID NO:1, and its amino acid sequence is shown in SEQ.ID NO:2. The specific structure of TNHH is: Met-NIF(257)-(Gly)5-FPRPGSGG-Hirugen(53-64). TNHH has a molecular weight of 31.5 KD, a pI of 4.5, contains 10 cysteine ​​residues, 5 disulfide bonds, and is non-glycosylated. Because of their long cysteine ​​side chains, cysteine ​​residues are prone to mismatches during translation and expression, leading to isoforms that reduce protein purity and bioactivity. Therefore, a TNHH mutant is needed to reduce mismatches during translation and expression, decrease isoform formation, improve protein purity, and ultimately enhance biological activity. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a mutant of recombinant leukocyte inhibitory factor and hirudin chimeric protein. This mutant is less prone to isoform generation during translational expression, resulting in higher product purity, and exhibits greater biological activity in inhibiting both leukocyte metastasis and thrombin aggregation.

[0007] This invention, based on recombinant leukocyte inhibitory factor and leech peptide chimeric protein TNHH (amino acid sequence shown in SEQ.ID NO:2), mutates cysteine, which is prone to translational mismatch, to obtain a TNHH mutant with higher purity. Furthermore, some nonpolar amino acids at the thrombin binding site are mutated to other amino acids, further enhancing the biological activity of the TNHH mutant.

[0008] In a first aspect, the present invention provides a mutant of recombinant leukocyte inhibitory factor and hirudin chimeric protein. Compared with the original TNHH, the amino acids of the TNHH mutant are mutated, resulting in a class of high-purity, high-biological-activity TNHH mutants that inhibit leukocyte adhesion. The mutations involve, on the one hand, mutating cysteine, which contains disulfide bonds prone to mismatch, to an amino acid with a shorter side chain; and on the other hand, mutating the nonpolar amino acid at the thrombin binding site to other amino acids.

[0009] A mutant of a recombinant leukocyte inhibitory factor and hirudin chimeric protein, the amino acid sequence of which is shown below;

[0010] Comprising:

[0011] Asn-Glu-His-Asn-Leu-Arg-Cys-Pro-Gln-Asn-Gly-Thr-Glu-Met-Pro-Gly-Phe-Asn-Asp-Ser-Ile-Arg-Leu-Gln-Phe-Leu-Ala-Met-His-Asn-Gly-Tyr-Arg-Ser-Lys-Leu-Ala-Leu-Gly-His-Ile-Ser-Ile-Thr-Glu-Glu-Ser-Glu-Ser-Asp-Asp-Asp-Asp-Asp-Phe-Gly-Phe-Leu-Pro-Asp-Phe-Ala-Pro-Arg-Ala-Ser-Lys-Met-Arg-Tyr-Leu-Glu-Tyr-Asp-Cys-Glu-Ala-Glu-Lys-Ser-Ala-Tyr-Met-Ser-Ala-Arg-Asn-Cys-Ser-Asp-Ser-Ser-Ser-Pro-Pro-Glu-Gly-Tyr-Asp-Glu-Asn-Lys-Tyr-Ile-Phe-Glu-Asn-Ser-Asn-Asn-Ile-Ser-Glu-Ala-Ala-Leu-Lys-Ala-Met-Ile-Ser-Trp-Ala-Lys-Glu-Ala-Phe-Asn-Leu-Asn-Lys-Thr-Lys-Glu-Gly-Glu-Gly-Val-Leu-Tyr-Arg-Ser-Asn-His-Asp-Ile-Ser-Asn-Phe-Ala-Asn-Leu-Ala-Trp-Asp-Ala-Arg-Glu-Lys-Phe-Gly-Xaa 162 -Ala-Val-Val-Asn-Cys-Pro-Leu-Gly-Glu-Ile-Asp-Asp-Glu-Thr-Asn-His-Asp-Gly-Glu-Thr-Tyr-Ala-Thr-Thr-Ile-His-Val-Val-Cys-His-Tyr-Pro-Lys-Ile-Asn-Lys-Thr-Glu-Gly-Gln-Pro-Ile-Tyr-Lys-Val-Gly-Thr-Pro-Xaa 211 -Asp-Asp-Xaa 214-Ser-Glu-Tyr-Thr-Lys-Lys-Ala-Asp-Asn-Thr-Thr-Ser-Ala-Asp-Pro-Val-Cys-Ile-Pro-Asp-Asp-Gly-Val-Cys-Phe-Ile-Gly-Ser-Lys-Ala -Asp-Tyr-Asp-Ser-Lys-Glu-Phe-Tyr-Arg-Phe-Arg-Glu-Leu-Gly-Gly-Gly-Gly-Gly-Phe-Pro-Arg-Pro-Gly-Ser-Gly-Gly-Asn-Gly-Asp-Xaa 274 -Glu-Glu-Xaa 277 -Xaa 278 -Glu-Glu-Tyr-Xaa 282 .

[0012] in,

[0013] Xaa 162 It is one of Cys, Ala, Leu, Gly, Ser, or Val;

[0014] Xaa 211 It is one of Cys, Ala, Leu, Gly, Ser, or Val;

[0015] Xaa 214 It is one of Ala, Leu, Gly, Ser, or Val;

[0016] Xaa 274 It is one of Phe, Asn, Cys, Glu, Gln, Thr, Tyr, Gly, or Ser;

[0017] Xaa 277 It is one of Ile, Asn, Cys, Glu, Gln, Thr, Tyr, Gly, or Ser;

[0018] Xaa 278 It is one of Pro, Asn, Cys, Glu, Gln, Thr, Tyr, Gly, or Ser;

[0019] Xaa 282 It is one of Leu, Asn, Cys, Glu, Gln, Thr, Tyr, Gly, or Ser.

[0020] Preferably,

[0021] Xaa 162 It is one of Cys, Ala, Ser, or Val;

[0022] Xaa 211 It is one of Cys, Ala, Gly, or Ser;

[0023] Xaa 214 It is either Ala or Gly;

[0024] Xaa 274 It is one of Phe, Asn, Glu, Thr, Gly, or Ser;

[0025] Xaa 277 It is one of Ile, Cys, Gln, Thr, Tyr, Gly, or Ser;

[0026] Xaa 278 It is one of Pro, Cys, Thr, Tyr, Gly, or Ser;

[0027] Xaa 282 It is one of Leu, Glu, Gln, Thr, Tyr, and Gly.

[0028] Further optimization,

[0029] Xaa 162 For Cys or Ala;

[0030] Xaa 211 For Cys or Ala;

[0031] Xaa 214 For Ala;

[0032] Xaa 274 For Phe or Ser;

[0033] Xaa 277 For Ile or Gly;

[0034] Xaa 278 For Pro or Thr;

[0035] Xaa 282 For Leu or Glu.

[0036] In one embodiment, the TNHH mutant

[0037] Xaa 162 For Ala;

[0038] Xaa 211 For Cys;

[0039] Xaa 214 For Ala;

[0040] Xaa274 For Phe;

[0041] Xaa 277 For Ile;

[0042] Xaa 278 For Thr;

[0043] Xaa 282 The amino acid sequence of the TNHH mutant is Glu; it is shown in SEQ.ID NO:3.

[0044] In another embodiment, the TNHH mutant;

[0045] Xaa 162 For Ala;

[0046] Xaa 211 For Cys;

[0047] Xaa 214 For Ala;

[0048] Xaa 274 For Ser;

[0049] Xaa 277 For Gly;

[0050] Xaa 278 For Pro;

[0051] Xaa 282 The amino acid sequence of the TNHH mutant is shown in SEQ.ID NO:4.

[0052] In another embodiment,

[0053] Xaa 162 For Cys;

[0054] Xaa 211 For Ala;

[0055] Xaa 214 For Ala;

[0056] Xaa 274 For Phe;

[0057] Xaa 277 For Ile;

[0058] Xaa 278 For Thr;

[0059] Xaa 282 The amino acid sequence of the TNHH mutant is Glu; it is shown in SEQ.ID NO:5.

[0060] In another embodiment,

[0061] Xaa 162 For Cys;

[0062] Xaa 211 For Ala;

[0063] Xaa 214 For Ala;

[0064] Xaa 274 For Ser;

[0065] Xaa 277 For Gly;

[0066] Xaa 278 For Pro;

[0067] Xaa 282 The amino acid sequence of the TNHH mutant is shown in SEQ.ID NO:6.

[0068] In a second aspect, the present invention provides a polynucleotide encoding the TNHH mutant described herein.

[0069] A third aspect of the present invention provides an expression vector comprising the polynucleotide described in the second aspect. In one embodiment, the expression vector is pET-3c.

[0070] In a fourth aspect, the present invention provides a host cell comprising the expression vector described in the third aspect, or the genome of the host cell having the polynucleotides described in the second aspect integrated therein.

[0071] In a preferred embodiment, the host cell is a prokaryotic cell, preferably Escherichia coli.

[0072] In a fifth aspect, the present invention provides a method for preparing a TNHH mutant.

[0073] To obtain the TNHH mutant, this invention employs the following steps: ① Referring to the original TNHH sequence SEQ.ID NO:2, identify amino acids prone to mismatch and those not functioning in the active site with leukocyte-inhibiting effects, determine the mutation site, and design primers. ② After preparing the template primers, obtain a complete plasmid using PCR amplification. Sequencing with an automated sequencer confirms that the base sequence of the artificially synthesized cDNA fragment is consistent with the design. ③ Prepare competent E. coli DH5α cells and transform the plasmid into DH5α cells. Sequencing of glycerol-containing bacteria confirms that the plasmid is consistent with the design. ④ Transform the recombinant plasmid into E. coli BL21(DE3)pLysS, plate it overnight, and the growth of a single colony indicates successful transformation. ⑤ Sequencing analysis of the successfully transformed E. coli pET-3c-BL21(DE3)pLysS shows that the target gene is correctly embedded in the vector and the target gene sequence is identical to the design. This enables the TNHH mutant protein to be expressed in the host cells. Finally, the TNHH mutant protein is extracted and purified from the bacterial cells.

[0074] In a sixth aspect, the invention provides the use of TNHH mutants in the preparation of drugs for treating cardiovascular and cerebrovascular diseases, particularly in cerebral ischemia, brain injury, or related complications.

[0075] A seventh aspect of the present invention provides a pharmaceutical composition comprising the TNHH mutant described in the first aspect and pharmaceutically acceptable excipients.

[0076] The pharmaceutical composition of this invention can be in any dosage form, such as an injectable formulation, including solutions and lyophilized powders. When preparing the injectable formulation, a certain amount of inorganic salts or amino acid buffers, such as phosphates, acetates, carbonates, citrates, glycine, and histidine, can be added to the TNHH of this invention. The salts primarily refer to sodium salts, and the ionic strength is 5-100 mmol / L. The pH of the pharmaceutical composition is maintained at 5.0-9.0. Protein protectants such as albumin, gelatin, polysaccharides, starch, and glycerol can also be added to the composition. The polysaccharides are preferably one or more of mannitol, sucrose, and trehalose.

[0077] Compared with the prior art, the present invention has the following advantages:

[0078] (1) Mutating some Cys in TNHH to Ala shortens the amino acid side chain, which can effectively reduce the probability of mismatch, reduce the content of isomers, and improve protein purity.

[0079] (2) The nonpolar amino acids of the leech peptide in the mutant TNHH gene sequence are replaced with other polar amino acids, which can improve its biological activity and enhance its ability to inhibit leukocyte adhesion.

[0080] (3) TNHH is a chimeric protein with dual functions. We solved two problems at the same time through gene site-directed mutagenesis technology. This not only reduced its isoform content and improved the protein purity, but also greatly improved its biological activity, which has great practical significance. Attached Figure Description

[0081] Figure 1 This is an SDS-PAGE electrophoresis image of TNHH and its mutants induced expression;

[0082] Where M: Protein Marker;

[0083] Lane 1: TNHH before IPTG induction;

[0084] Lane 2: TNHH;

[0085] Lane 3: Mutant TNHH-M1;

[0086] Lane 4: Mutant TNHH-M2;

[0087] Lane 5: Mutant TNHH-M3;

[0088] Lane 6: Mutant TNHH-M4.

[0089] Figure 2 This is an SDS-PAGE electrophoresis image of the purified TNHH mutant.

[0090] Where M: Protein Marker;

[0091] Lane 1: Purified TNHH;

[0092] Lane 2: Purified mutant TNHH-M1;

[0093] Lane 3: Purified mutant TNHH-M2;

[0094] Lane 4: Purified mutant TNHH-M3;

[0095] Lane 5: Purified mutant TNHH-M4.

[0096] Figure 3 This is the HPLC chromatogram of TNHH.

[0097] Figure 4 This is the HPLC chromatogram of the mutant TNHH-M1.

[0098] Figure 5 This is the HPLC chromatogram of the mutant TNHH-M2.

[0099] Figure 6This is the HPLC chromatogram of the mutant TNHH-M3.

[0100] Figure 7 This is the HPLC chromatogram of the mutant TNHH-M4. Detailed Implementation

[0101] Site-directed mutagenesis

[0102] Targeted mutagenesis is a protein engineering technique that involves replacing, inserting, or deleting specific amino acids in a known DNA sequence, based on the known structure and function of the protein, to produce mutant protein molecules with new traits.

[0103] Targeted mutagenesis can alter the physicochemical properties of proteins, such as improving the stability of protein drugs; enhancing the solubility of proteins and drugs; and improving biological properties, including but not limited to altering the specificity of enzymes to substrates, increasing enzyme activity, and improving affinity and specificity.

[0104] Site-directed mutagenesis can enhance or eliminate the binding activity between ligands and receptors, or enzymes and substrates, by mutating amino acids in the binding domain. This mutation specifically alters the secondary or higher-order structure and charge of the protein. If the mutated amino acid is located at a critical antigen-antibody interaction site, this amino acid change is likely to alter the charge and secondary or higher-order structure of that site, preventing the binding of ligands and receptors, or enzymes and substrates, thus achieving the mutagenesis goal. Simultaneously, it prevents the original antibody from recognizing this site, forming a new antigen. This is an inevitable consequence of this traditional mutagenesis method.

[0105] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the raw materials and other materials used in the embodiments can be obtained commercially. Experimental methods not specified in the embodiments can be implemented by conventional techniques in the art or methods recommended by the manufacturer.

[0106] Example 1: Obtaining the TNHH gene mutant

[0107] I. Primer Synthesis

[0108] Based on the TNHH mutation sites shown in Table 1, primers required for the mutation were designed using Primer software and then designed and synthesized by Shanghai Sangon Biotech. Primers required for the TNHH mutant are listed in Table 2.

[0109] Table 1 Mutation sites of different TNHH mutants

[0110] mutant mutation site TNHH-M1 278th position: Pro → Thr; 282nd position: Leu → Glu; 162nd and 214th positions: Cys → Ala TNHH-M2 274th position: Phe → Ser; 277th position: Ile → Gly; 162nd and 214th positions: Cys → Ala TNHH-M3 278th position: Pro → Thr; 282nd position: Leu → Glu; 211th and 214th positions: Cys → Ala TNHH-M4 274th position: Phe → Ser; 277th position: Ile → Gly; 211th and 214th positions: Cys → Ala

[0111] Table 2 Primers required for TNHH mutants

[0112]

[0113]

[0114] II. PCR reaction

[0115] The primers, template DNA, and buffer system required to mutate the 162nd amino acid of the original TNHH sequence were mixed according to the following reaction system.

[0116]

[0117] After mixing, perform PCR amplification using the following procedure:

[0118]

[0119] The first site mutation of pET-3c-TNHH-M1 was completed in the plasmid. Then, according to the primers required in Table 2, the above steps were repeated until all four sites of the pET-3c-TNHH-M1 mutant were mutated, resulting in the complete pET-3c-TNHH-M1 mutant plasmid, labeled as pET-3c-TNHH-M1. The sample was sent to Shanghai Sangon Biotech for sequencing analysis, confirming that the amino acid sequence of the obtained mutant plasmid was consistent with the design. The amino acid sequence of the TNHH mutant in this invention is shown in SEQ.ID NO:3-6, and the nucleotide sequence is shown in SEQ.ID NO:7-10.

[0120] III. Transformation of mutant plasmids into DH5α

[0121] Preparation of DH5α competent cells:

[0122] 1. Streak DH5α glycerol bacteria on LB agar plates and incubate overnight at 37°C;

[0123] 2. Pick a single colony and inoculate it into 5 ml of LB liquid medium. Incubate overnight at 37°C and 150 rpm. Transfer the inoculum to 5 ml of LB medium at a 1% inoculation rate and incubate at 37°C and 150 rpm for 2-3 hours. Measure the OD. 600 It ranges from 0.4 to 0.6;

[0124] 3. Place the culture medium in an ice bath or pre-cool it in a 4°C refrigerator. Take 1 ml of the culture medium and centrifuge at 4°C, 6000 rpm for 5 min to collect the bacterial cells.

[0125] 4. Wash the bacterial cells twice with 1 ml of pre-cooled sterile 0.1 mol / L CaCl2 solution;

[0126] 5. Resuspend the cells in 100 μL of pre-cooled 0.1 mol / L CaCl2 solution and store at 4°C for later use.

[0127] Plasmid transformation of competent cells:

[0128] 1. Transform the mutant product M1 into competent cells, mix gently, and incubate on ice for 30 minutes;

[0129] 2. Place the EP tube in a water bath preheated to 42°C for 90 seconds, without moving the EP tube;

[0130] 3. Quickly remove the EP tube and cool it in an ice bath for 3-5 minutes;

[0131] 4. Add 800 μL of SOC medium to each tube and incubate at 37°C and 100 rpm for 45 min;

[0132] 5. Spread 100 μL of culture medium onto an LB ampicillin plate and incubate overnight at 37°C.

[0133] Pick a single colony from the plate and inoculate it into 5 ml of LB liquid medium containing 100 μg / ml ampicillin. Incubate overnight at 37°C and 150 rpm.

[0134] Centrifuge 4 ml of bacterial culture to collect bacterial cells, extract plasmids using the Dalian Baosheng plasmid extraction kit, elute with 60 μL of sterile water, label, and store at -20℃.

[0135] IV. Transformation of recombinant plasmid into E. coli BL219(DE3)pLysS

[0136] Preparation of BL21(DE3)pLysS competent cells:

[0137] 1. Streak BL21(DE3)pLysS glycerol bacteria on LB agar plates and incubate overnight at 37°C;

[0138] 2. Pick a single colony and inoculate it into 5 ml of LB liquid medium. Incubate overnight at 37°C and 150 rpm. Transfer the inoculum to 5 ml of LB medium at a 1% inoculation rate and incubate at 37°C and 150 rpm for 2-3 hours. Measure the OD. 600 It ranges from 0.4 to 0.6;

[0139] 3. Place the culture solution in an ice bath or pre-cool it in a 4°C refrigerator. Take 1 ml of the culture solution and centrifuge at 4°C, 6000 rpm for 5 min to collect the bacterial cells.

[0140] 4. Wash the bacterial cells twice with 1 ml of pre-cooled and sterilized 0.1 mol / L CaCl2 solution;

[0141] 5. Resuspend the cells in 100 μL of pre-cooled 0.1 mol / L CaCl2 solution and store at 4°C for later use.

[0142] Plasmid transformation of competent cells:

[0143] 1. Transform the mutant plasmid M1 into competent cells, mix gently, and incubate on ice for 30 minutes;

[0144] 2. Place the EP tube in a water bath preheated to 42°C for 90 seconds, without moving the EP tube;

[0145] 3. Quickly remove the EP tube and cool it in an ice bath for 3-5 minutes;

[0146] 4. Add 800 μL of SOC medium to each tube and incubate at 37°C and 100 rpm for 45 min;

[0147] 5. Spread 100 μL of culture medium onto an LB ampicillin plate and incubate overnight at 37°C.

[0148] A single colony growing on the plate indicates successful transformation, which is the TNHH mutant strain pET-3c-TNHH / BL21(DE3)pLysS-M1.

[0149] The same method was used to prepare strains pET-3c-TNHH / BL21(DE3)pLysS-M2, pET-3c-TNHH / BL21(DE3)pLysS-M3, and pET-3c-TNHH / BL21(DE3)pLysS-M4.

[0150] Example 2: Shake-flask expression of mutant strains pET-3c-TNHH / BL21(DE3)pLysS-M1 to pET-3c-TNHH / BL21(DE3)pLysS-M4

[0151] Preliminary expression studies were conducted on four mutant strains and the original *E. coli* strain pET-3c-TNHH / BL21(DE3)pLysS in shake flasks. First, single colonies were picked from the corresponding plates and inoculated into LB ampicillin medium (0.5% yeast extract, 1% peptone, 1% sodium chloride, 100 mg / L ampicillin), with two flasks inoculated per strain. The cultures were incubated at 37°C for 10 h, and OD was measured. 600 Expression was induced using a final concentration of 0.4 mol / L IPTG between 0.8 and 1.2, and the culture was terminated after 4 hours. The bacterial culture was centrifuged at 12000 rpm for 3 minutes, the supernatant was discarded, and the expression level was analyzed by SDS-PADE. The results are shown below. Figure 1 ,from Figure 1 It can be seen that the protein expression level after gene mutation is increased compared with the initial TNHH expression level.

[0152] Example 3: Fermentation purification of TNHH and mutants TNHHM1-M4

[0153] I. Fermentation-induced expression of the original strain pET-3c-TNHH / BL21(DE3)pLysS and the mutant strains pET-3c-TNHH / BL21(DE3)pLysS-M1~pET-3c-TNHH / BL21(DE3)pLysS-M4 in fermenters

[0154] 1. Seed culture:

[0155] Single colonies of the original strain pET-3c-TNHH / BL21(DE3)pLysS and the mutant strains pET-3c-TNHH / BL21(DE3)pLysS-M1~pET-3c-TNHH / BL21(DE3)pLysS-M4 were picked and inoculated into LB medium containing 100 mg / L ampicillin. They were cultured at 37℃ and 150 rpm for 10 h to obtain the seed culture for fermentation.

[0156] 2. Fermentation in a 30L fermentation tank:

[0157] Add 10L of basal fermentation medium (0.3% yeast extract, 0.5% peptone, 3.5% dodecahydrate and disodium hydrogen phosphate, 0.5% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.1% sodium chloride, 0.1% magnesium sulfate, 1% glucose) to a 30L fermenter. Autoclave the medium. After the medium cools to 37℃, adjust the pH to 7.0 with ammonia. Inoculate the culture, adjust the aeration speed and ventilation rate to maintain dissolved oxygen at 30-70%. After 6 hours of cultivation, when the dissolved oxygen recovers to 90%, start feeding (0.3% yeast extract, 0.5% peptone, 2% glucose). After 2 hours of feeding, induce expression using IPTG. Induce fermentation for 12 hours and then end the fermentation. Centrifuge the fermentation broth at 12000rpm for 10 minutes, discard the supernatant, collect the cells, and store at 4℃ for later use.

[0158] II. Purification of TNHH and its mutant TNHH-M1-M4

[0159] 1. Pressure homogenization and sterilization

[0160] Add 1L of lysis buffer to every 100g of pET-3c-TNHH / BL21(DE3)pLysS-M1 cells, and homogenize 2-3 times at 40MPa. Centrifuge at 12000rpm for 15min at 4℃, and retain the precipitate.

[0161] 2. Washing inclusion bodies

[0162] Add the same volume of wash buffer as above, stir thoroughly to dissolve, centrifuge at 4°C, 12000 rpm, for 15 min, and retain the precipitate. Then dissolve thoroughly with the same volume of dissolving buffer as above.

[0163] 3. Refolding and Concentration

[0164] Refold using the same volume of refolding solution at approximately 10°C, with an ultrafiltration concentration 20-30 times higher than the 3KD molecular weight cutoff.

[0165] 4. Purification using Butyl Sepharose hydrophobic chromatography column

[0166] Connect the hydrophobic chromatography column to the AKTA chromatography system and equilibrate it with pH 8.0 equilibration buffer (25 mmol / L Tris, 0.3 M (NH4)2SO4, 0.5 mol / L NaCl). Start loading the sample, and after equilibration, elute with the equilibration buffer to collect the permeate peak. Elute any other proteins with pH 8.0 25 mmol / L Tris. Adjust the pH to 3.0–4.0 with hydrochloric acid to precipitate the target protein, centrifuge at 12,000 rpm for 15 min at 4°C, and retain the precipitate.

[0167] 5. Purification using Source 30Q ion exchange chromatography column

[0168] The precipitate was dissolved in 20 mmol / L PB and filtered through a 0.45 μm filter. Ion exchange chromatography was connected to the AKTA chromatography system, equilibrated with pH 8.0 equilibration buffer (20 mmol / L PB, 0.2 mol / L NaCl), and sample loading began. After equilibration, the permeate peak was collected with pH 8.0 elution buffer (20 mmol / L PB, 0.5 mol / L NaCl). The pH was adjusted to 3.0–4.0 with phosphate to precipitate the target protein. The precipitate was centrifuged at 12,000 rpm for 15 min at 4 °C and retained.

[0169] 6. Purification using Superdex 75 chromatography column

[0170] Connect the gel chromatography to the AKTA chromatography instrument, equilibrate with pH 6.5 10 mmol / L PB, start loading the sample, and after completion, elute with pH 6.5 10 mmol / L PB. The obtained target protein solution is the mutant TNHH-M1.

[0171] The same method was used to obtain TNHH protein solution and mutant TNHH-M2~M4 protein solution.

[0172] The purified TNHH and mutant TNHH-M1~M4 protein solutions were subjected to SDS-PAGE electrophoresis. The electrophoresis images are shown below. Figure 2The results showed that only a single target band was observed in each lane; the purity of each protein solution was determined by HPLC, and the HPLC chromatogram is shown below. Figures 3-7 Based on the area normalization method, the purity of the original TNHH protein solution was 95.8%; an impurity with a retention time of 8.760 min was observed before the TNHH chromatographic peak. Figure 4-7 The purity of mutant TNHH-M1 was 98.3%. The purity of mutant TNHH-M2 was 98.77%. The purity of mutant TNHH-M3 was 99.01%. The purity of mutant TNHH-M4 was 98.94%. Compared with the original TNHH protein solution (95.8%), mutants TNHH-M1–M4 showed a significant decrease in isoform content and a significant increase in HPLC purity.

[0173] Example 5: In vivo bioactivity assay

[0174] Ninety Wistar rats were selected and fed for one week. Fifty rats weighing between 200 and 300 g were randomly divided into five groups: TNHH group, M1 group, M2 group, M3 group, and M4 group. An ischemic brain injury model was established. After successful modeling, treatment was administered via tail vein injection. The TNHH group received 6.5 mg / kg, 0.5 ml / rat; the M1-M4 groups received the mutant TNHH-M1-M4 at 6.0 mg / kg, 0.5 ml / rat, respectively. Administered every 12 hours for 14 consecutive days. Rats were then sacrificed, and the infarct area, brain tissue IL-10 content, serum TNF-α content, and MPO activity were measured. The results are shown in Table 3.

[0175] Table 3. In vivo bioactivity assay of TNHH and mutants

[0176] Group Cerebral infarction area (%) IL-10 TNF-α (ng / ml) MPO activity (U / g) TNHH group 24.51±6.61 23.11±3.18 1.035±0.131 0.168±0.019 TNHH-M1 group 22.85±5.77 20.74±3.14 1.003±0.075 0.155±0.017 TNHH-M2 group 21.63±3.50 22.97±3.41 0.903±0.115 0.161±0.014 TNHH-M3 group 19.88±4.79 20.57±2.33 0.871±0.092 0.147±0.012 TNHH-M4 group 21.34±3.27 21.52±2.47 0.984±0.147 0.154±0008

[0177] As shown in Table 3, compared with TNHH, the mutants TNHH-M1 to M4 can more effectively reduce the cerebral infarction area in rats, decrease the serum TNF-α content, and increase MPO activity, indicating stronger anti-inflammatory ability.

[0178] Example 6 Anti-leukocyte adhesion assay

[0179] I. Cell passage

[0180] Human promyelocytic leukemia cells HL-60 were cultured and passaged using complete culture medium (RPMI 1640 medium containing 20% ​​fetal bovine serum).

[0181] II. Experiment on inhibition of leukocyte adhesion

[0182] 1. Centrifuge the above cells, discard the supernatant, resuspend in complete culture medium, and count the cells at 1.0 × 10⁻⁶. 6 Cells were seeded at a concentration of 10 ng / ml into culture flasks, and PMA inducer was added to a final concentration of 10 ng / ml. The flasks were then incubated at 37°C in a 5% CO2 incubator for 24-36 hours. Cell adhesion was considered a successful induction.

[0183] 2. Discard the cell culture medium, wash three times with complete culture medium, gently scrape off the cells using a cell scraper, add 2 ml of complete culture medium and mix well. Transfer to a 4 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend in cell maintenance medium (RPMI 1640 medium containing 5% fetal bovine serum) and dilute to 1.0–1.5 × 10⁻⁶. 6 Induction was performed by adding PMA inducer at a final concentration of 1 μM per ml.

[0184] 3. Add 50 μL / well of TNHH and mutant TNHH-M1~M4 samples to a 96-well high-efficiency adsorption cell culture plate, with 3 replicates per sample. Add 100 μL / well of HL-60 cells and incubate at 37℃ in a 5% CO2 incubator for 3.5 h.

[0185] 4. Aspirate and discard the unattached cells from the upper layer. Wash the 96-well plate three times with cell maintenance medium.

[0186] 5. Add 100 μL / well of CCK-8 chromogenic solution, incubate in a 37℃, 5% CO2 incubator for 3-5 h in the dark, and then measure the OD value at 450 nm using a microplate reader. The results are shown in Table 4.

[0187] Table 4. Determination of TNHH and mutant activity

[0188] Product Name Anti-leukocyte adhesion activity (U / mg) TNHH 6.0 TNHH-M1 8.0 TNHH-M2 8.2 TNHH-M3 9.4 TNHH-M4 9.1

[0189] As shown in Table 4, the mutants TNHH-M1 to M4 exhibited better anti-leukocyte adhesion ability than TNHH, with all of them showing higher activity, reaching 8.0–9.4 × 10⁻⁶. 4 U / mg. SEQUENCE LISTING <110> Lunan Pharmaceutical Group Co., Ltd. <120> A recombinant leukocyte inhibitor and leech peptide chimeric protein mutant <130> 2020 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 846 <212> DNA <213> Artificial sequence <400> 1 aacgaacaca acttgagatg tccacaaaac ggtactgaaa tgccaggttt caacgactcc 60 atcagattgc aattcttggc tatgcacaac ggttacagat ccaagttggc tttgggtcac 120 atctccatca ctgaagaatc cgaatccgac gacgacgacg acttcggttt cttgccagac 180 ttcgctccaa gagcttccaa gatgagatac ttggaatacg actgtgaagc tgaaaagtcc 240 gcttacatgt ccgctagaaa ctgttccgac tcctcctccc caccagaagg ttacgacgaa 300 aacaagtaca tcttcgaaaa ctccaacaac atctccgaag ctgctttgaa ggctatgatc 360 tcctgggcta aggaagcttt caacttgaac aagactaagg aaggtgaagg tgttttgtac 420 agatccaacc acgacatctc caacttcgct aacttggctt gggacgctag agaaaagttc 480 ggttgtgctg ttgttaactg tccattgggt gaaatcgacg acgaaactaa ccacgacggt 540 gaaacttacg ctactactat ccacgttgtt tgtcactacc caaagatcaa caagactgaa 600 ggtcaaccaa tctacaaggt tggtactcca tgtgacgact gttccgaata cactaagaag 660 gctgacaaca ctacttccgc tgacccagtt tgtatcccag acgacggtgt ttgtttcatc 720 ggttccaagg ctgactacga ctccaaggag ttctacagat tcagagaatt gggcggtggc 780 ggtggcttcc caagaccagg tagcggtggc aacggtgact tcgaagaaat cccagaagaa 840 tacttg 846 <210> 2 <211> 282 <212> PRT <213> Artificial sequence <400> 2 Asn Glu His Asn Leu Arg Cys Pro Gln Asn Gly Thr Glu Met Pro Gly 1 5 10 15 Phe Asn Asp Ser Ile Arg Leu Gln Phe Leu Ala Met His Asn Gly Tyr 20 25 30 Arg Ser Lys Leu Ala Leu Gly His Ile Ser Ile Thr Glu Glu Ser Glu 35 40 45 Ser Asp Asp Asp Asp Asp Phe Gly Phe Leu Pro Asp Phe Ala Pro Arg 50 55 60​​​​​​​​​Gly Tyr Asp Glu Asn Lys Tyr Ile Phe Glu Asn Ser Asn Asn Ile Ser 100 105 110 Glu Ala Ala Leu Lys Ala Met Ile Ser Trp Ala Lys Glu Ala Phe Asn 115 120 125 Leu Asn Lys Thr Lys Glu Gly Glu Gly Val Leu Tyr Arg Ser Asn His 130 135 140 Asp Ile Ser Asn Phe Ala Asn Leu Ala Trp Asp Ala Arg Glu Lys Phe 145 150 155 160 Gly Cys Ala Val Val Asn Cys Pro Leu Gly Glu Ile Asp Asp Glu Thr 165 170 175 Asn His Asp Gly Glu Thr Tyr Ala Thr Thr Ile His Val Val Cys His 180 185 190 Tyr Pro Lys Ile Asn Lys Thr Glu Gly Gln Pro Ile Tyr Lys Val Gly 195 200 205 Thr Pro Cys Asp Asp Cys Ser Glu Tyr Thr Lys Lys Ala Asp Asn Thr 210 215 220 Thr Ser Ala Asp Pro Val Cys Ile Pro Asp Asp Gly Val Cys Phe Ile 225 230 235 240 Gly Ser Lys Ala Asp Tyr Asp Ser Lys Glu Phe Tyr Arg Phe Arg Glu 245 250 255 Leu Gly Gly Gly Gly Gly Phe Pro Arg Pro Gly Ser Gly Gly Asn Gly 260 265 270 Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu 275 280 <210> 3 <211> 282 <212> PRT <213> Artificial sequence <400> 3 Asn Glu His Asn Leu Arg Cys Pro Gln Asn Gly Thr Glu Met Pro Gly 1 5 10 15 Phe Asn Asp Ser Ile Arg Leu Gln Phe Leu Ala Met His Asn Gly Tyr 20 25 30 Arg Ser Lys Leu Ala Leu Gly His Ile Ser Ile Thr Glu Glu Ser Glu 35 40 45 Ser Asp Asp Asp Asp Asp Phe Gly Phe Leu Pro Asp Phe Ala Pro Arg 50 55 60 Ala Ser Lys Met Arg Tyr Leu Glu Tyr Asp Cys Glu Ala Glu Lys Ser 65 70 75 80 Ala Tyr Met Ser Ala Arg Asn Cys Ser Asp Ser Ser Ser Pro Pro Glu 85 90 95 Gly Tyr Asp Glu Asn Lys Tyr Ile Phe Glu Asn Ser Asn Asn Ile Ser 100 105 110 Glu Ala Ala Leu Lys Ala Met Ile Ser Trp Ala Lys Glu Ala Phe Asn 115 120 125 Leu Asn Lys Thr Lys Glu Gly Glu Gly Val Leu Tyr Arg Ser Asn His 130 135 140 Asp Ile Ser Asn Phe Ala Asn Leu Ala Trp Asp Ala Arg Glu Lys Phe 145 150 155 160 Gly Ala Ala Val Val Asn Cys Pro Leu Gly Glu Ile Asp Asp Glu Thr 165 170 175 Asn His Asp Gly Glu Thr Tyr Ala Thr Thr Ile His Val Val Cys His 180 185 190 Tyr Pro Lys Ile Asn Lys Thr Glu Gly Gln Pro Ile Tyr Lys Val Gly 195 200 205 Thr Pro Cys Asp Asp Ala Ser Glu Tyr Thr Lys Lys Ala Asp Asn Thr 210 215 220 Thr Ser Ala Asp Pro Val Cys Ile Pro Asp Asp Gly Val Cys Phe Ile 225 230 235 240 Gly Ser Lys Ala Asp Tyr Asp Ser Lys Glu Phe Tyr Arg Phe Arg Glu 245 250 255 Leu Gly Gly Gly Gly Gly Phe Pro Arg Pro Gly Ser Gly Gly Asn Gly 260 265 270 Asp Phe Glu Glu Ile Thr Glu Glu Tyr Glu 275 280 <210> 4 <211> 282 <212> PRT <213> Artificial sequence <400> 4 Asn Glu His Asn Leu Arg Cys Pro Gln Asn Gly Thr Glu Met Pro Gly 1 5 10 15 Phe Asn Asp Ser Ile Arg Leu Gln Phe Leu Ala Met His Asn Gly Tyr 20 25 30 Arg Ser Lys Leu Ala Leu Gly His Ile Ser Ile Thr Glu Glu Ser Glu 35 40 45 Ser Asp Asp Asp Asp Asp Phe Gly Phe Leu Pro Asp Phe Ala Pro Arg 50 55 60 Ala Ser Lys Met Arg Tyr Leu Glu Tyr Asp Cys Glu Ala Glu Lys Ser 65 70 75 80 Ala Tyr Met Ser Ala Arg Asn Cys Ser Asp Ser Ser Ser Pro Pro Glu 85 90 95 Gly Tyr Asp Glu Asn Lys Tyr Ile Phe Glu Asn Ser Asn Asn Ile Ser 100 105 110 Glu Ala Ala Leu Lys Ala Met Ile Ser Trp Ala Lys Glu Ala Phe Asn 115 120 125 Leu Asn Lys Thr Lys Glu Gly Glu Gly Val Leu Tyr Arg Ser Asn His 130 135 140 Asp Ile Ser Asn Phe Ala Asn Leu Ala Trp Asp Ala Arg Glu Lys Phe 145 150 155 160 Gly Ala Ala Val Val Asn Cys Pro Leu Gly Glu Ile Asp Asp Glu Thr 165 170 175 Asn His Asp Gly Glu Thr Tyr Ala Thr Thr Ile His Val Val Cys His 180 185 190 Tyr Pro Lys Ile Asn Lys Thr Glu Gly Gln Pro Ile Tyr Lys Val Gly 195 200 205 Thr Pro Cys Asp Asp Ala Ser Glu Tyr Thr Lys Lys Ala Asp Asn Thr 210 215 220 Thr Ser Ala Asp Pro Val Cys Ile Pro Asp Asp Gly Val Cys Phe Ile 225 230 235 240 Gly Ser Lys Ala Asp Tyr Asp Ser Lys Glu Phe Tyr Arg Phe Arg Glu 245 250 255 Leu Gly Gly Gly Gly Gly Phe Pro Arg Pro Gly Ser Gly Gly Asn Gly 260 265 270 Asp Ser Glu Glu Gly Pro Glu Glu Tyr Leu 275 280 <210> 5 <211> 282 <212> PRT <213> Artificial Sequence <400> 5 Asn Glu His Asn Leu Arg Cys Pro Gln Asn Gly Thr Glu Met Pro Gly 1 5 10 15 Phe Asn Asp Ser Ile Arg Leu Gln Phe Leu Ala Met His Asn Gly Tyr 20 25 30 Arg Ser Lys Leu Ala Leu Gly His Ile Ser Ile Thr Glu Glu Ser Glu 35 40 45 Ser Asp Asp Asp Asp Asp Phe Gly Phe Leu Pro Asp Phe Ala Pro Arg 50 55 60 Ala Ser Lys Met Arg Tyr Leu Glu Tyr Asp Cys Glu Ala Glu Lys Ser 65 70 75 80 Ala Tyr Met Ser Ala Arg Asn Cys Ser Asp Ser Ser Ser Pro Pro Glu 85 90 95 Gly Tyr Asp Glu Asn Lys Tyr Ile Phe Glu Asn Ser Asn Asn Ile Ser 100 105 110 Glu Ala Ala Leu Lys Ala Met Ile Ser Trp Ala Lys Glu Ala Phe Asn 115 120 125 Leu Asn Lys Thr Lys Glu Gly Glu Gly Val Leu Tyr Arg Ser Asn His 130 135 140 Asp Ile Ser Asn Phe Ala Asn Leu Ala Trp Asp Ala Arg Glu Lys Phe 145 150 155 160 Gly Cys Ala Val Val Asn Cys Pro Leu Gly Glu Ile Asp Asp Glu Thr 165 170 175 Asn His Asp Gly Glu Thr Tyr Ala Thr Thr Ile His Val Val Cys His 180 185 190 Tyr Pro Lys Ile Asn Lys Thr Glu Gly Gln Pro Ile Tyr Lys Val Gly 195 200 205 Thr Pro Ala Asp Asp Ala Ser Glu Tyr Thr Lys Lys Ala Asp Asn Thr 210 215 220 Thr Ser Ala Asp Pro Val Cys Ile Pro Asp Asp Gly Val Cys Phe Ile 225 230 235 240 Gly Ser Lys Ala Asp Tyr Asp Ser Lys Glu Phe Tyr Arg Phe Arg Glu 245 250 255 Leu Gly Gly Gly Gly Gly Phe Pro Arg Pro Gly Ser Gly Gly Asn Gly 260 265 270 Asp Phe Glu Glu Ile Thr Glu Glu Tyr Glu 275 280 <210> 6 <211> 282 <212> PRT <213> Artificial Sequence <400> 6 Asn Glu His Asn Leu Arg Cys Pro Gln Asn Gly Thr Glu Met Pro Gly 1 5 10 15 Phe Asn Asp Ser Ile Arg Leu Gln Phe Leu Ala Met His Asn Gly Tyr 20 25 30 Arg Ser Lys Leu Ala Leu Gly His Ile Ser Ile Thr Glu Glu Ser Glu 35 40 45 Ser Asp Asp Asp Asp Asp Phe Gly Phe Leu Pro Asp Phe Ala Pro Arg 50 55 60 Ala Ser Lys Met Arg Tyr Leu Glu Tyr Asp Cys Glu Ala Glu Lys Ser 65 70 75 80 Ala Tyr Met Ser Ala Arg Asn Cys Ser Asp Ser Ser Ser Pro Pro Glu 85 90 95 Gly Tyr Asp Glu Asn Lys Tyr Ile Phe Glu Asn Ser Asn Asn Ile Ser 100 105 110 Glu Ala Ala Leu Lys Ala Met Ile Ser Trp Ala Lys Glu Ala Phe Asn 115 120 125 Leu Asn Lys Thr Lys Glu Gly Glu Gly Val Leu Tyr Arg Ser Asn His 130 135 140 Asp Ile Ser Asn Phe Ala Asn Leu Ala Trp Asp Ala Arg Glu Lys Phe 145 150 155 160 Gly Cys Ala Val Val Asn Cys Pro Leu Gly Glu Ile Asp Asp Glu Thr 165 170 175 Asn His Asp Gly Glu Thr Tyr Ala Thr Thr Ile His Val Val Cys His 180 185 190 Tyr Pro Lys Ile Asn Lys Thr Glu Gly Gln Pro Ile Tyr Lys Val Gly 195 200 205 Thr Pro Ala Asp Asp Ala Ser Glu Tyr Thr Lys Lys Ala Asp Asn Thr 210 215 220 Thr Ser Ala Asp Pro Val Cys Ile Pro Asp Asp Gly Val Cys Phe Ile 225 230 235 240 Gly Ser Lys Ala Asp Tyr Asp Ser Lys Glu Phe Tyr Arg Phe Arg Glu 245 250 255 Leu Gly Gly Gly Gly Gly Phe Pro Arg Pro Gly Ser Gly Gly Asn Gly 260 265 270 Asp Ser Glu Glu Gly Pro Glu Glu Tyr Leu 275 280 <210> 7 <211> 846 <212> DNA <213> Artificial Sequence <400> 7 aacgaacaca acttgagatg tccacaaaac ggtactgaaa tgccaggttt caacgactcc atcagattgc aattcttggc tatgcacaac ggttacagat ccaagttggc tttgggtcac 120 atctccatca ctgaagaatc cgaatccgac gacgacgacg acttcggttt cttgccagac 180 ttcgctccaa gagcttccaa gatgagatac ttggaatacg actgtgaagc tgaaaagtcc 240 gcttacatgt ccgctagaaa ctgttccgac tcctcctccc caccagaagg ttacgacgaa 300 aacaagtaca tcttcgaaaa ctccaacaac atctccgaag ctgctttgaa ggctatgatc 360 tcctgggcta aggaagcttt caacttgaac aagactaagg aaggtgaagg tgttttgtac 420 agatccaacc acgacatctc caacttcgct aacttggctt gggacgctag agaaaagttc 480 ggtgctgctg ttgttaactg tccattgggt gaaatcgacg acgaaactaa ccacgacggt 540 gaaacttacg ctactactat ccacgttgtt tgtcactacc caaagatcaa caagactgaa 600 ggtcaaccaa tctacaaggt tggtactcca tgtgacgacg cttccgaata cactaagaag 660 gctgacaaca ctacttccgc tgacccagtt tgtatcccag acgacggtgt ttgtttcatc 720 ggttccaagg ctgactacga ctccaaggag ttctacagat tcagagaatt gggcggtggc 780 ggtggcttcc caagaccagg tagcggtggc aacggtgact tcgaagaaat cactgaagaa 840 tacgaa 846 <210> 8 <211> 846 <212> DNA <213> Artificial sequence <400> 8 aacgaacaca acttgagatg tccacaaaac ggtactgaaa tgccaggttt caacgactcc 60 atcagattgc aattcttggc tatgcacaac ggttacagat ccaagttggc tttgggtcac 120 atctccatca ctgaagaatc cgaatccgac gacgacgacg acttcggttt cttgccagac 180 ttcgctccaa gagcttccaa gatgagatac ttggaatacg actgtgaagc tgaaaagtcc 240 gcttacatgt ccgctagaaa ctgttccgac tcctcctccc caccagaagg ttacgacgaa 300 aacaagtaca tcttcgaaaa ctccaacaac atctccgaag ctgctttgaa ggctatgatc 360 tcctgggcta aggaagcttt caacttgaac aagactaagg aaggtgaagg tgttttgtac 420 agatccaacc acgacatctc caacttcgct aacttggctt gggacgctag agaaaagttc 480 ggtgctgctg ttgttaactg tccattgggt gaaatcgacg acgaaactaa ccacgacggt 540 gaaacttacg ctactactat ccacgttgtt tgtcactacc caaagatcaa caagactgaa 600 ggtcaaccaa tctacaaggt tggtactcca tgtgacgacg cttccgaata cactaagaag 660 gctgacaaca ctacttccgc tgacccagtt tgtatcccag acgacggtgt ttgtttcatc 720 ggttccaagg ctgactacga ctccaaggag ttctacagat tcagagaatt gggcggtggc 780 ggtggcttcc caagaccagg tagcggtggc aacggtgact ccgaagaagg tccagaagaa 840 tacttg 846 <210> 9 <211> 846 <212> DNA <213> Artificial sequence <400> 9 aacgaacaca acttgagatg tccacaaaac ggtactgaaa tgccaggttt caacgactcc 60 atcagattgc aattcttggc tatgcacaac ggttacagat ccaagttggc tttgggtcac 120 atctccatca ctgaagaatc cgaatccgac gacgacgacg acttcggttt cttgccagac 180 ttcgctccaa gagcttccaa gatgagatac ttggaatacg actgtgaagc tgaaaagtcc 240 gcttacatgt ccgctagaaa ctgttccgac tcctcctccc caccagaagg ttacgacgaa 300 aacaagtaca tcttcgaaaa ctccaacaac atctccgaag ctgctttgaa ggctatgatc 360 tcctgggcta aggaagcttt caacttgaac aagactaagg aaggtgaagg tgttttgtac 420 agatccaacc acgacatctc caacttcgct aacttggctt gggacgctag agaaaagttc 480 ggttgtgctg ttgttaactg tccattgggt gaaatcgacg acgaaactaa ccacgacggt 540 gaaacttacg ctactactat ccacgttgtt tgtcactacc caaagatcaa caagactgaa 600 ggtcaaccaa tctacaaggt tggtactcca gctgacgacg cttccgaata cactaagaag 660 gctgacaaca ctacttccgc tgacccagtt tgtatcccag acgacggtgt ttgtttcatc 720 ggttccaagg ctgactacga ctccaaggag ttctacagat tcagagaatt gggcggtggc 780 ggtggcttcc caagaccagg tagcggtggc aacggtgact tcgaagaaat cactgaagaa 840 tacgaa 846 <210> 10 <211> 846 <212> DNA <213> Artificial sequence <400> 10 aacgaacaca acttgagatg tccacaaaac ggtactgaaa tgccaggttt caacgactcc 60 atcagattgc aattcttggc tatgcacaac ggttacagat ccaagttggc tttgggtcac 120 atctccatca ctgaagaatc cgaatccgac gacgacgacg acttcggttt cttgccagac 180 ttcgctccaa gagcttccaa gatgagatac ttggaatacg actgtgaagc tgaaaagtcc 240 gcttacatgt ccgctagaaa ctgttccgac tcctcctccc caccagaagg ttacgacgaa 300 aacaagtaca tcttcgaaaa ctccaacaac atctccgaag ctgctttgaa ggctatgatc 360 tcctgggcta aggaagcttt caacttgaac aagactaagg aaggtgaagg tgttttgtac 420 agatccaacc acgacatctc caacttcgct aacttggctt gggacgctag agaaaagttc 480 ggttgtgctg ttgttaactg tccattgggt gaaatcgacg acgaaactaa ccacgacggt 540 gaaacttacg ctactactat ccacgttgtt tgtcactacc caaagatcaa caagactgaa 600 ggtcaaccaa tctacaaggt tggtactcca gctgacgacg cttccgaata cactaagaag 660 gctgacaaca ctacttccgc tgacccagtt tgtatcccag acgacggtgt ttgtttcatc 720 ggttccaagg ctgactacga ctccaaggag ttctacagat tcagagaatt gggcggtggc 780 ggtggcttcc caagaccagg tagcggtggc aacggtgact ccgaagaagg tccagaagaa 840 tacttg 846

Claims

1. A recombinant leukocyte inhibitor and leech peptide chimeric protein mutant, characterized in that, Its amino acid sequence is shown below; Asn-Glu-His-Asn-Leu-Arg-Cys-Pro-Gln-Asn-Gly-Thr-Glu-Met-Pro-Gly-Phe-Asn- Asp-Ser-Ile-Arg-Leu-Gln-Phe-Leu-Ala-Met-His-Asn-Gly-Tyr-Arg-Ser-Lys-Leu-Ala-Leu-Gly-His-Ile-Ser-Ile-Thr-Glu-Glu-Ser-Glu-Ser-Asp-Asp-Asp-Asp-Asp-Phe-Gly- Phe-Leu-Pro-Asp-Phe-Ala-Pro-Arg-Ala-Ser-Lys-Met-Arg-Tyr-Leu-Glu-Tyr-Asp-Cys-Glu-Ala-Glu-Lys-Ser-Ala-Tyr-Met-Ser-Ala-Arg-Asn-Cys-Ser-Asp-Ser-Ser-Ser-Pro- Pro-Glu-Gly-Tyr-Asp-Glu-Asn-Lys-Tyr-Ile-Phe-Glu-Asn-Ser-Asn-Asn-Ile-Ser-Glu- Ala-Ala-Leu-Lys-Ala-Met-Ile-Ser-Trp-Ala-Lys-Glu-Ala-Phe-Asn-Leu-Asn-Lys-Thr-Lys-Glu-Gly-Glu-Gly-V al-Leu-Tyr-Arg-Ser-Asn-His-Asp-Ile-Ser-Asn-Phe-Ala-Asn-Leu-Ala-Trp-Asp-Ala-Arg-Glu-Lys-Phe-Gly-Xaa 162 -Ala-Val-Val-Asn-Cys-Pro-Leu- Gly-Glu-Ile-Asp-Asp-Glu-Thr-Asn-His-Asp-Gly-Glu-Thr-Tyr-Ala-Thr-Thr-Ile-His- Val-Val-Cys-His-Tyr-Pro-Lys-Ile-Asn-Lys-Thr-Glu-Gly-Gln-Pro-Ile-Tyr-Lys-Val-Gly-Thr-Pro-Xaa 211 -Asp-Asp-Xaa 214 -Ser-Glu-Tyr-Thr-Lys-Lys-Ala-Asp-Asn-Thr-Thr- Ser-Ala-Asp-Pro-Val-Cys-Ile-Pro-Asp-Asp-Gly-Val-Cys-Phe-Ile-Gly-Ser-Lys-Ala- Asp-Tyr-Asp-Ser-Lys-Glu-Phe-Tyr-Arg-Phe-Arg-Glu-Leu-Gly-Gly-Gly-Gly-Gly-Phe-Pro-Arg-Pro-Gly-Ser-Gly-Gly-Asn-Gly-Asp-Xaa 274 -Glu-Glu-Xaa 277 -Xaa 278 -Glu-Glu-Tyr-Xaa 282 ; in, Xaa 162 is Ala; Xaa 211 For Cys; Xaa 214 is Ala; Xaa 274 For Phe; Xaa 277 For Ile; Xaa 278 For Thr; Xaa 282 is Glu; or Xaa 162 For Ala; Xaa 211 For Cys; Xaa 214 is Ala; Xaa 274 For Ser; Xaa 277 For Gly; Xaa 278 For Pro; Xaa 282 For Leu; or Xaa 162 For Cys; Xaa 211 is Ala; Xaa 214 is Ala; Xaa 274 For Phe; Xaa 277 For Ile; Xaa 278 For Thr; Xaa 282 is Glu; or Xaa 162 For Cys; Xaa 211 is Ala; Xaa 214 is Ala; Xaa 274 For Ser; Xaa 277 For Gly; Xaa 278 For Pro; Xaa 282 For Leu.

2. A polynucleotide encoding the TNHH mutant of claim 1.

3. An expression vector comprising the polynucleotide of claim 2.

4. A host cell comprising the expression vector of claim 3, or the genome of the host cell having the polynucleotide of claim 2 integrated therein.

5. The use of the mutant of claim 1 in the preparation of a drug for treating ischemic brain injury.

6. A pharmaceutical composition comprising the mutant of claim 1 and pharmaceutically acceptable excipients.

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