阅读说明：本技术 一种人粒细胞集落刺激因子突变体重组融合蛋白及其制备方法和应用 (Human granulocyte colony stimulating factor mutant recombinant fusion protein and preparation method and application thereof ) 是由 许娜 孙成彪 石晶 王燕 于 2021-08-17 设计创作，主要内容包括：一种人粒细胞集落刺激因子突变体重组融合蛋白及其制备方法和应用,涉及生物基因工程领域,该重组融合蛋白由人粒细胞集落刺激因子突变体与蓖麻毒素B链截短肽经柔性linker连接而形成,柔性linker为(Gly-4Ser)-3连接肽。本发明的一种人粒细胞集落刺激因子突变体重组融合蛋白的制备方法包括：人粒细胞集落刺激因子突变表达载体构建；大肠杆菌重组表达载体构建；重组hG-CSF-Mut/RTBD1融合蛋白表达；重组hG-CSF-Mut/RTBD1融合蛋白纯化与复性。该重组融合蛋白半衰期长、生物学活性高,延长了半衰期,提高了生物活性,降低了粒细胞集落刺激因子制剂的生产成本,可应用于制备治疗白细胞减少症药物。(A recombinant fusion protein of human granulocyte colony stimulating factor mutant is prepared through connecting human granulocyte colony stimulating factor mutant and ricin B chain truncated peptide by flexible linker (Gly) 4 Ser) 3 A linker peptide. The preparation method of the human granulocyte colony stimulating factor mutant recombinant fusion protein comprises the following steps: constructing a human granulocyte colony stimulating factor mutation expression vector; constructing an escherichia coli recombinant expression vector; expressing recombinant hG-CSF-Mut/RTBD1 fusion protein; purifying and renaturing the recombinant hG-CSF-Mut/RTBD1 fusion protein. The recombinant fusion protein has long half-life and high biological activity, prolongs the half-life, improves the biological activity, reduces the production cost of the granulocyte colony stimulating factor preparation, and can be applied to the preparation of the medicines for treating leukopenia.)

1. The recombinant fusion protein is characterized in that the recombinant fusion protein is formed by connecting a human granulocyte colony stimulating factor mutant and ricin B chain truncated peptide through a flexible linker.

2. The recombinant fusion protein of claim 1, wherein the human granulocyte colony stimulating factor mutant is prepared by the following steps: designing a site-directed mutagenesis primer, mutating the 19 th leucine to tryptophan and the 28 th aspartic acid to arginine in a human granulocyte colony stimulating factor sequence by taking a recombinant plasmid pUC57-hG-CSF as a template, and naming the mutated sequence as hG-CSF-Mut;

the sequence of the site-directed mutagenesis primer is as follows:

SiteⅠ

Forward：5'-GAGTTTTCTGCTGAAATGCTGGGAACAGGTTCGTAAAATTC-3'；

Reverse：5'-GAATTTTACGAACCTGTTCCCAGCATTTCAGCAGAAAACTC-3'；

SiteⅡ

Forward：5'-GTAAAATTCAGGGTAGGGGCGCAGCCCTGCAG-3'；

Reverse：5'-CTGCAGGGCTGCGCCCCTACCCTGAATTTTAC-3'。

3. the recombinant fusion protein of claim 2, wherein the mutated hG-CSF-Mut sequence and the ricin B chain truncated peptide sequence are linked by a flexible linker to form a hG-CSF-Mut/RTBD1 sequence, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of which is shown in SEQ ID NO. 2.

4. The recombinant fusion protein of claim 1, wherein the flexible linker is (Gly)₄Ser)₃A linker peptide.

5. The method for preparing human granulocyte colony stimulating factor mutant recombinant fusion protein of any one of claims 1-4, comprising the steps of:

step one, constructing a human granulocyte colony stimulating factor mutation expression vector;

constructing an escherichia coli recombinant expression vector;

step three, expressing the recombinant hG-CSF-Mut/RTBD1 fusion protein;

and step four, purifying and renaturing the recombinant hG-CSF-Mut/RTBD1 fusion protein.

6. The method for preparing human granulocyte colony stimulating factor mutant recombinant fusion protein of claim 5, wherein the first step specifically comprises the following steps:

(1) carrying out homologous modeling on human granulocyte colony stimulating factor molecules and human granulocyte colony stimulating factor receptor molecules through a SWISS-MODEL online platform; docking the human granulocyte colony stimulating factor molecules and the human granulocyte colony stimulating factor receptor molecules by a Z-DOCK method to obtain a compound structure model; mutating the full-length sequence of the compound structure model by alanine scanning to obtain potential mutation sites; calculating intermolecular binding force change before and after mutation on a single mutation site through virtual saturation mutation;

(2) preparation of recombinant plasmid pUC 57-hG-CSF:

inquiring a human granulocyte colony stimulating factor gene sequence through an NCBI Genbank database, carrying out whole-gene synthesis on the obtained sequence, carrying out codon optimization by using codon preference of escherichia coli in the synthesis process, and cloning the sequence into an escherichia coli cloning vector pUC57 to obtain an escherichia coli recombinant cloning vector pUC 57-hG-CSF;

(3) designing a site-directed mutagenesis primer, wherein the sequence of the site-directed mutagenesis primer is as follows:

SiteⅠ

Forward：5'-GAGTTTTCTGCTGAAATGCTGGGAACAGGTTCGTAAAATTC-3'；

Reverse：5'-GAATTTTACGAACCTGTTCCCAGCATTTCAGCAGAAAACTC-3'；

SiteⅡ

Forward：5'-GTAAAATTCAGGGTAGGGGCGCAGCCCTGCAG-3'；

Reverse：5'-CTGCAGGGCTGCGCCCCTACCCTGAATTTTAC-3'；

(4) the 19 th leucine in the sequence of the human granulocyte colony-stimulating factor is mutated into tryptophan and the 28 th aspartic acid in the sequence of the human granulocyte colony-stimulating factor is mutated into arginine by taking the recombinant plasmid pUC57-hG-CSF as a template, and the mutated sequence is named as hG-CSF-Mut; the mutated hG-CSF-Mut sequence and the ricin B chain truncated peptide sequence are connected through a flexible linker to form an hG-CSF-Mut/RTBD1 sequence, the nucleotide sequence of the hG-CSF-Mut/RTBD1 sequence is shown as SEQ ID NO. 1, the amino acid sequence of the hG-CSF-Mut/RTBD1 sequence is shown as SEQ ID NO. 2, the hG-CSF-Mut/RTBD1 sequence is inserted into a pMD18T vector, and a product which is correctly sequenced is named as a pMD18T-hG-CSF-Mut/RTBD1 plasmid and serves as an hG-CSF mutation expression vector.

7. The method for preparing human granulocyte colony stimulating factor mutant recombinant fusion protein of claim 6, wherein the second step comprises the following steps:

carrying out double enzyme digestion on the pMD18T-hG-CSF-Mut/RTBD1 plasmid and the pET30a vector with correct sequencing respectively, wherein the reaction system and conditions are shown in Table 1, and carrying out gel recovery on double enzyme digestion products; connecting the two products obtained after glue recovery by using T4 ligase, wherein the connection system and conditions are shown in Table 1;

TABLE 1

Name of reagent Dosage of T4DNALigase 1μL 10×T4DNALigaseBμffer 4μL Recovered hG-CSF target fragment 4μL Recovered pET30a vector fragment 1μL Reaction conditions 16 ℃ overnight

Transforming the ligation product into a Trans10 competent cell; after transformation, the cells were plated to a final concentration of 100. mu.g/mLKan⁺After overnight culture, selecting a plurality of white single colonies with good growth vigor, carrying out amplification culture and carrying out PCR; the positive plasmids identified by PCR were subjected to sequencing analysis.

8. The method for preparing human granulocyte colony stimulating factor mutant recombinant fusion protein of claim 7, wherein the third step comprises the following steps:

inoculating correctly sequenced positive bacteria BL21(DE3)/pET30a-hG-CSF-Mut/RTBD1 into 5mL LB culture medium with concentration of 100 mug/mLKan according to the proportion of 1:100v/v, carrying out constant temperature shaking culture at 180rpm at 37 ℃ until OD600 is 0.6, transferring the positive bacteria to 500mL of the same LB culture medium according to the proportion of 1:100v/v, supplementing Kan until the final concentration is 100 mug/mL, and carrying out constant temperature shaking culture at 180rpm at 37 ℃ until OD600 is 0.6; adding IPTG to the induction group until the final concentration is 1mmol/L, and oscillating at constant temperature of 180rpm at 37 ℃ for induction for 16 h; after 16h of induction, the bacterial pellet rhG-CSF-Mut/RTBD1 is obtained by centrifugation at 8000rpm and 4 ℃.

9. The method for preparing human granulocyte colony stimulating factor mutant recombinant fusion protein of claim 8, wherein the fourth step comprises the following steps:

(1) ion exchange chromatography: balancing a Q column by using a protein purification A liquid, dissolving rhG-CSF/RTBD1 inclusion bodies in a protein purification A liquid in a denaturing manner, loading a dissolving liquid on the column, collecting a flow-through liquid, wherein the target protein exists in the flow-through liquid; adjusting the salt ion concentration of the flow-through solution to 0.5mol/L for the next step of metal chelating chromatography;

(2) metal chelating chromatography: pair of Ni-Supported proteins with protein purification solution B²⁺Balancing by using chemical Sepharose; loading rhG-CSF-Mut/RTBD1 solution purified by ion exchange chromatography to a column, mixing protein purification solution C and protein purification solution D through a gradient mixing module in a protein purification system, eluting chemical Sepharose with imidazole with final concentration of 50mmol/L and 250mmol/L respectively, and collecting protein flow-through solution, hybrid protein solution and target protein solution respectively; performing SDS-PAGE analysis on the protein solution collected in each step, wherein the gel concentration is 12%;

(3) protein renaturation of rhG-CSF-Mut/RTBD 1: diluting the rhG-CSF-Mut/RTBD1 protein solution purified by the two steps by using protein renaturation A liquid until the concentration of the rhG-CSF-Mut/RTBD1 is 0.1mg/mL, filling the rhG-CSF-Mut/RTBD1 into a protein ultrafiltration system with the molecular weight cutoff of 3000 for dialysis renaturation, and adding protein renaturation B liquid into the protein ultrafiltration system; regulating the rotating speed of a peristaltic pump in the protein ultrafiltration system and the tightness degree of a liquid outlet valve to keep the protein concentration speed consistent with the liquid inlet speed of the liquid B; the protein renaturation process is carried out at 4 ℃, and the whole process is carried out for 48-72 h, so as to meet the requirements of thorough removal of urea and slow and sufficient folding of rhG-CSF-Mut/RTBD1 protein; after the protein renaturation B liquid is drained, introducing a protein renaturation C liquid to remove residual cane sugar and glycerin components in the renaturation process, concentrating rhG-CSF-Mut/RTBD1 protein until the concentration is 1-1.5 mg/mL, and obtaining recombinant hG-CSF-Mut/RTBD1 fusion protein;

the composition of the protein purification solution A is as follows: 50mmol/L PB, 8mol/LUrea, pH 6.0;

the composition of the protein purification solution B is as follows: 50mmol/LPB, 8mol/LUrea, 0.5mol/LNaClpH 6.0;

the composition of the protein purification solution C is as follows: 50mmol/LTris, 8mol/LUrea, 0.5mol/LNaCl, pH 8.0;

the composition of the protein purification solution D is as follows: 50mmol/L Tris, 8mol/L Urea, 0.5mol/L NaCl, 500mmol/L Imidazole, pH 8.0;

the composition of the protein renaturation A solution is as follows: 50mmol/L Tris, 8mol/LUrea, 10% m/v sucrose, 10% v/v glycerol, 5mmol/L DTT, pH 8.0;

the composition of the protein renaturation B liquid is as follows: 50mmol/L Tris, 10% m/v sucrose, 10% v/v glycerol, pH 8.0;

the composition of the protein renaturation C liquid is as follows: 50mmol/LTris, pH 8.0.

10. Use of the human granulocyte colony stimulating factor mutant recombinant fusion protein of any one of claims 1-4 in the preparation of a medicament for treating leukopenia.

Technical Field

The invention relates to the technical field of biological genetic engineering, in particular to a human granulocyte colony stimulating factor mutant recombinant fusion protein and a preparation method and application thereof.

Background

Granulocyte colony stimulating factor (G-CSF) is a glycoprotein that primarily acts on the proliferation, differentiation and activation of hematopoietic cells of the neutrophil lineage (linkage). The recombinant human granulocyte colony stimulating factor (rhG-CSF-Mut/RTBD1) acts on hematopoietic progenitor cells to promote the proliferation and differentiation of the hematopoietic progenitor cells, and has the important functions of stimulating the maturation of granulocyte and monocyte macrophages, promoting the release of the mature cells to peripheral blood and promoting multiple functions of the macrophages and the eosinophilic cells. The G-CSF is mainly used for clinically preventing and treating leukopenia caused by tumor radiotherapy or chemotherapy, treating myelohematopoietic dysfunction and myelodysplastic syndrome, preventing infection complications possibly caused by leukopenia and accelerating recovery of neutropenia caused by infection. However, the granulocyte colony stimulating factor has the problems of short half-life period, easy degradation in vivo and the like in clinic, and the clinical application of the granulocyte colony stimulating factor is limited.

Ricin (RT) is covalently linked by two peptide chains, RTA and RTB, with a disulfide bond. Wherein RTB consists of 262 amino acids. RTB alone is not toxic. RTB is currently believed to have two main functions: 1. the RTA is facilitated to be transported into cytoplasm, and the protein synthesis is inhibited; 2. RTB enhances the immune response of the body.

At present, the research of improving the activity of the recombinant fusion protein for treating leukopenia and prolonging the half life of the recombinant fusion protein by combining the human G-CSF mutant and the ricin B chain truncated peptide into the recombinant fusion protein has not been reported.

Disclosure of Invention

The invention provides a human granulocyte colony stimulating factor mutant recombinant fusion protein, a preparation method and application thereof, which are used for improving the activity of the recombinant fusion protein in treating leukopenia, prolonging the half-life period of the recombinant fusion protein and improving the treatment effect of the leukopenia.

The technical scheme adopted by the invention for solving the technical problem is as follows:

the invention relates to a human granulocyte colony stimulating factor mutant recombinant fusion protein which is formed by connecting a human granulocyte colony stimulating factor mutant and ricin B chain truncated peptide through a flexible linker.

As a preferred embodiment, the human granulocyte colony stimulating factor mutant is prepared by the following steps: designing a site-directed mutagenesis primer, mutating the 19 th leucine to tryptophan and the 28 th aspartic acid to arginine in a human granulocyte colony stimulating factor sequence by taking a recombinant plasmid pUC57-hG-CSF as a template, and naming the mutated sequence as hG-CSF-Mut;

the sequence of the site-directed mutagenesis primer is as follows:

SiteⅠ

Forward：5'-GAGTTTTCTGCTGAAATGCTGGGAACAGGTTCGTAAAATT C-3'；

Reverse：5'-GAATTTTACGAACCTGTTCCCAGCATTTCAGCAGAAAACTC-3'；

SiteⅡ

Forward：5'-GTAAAATTCAGGGTAGGGGCGCAGCCCTGCAG-3'；

Reverse：5'-CTGCAGGGCTGCGCCCCTACCCTGAATTTTAC-3'。

as a preferred embodiment, the mutated hG-CSF-Mut sequence and the ricin B chain truncated peptide sequence are connected by a flexible linker to form the hG-CSF-Mut/RTBD1 sequence, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the amino acid sequence of which is shown as SEQ ID NO. 2.

As a preferred embodiment, the flexible linker is (Gly)₄Ser)₃A linker peptide.

The invention relates to a preparation method of a human granulocyte colony stimulating factor mutant recombinant fusion protein, which comprises the following steps:

step one, constructing a human granulocyte colony stimulating factor mutation expression vector;

constructing an escherichia coli recombinant expression vector;

step three, expressing the recombinant hG-CSF-Mut/RTBD1 fusion protein;

and step four, purifying and renaturing the recombinant hG-CSF-Mut/RTBD1 fusion protein.

As a preferred embodiment, the step one specifically comprises the following steps:

(1) carrying out homologous modeling on human granulocyte colony stimulating factor molecules and human granulocyte colony stimulating factor receptor molecules through a SWISS-MODEL online platform; docking the human granulocyte colony stimulating factor molecules and the human granulocyte colony stimulating factor receptor molecules by a Z-DOCK method to obtain a compound structure model; mutating the full-length sequence of the compound structure model by alanine scanning to obtain potential mutation sites; calculating intermolecular binding force change before and after mutation on a single mutation site through virtual saturation mutation;

(2) preparation of recombinant plasmid pUC 57-hG-CSF:

inquiring a human granulocyte colony stimulating factor gene sequence through an NCBI Genbank database, carrying out whole-gene synthesis on the obtained sequence, carrying out codon optimization by using codon preference of escherichia coli in the synthesis process, and cloning the sequence into an escherichia coli cloning vector pUC57 to obtain an escherichia coli recombinant cloning vector pUC 57-hG-CSF;

(3) designing a site-directed mutagenesis primer, wherein the sequence of the site-directed mutagenesis primer is as follows:

SiteⅠ

Forward：5'-GAGTTTTCTGCTGAAATGCTGGGAACAGGTTCGTAAAATT C-3'；

Reverse：5'-GAATTTTACGAACCTGTTCCCAGCATTTCAGCAGAAAACTC-3'；

SiteⅡ

Forward：5'-GTAAAATTCAGGGTAGGGGCGCAGCCCTGCAG-3'；

Reverse：5'-CTGCAGGGCTGCGCCCCTACCCTGAATTTTAC-3'；

(4) the 19 th leucine in the sequence of the human granulocyte colony-stimulating factor is mutated into tryptophan and the 28 th aspartic acid in the sequence of the human granulocyte colony-stimulating factor is mutated into arginine by taking the recombinant plasmid pUC57-hG-CSF as a template, and the mutated sequence is named as hG-CSF-Mut; the mutated hG-CSF-Mut sequence and the ricin B chain truncated peptide sequence are connected through a flexible linker to form an hG-CSF-Mut/RTBD1 sequence, the nucleotide sequence of the hG-CSF-Mut/RTBD1 sequence is shown as SEQ ID NO. 1, the amino acid sequence of the hG-CSF-Mut/RTBD1 sequence is shown as SEQ ID NO. 2, the hG-CSF-Mut/RTBD1 sequence is inserted into a pMD18T vector, and a product which is correctly sequenced is named as a pMD18T-hG-CSF-Mut/RTBD1 plasmid and serves as an hG-CSF mutation expression vector.

As a preferred embodiment, the second step specifically comprises the following steps:

carrying out double enzyme digestion on the pMD18T-hG-CSF-Mut/RTBD1 plasmid and the pET30a vector with correct sequencing respectively, wherein the reaction system and conditions are shown in Table 1, and carrying out gel recovery on double enzyme digestion products; connecting the two products obtained after glue recovery by using T4 ligase, wherein the connection system and conditions are shown in Table 1;

TABLE 1

Name of reagent	Dosage of
		T4 DNA Ligase	1μL
10×T4 DNA Ligase Bμffer	4μL
		Recovered hG-CSF target fragment	4μL
Recovered pET30a vector fragment	1μL
		Reaction conditions	16 ℃ overnight

Transforming the ligation product into a Trans10 competent cell; after conversion, coated to a final concentration of 100. mu.g/mL Kan⁺After overnight culture, selecting a plurality of white single colonies with good growth vigor, carrying out amplification culture and carrying out PCR; the positive plasmids identified by PCR were subjected to sequencing analysis.

As a preferred embodiment, the step three specifically includes the following steps:

inoculating correctly sequenced positive bacteria BL21(DE3)/pET30a-hG-CSF-Mut/RTBD1 into 5mL LB culture medium containing 100 mug/mL Kan according to the proportion of 1:100v/v, carrying out constant temperature shaking culture at 37 ℃ and 180rpm until OD600 is 0.6, then transferring the positive bacteria to 500mL of the same LB culture medium according to the proportion of 1:100v/v, supplementing Kan until the final concentration is 100 mug/mL, and carrying out constant temperature shaking culture at 37 ℃ and 180rpm until OD600 is 0.6; adding IPTG to the induction group until the final concentration is 1mmol/L, and oscillating at constant temperature of 180rpm at 37 ℃ for induction for 16 h; after 16h of induction, the bacterial pellet rhG-CSF-Mut/RTBD1 is obtained by centrifugation at 8000rpm and 4 ℃.

As a preferred embodiment, the step four specifically includes the following steps:

(1) ion exchange chromatography: balancing a Q column by using a protein purification A liquid, dissolving rhG-CSF/RTBD1 inclusion bodies in a protein purification A liquid in a denaturing manner, loading a dissolving liquid on the column, collecting a flow-through liquid, wherein the target protein exists in the flow-through liquid; adjusting the salt ion concentration of the flow-through solution to 0.5mol/L for the next step of metal chelating chromatography;

(2) metal chelating chromatography: pair of Ni-Supported proteins with protein purification solution B²⁺Balancing by using chemical Sepharose; loading rhG-CSF-Mut/RTBD1 solution purified by ion exchange chromatography to a column, mixing protein purification solution C and protein purification solution D through a gradient mixing module in a protein purification system, eluting chemical Sepharose with imidazole with final concentration of 50mmol/L and 250mmol/L respectively, and collecting protein flow-through solution, hybrid protein solution and target protein solution respectively; performing SDS-PAGE analysis on the protein solution collected in each step, wherein the gel concentration is 12%;

(3) protein renaturation of rhG-CSF-Mut/RTBD 1: diluting the rhG-CSF-Mut/RTBD1 protein solution purified by the two steps by using protein renaturation A liquid until the concentration of the rhG-CSF-Mut/RTBD1 is 0.1mg/mL, filling the rhG-CSF-Mut/RTBD1 into a protein ultrafiltration system with the molecular weight cutoff of 3000 for dialysis renaturation, and adding protein renaturation B liquid into the protein ultrafiltration system; regulating the rotating speed of a peristaltic pump in the protein ultrafiltration system and the tightness degree of a liquid outlet valve to keep the protein concentration speed consistent with the liquid inlet speed of the liquid B; the protein renaturation process is carried out at 4 ℃, and the whole process is carried out for 48-72 h, so as to meet the requirements of thorough removal of urea and slow and sufficient folding of rhG-CSF-Mut/RTBD1 protein; after the protein renaturation B liquid is drained, introducing a protein renaturation C liquid to remove residual cane sugar and glycerin components in the renaturation process, concentrating rhG-CSF-Mut/RTBD1 protein until the concentration is 1-1.5 mg/mL, and obtaining recombinant hG-CSF-Mut/RTBD1 fusion protein;

the composition of the protein purification solution A is as follows: 50mmol/L PB, 8mol/L Urea, pH 6.0;

the composition of the protein purification solution B is as follows: 50mmol/L PB, 8mol/L Urea, 0.5mol/L NaCl pH 6.0;

the composition of the protein purification solution C is as follows: 50mmol/L Tris, 8mol/L Urea, 0.5mol/L NaCl, pH 8.0;

the composition of the protein purification solution D is as follows: 50mmol/L Tris, 8mol/L Urea, 0.5mol/L NaCl, 500mmol/L Imidazole, pH 8.0;

the composition of the protein renaturation A solution is as follows: 50mmol/L Tris, 8mol/L Urea, 10% m/v sucrose, 10% v/v glycerol, 5mmol/L DTT, pH 8.0;

the composition of the protein renaturation B liquid is as follows: 50mmol/L Tris, 10% m/v sucrose, 10% v/v glycerol, pH 8.0;

the composition of the protein renaturation C liquid is as follows: 50mmol/L Tris, pH 8.0.

The invention relates to an application of a human granulocyte colony stimulating factor mutant recombinant fusion protein in preparing a medicament for treating leukopenia.

The invention has the beneficial effects that:

the invention relates to a human granulocyte colony stimulating factor mutant recombinant fusion protein which is formed by connecting a human granulocyte colony stimulating factor mutant and ricin B chain truncated peptide through a flexible linker. Wherein the flexible linker is (Gly)₄Ser)₃A linker peptide. Compared with the common human granulocyte colony stimulating factor, the human granulocyte colony stimulating factor mutant recombinant fusion protein has the advantages of long half-life period, high biological activity and the like.

The human granulocyte colony stimulating factor mutant recombinant fusion protein can be applied to preparing medicaments for treating leukopenia, and has great potential and value in development of medicaments for treating leukopenia and veterinary clinical application.

Drawings

FIG. 1 is a schematic diagram of human granulocyte colony-stimulating factor (hG-CSF) before and after mutation.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 preparation of recombinant hG-CSF-Mut/RTBD1 fusion protein

1. Construction of human granulocyte colony-stimulating factor (hG-CSF) mutant expression vector

(1) Carrying out homologous modeling on human granulocyte colony stimulating factor molecules and human granulocyte colony stimulating factor receptor molecules through a SWISS-MODEL online platform; docking the human granulocyte colony stimulating factor molecules and the human granulocyte colony stimulating factor receptor molecules by a Z-DOCK method to obtain a compound structure model; mutating the full-length sequence of the compound structure model by alanine scanning to obtain potential mutation sites; the intermolecular binding force changes before and after mutation were calculated for a single mutation site by virtual saturation mutation.

(2) Preparation of recombinant plasmid pUC 57-hG-CSF:

querying a human granulocyte colony stimulating factor gene sequence through an NCBI Genbank database, carrying out whole-gene synthesis on the obtained sequence, carrying out codon optimization by using codon preference of escherichia coli in the synthesis process, and cloning the obtained sequence into an escherichia coli cloning vector pUC57 to obtain an escherichia coli recombinant cloning vector pUC 57-hG-CSF.

(3) Designing a site-directed mutagenesis primer, wherein the sequence of the site-directed mutagenesis primer is as follows:

SiteⅠ

Forward：5'-GAGTTTTCTGCTGAAATGCTGGGAACAGGTTCGTAAAATT C-3'；

Reverse：5'-GAATTTTACGAACCTGTTCCCAGCATTTCAGCAGAAAACTC-3'；

Site Ⅱ

Forward：5'-GTAAAATTCAGGGTAGGGGCGCAGCCCTGCAG-3'；

Reverse：5'-CTGCAGGGCTGCGCCCCTACCCTGAATTTTAC-3'。

(4) as shown in figure 1, recombinant plasmid pUC57-hG-CSF is used as a template, leucine at position 19 in a human granulocyte colony stimulating factor sequence is mutated into tryptophan, aspartic acid at position 28 in the human granulocyte colony stimulating factor sequence is mutated into arginine, so that the stability and the antiviral activity of the human granulocyte colony stimulating factor/RTBD 1 fusion protein are enhanced, and the mutated sequence is named as hG-CSF-Mut. Connecting the mutated hG-CSF-Mut sequence with a ricin B chain truncated peptide sequence (RTBD1 sequence, the nucleotide sequence of which is shown as SEQ ID NO:3 in the sequence table, and the amino acid sequence of which is shown as SEQ ID NO:4 in the sequence table) by a flexible linker to form an hG-CSF-Mut/RTBD1 sequence, the nucleotide sequence of which is shown as SEQ ID NO:1 and the amino acid sequence of which is shown as SEQ ID NO:2, inserting the hG-CSF-Mut/RTBD1 sequence into a pMD18T vector, and after sequencing, naming a product with correct sequencing as: pMD18T-hG-CSF-Mut/RTBD1 plasmid, as hG-CSF mutation expression vector for downstream genetic engineering operations. Wherein the adopted flexible linker is (Gly)₄Ser)₃A linker peptide.

2. Construction of recombinant expression vector for Escherichia coli

The pMD18T-hG-CSF-Mut/RTBD1 plasmid and the pET30a vector which are correctly sequenced are subjected to double enzyme digestion respectively, the reaction system and conditions are shown in Table 1, and the double enzyme digestion products are subjected to gel recovery. The two products after gel recovery were ligated using T4 ligase, and the ligation system and conditions are shown in table 1.

TABLE 1

Name of reagent	Dosage of
		T4 DNA Ligase	1μL
10×T4 DNA Ligase Bμffer	4μL
		Recovered hG-CSF target fragment	4μL
Recovered pET30a vector fragment	1μL
		Reaction conditions	16 ℃ overnight

The ligation product was transformed into Trans10 competent cells. After conversion, coated to a final concentration of 100. mu.g/mL Kan⁺After overnight incubation, multiple white single colonies with good growth were picked, expanded and subjected to PCR. And (3) sending the positive plasmid which is correctly identified by the PCR to Shanghai biological engineering technical service company Limited for sequencing analysis.

3. Recombinant hG-CSF-Mut/RTBD1 fusion protein expression

Inoculating the positive bacterium BL21(DE3)/pET30a-hG-CSF-Mut/RTBD1 with correct sequencing into 5mL LB culture medium containing 100 ug/mL Kan according to the ratio of 1:100v/v, carrying out shaking culture at constant temperature of 180rpm at 37 ℃ until OD600 is 0.6, transferring the culture medium to 500mL of the same LB culture medium according to the ratio of 1:100v/v, supplementing Kan until the final concentration is 100 ug/mL, and carrying out shaking culture at constant temperature of 180rpm at 37 ℃ until OD600 is 0.6; adding IPTG to the induction group until the final concentration is 1mmol/L, and oscillating at constant temperature of 180rpm at 37 ℃ for induction for 16 h; after 16h of induction, the bacterial pellet rhG-CSF-Mut/RTBD1 is obtained by centrifugation at 8000rpm and 4 ℃.

4. Purification and renaturation of recombinant hG-CSF-Mut/RTBD1 fusion protein

The cell pellet rhG-CSF-Mut/RTBD1 was purified using a two-step purification method. The purification method is carried out in the order of ion exchange chromatography and metal chelating chromatography, and comprises the following steps:

(1) ion exchange chromatography: a Q column was equilibrated with protein purification A solution (50mmol/L PB, 8mol/L Urea, pH 6.0), inclusion bodies of rhG-CSF/RTBD1 were denatured with the protein purification A solution and the lysate was applied to the column, and the flow-through solution in which the protein of interest was mainly present was collected. The salt ion concentration of the flow-through was adjusted to 0.5mol/L for the next step of metal chelate chromatography.

(2) Metal chelating chromatography: loading with Ni was performed using protein purification B solution (50mmol/L PB, 8mol/L Urea, 0.5mol/L NaCl pH 6.0)²⁺The equilibration was carried out on a chemical Sepharose. Loading rhG-CSF-Mut/RTBD1 solution purified by ion exchange chromatography to a column, mixing protein purification solution C (50mmol/L Tris, 8mol/L Urea, 0.5mol/L NaCl, pH 8.0) and protein purification solution D (50mmol/L Tris, 8mol/L Urea, 0.5mol/L NaCl, 500mmol/L Imidazole, pH 8.0) by a gradient mixing module in a protein purification system, eluting chelating Sepharose by using Imidazole with final concentration of 50mmol/L and 250mmol/L respectively, and collecting protein flow-through solution, hetero-protein solution and target protein solution respectively. The protein solution collected at each step was subjected to SDS-PAGE analysis at a gel concentration of 12%.

(3) Protein renaturation of rhG-CSF-Mut/RTBD 1: the rhG-CSF-Mut/RTBD1 protein solution purified in two steps was diluted with protein renaturation A solution (50mmol/L Tris, 8mol/L Urea, 10% m/v sucrose, 10% v/v glycerol, 5mmol/L DTT, pH 8.0) to a concentration of 0.1mg/mL rhG-CSF-Mut/RTBD1 and loaded into a protein ultrafiltration system having a molecular weight cut-off of 3000 for renaturation by dialysis, and protein renaturation B solution (50mmol/L Tris, 10% m/v sucrose, 10% v/v glycerol, pH 8.0) was added to the protein ultrafiltration system. The rotating speed of a peristaltic pump in the protein ultrafiltration system and the tightness degree of a liquid outlet valve are adjusted, so that the protein concentration speed is consistent with the liquid inlet speed of the liquid B. The protein renaturation process is carried out at 4 ℃, and the whole process is carried out for 48-72 h, so as to meet the requirements of thorough removal of urea and slow and sufficient folding of rhG-CSF-Mut/RTBD1 protein. And after the protein renaturation B liquid flows out, introducing a protein renaturation C liquid (50mmol/L Tris, pH 8.0) to remove components such as sucrose and glycerol remained in the renaturation process, concentrating the rhG-CSF-Mut/RTBD1 protein until the concentration is 1-1.5 mg/mL, and obtaining the rhG-CSF-Mut/RTBD1 fusion protein.

EXAMPLE 2 pharmacodynamic assay of recombinant hG-CSF-Mut/RTBD1 fusion proteins

1. Construction of leukopenia mouse model

18 mice were selected and were injected intraperitoneally with 2 mg/day cyclophosphamide for three consecutive days. On the fourth day, whole blood from the mice was routinely assayed for white blood cell count (WBC). The model success criteria used in this test were: the number of leukocytes is lower than the lowest value of the reference range by 0.8X 10⁹And L. The mice meeting the conditions prove that the preparation of the leukopenia mouse model is successful. The normal group and the model control group were subcutaneously administered with physiological saline the day after the molding, and the administration group was subcutaneously administered with 0.2. mu.g of rhG-CSF-Mut/RTBD1 fusion protein to the back and neck of the mice the day after the molding.

TABLE 2 treatment of leukopenia animal groupings with recombinant hG-CSF-Mut/RTBD1 fusion protein

Grouping animals	Number of animals	Group of	Mode of administration	Administration of drugs
					1	6	Normal group	Subcutaneous injection	Physiological saline
2	6	Model control group	Subcutaneous injection	Physiological saline
					3	6	Administration set	Subcutaneous injection	rhG-CSF-Mut/RTBD1

2. Routine blood test

The number of leukocytes in the blood of the mice was measured using a blood-based analyzer, and the results are shown in Table 3.

TABLE 3 number of leukocytes in each group (n ═ 6)

As a result, the number of leukocytes was significantly increased compared with the model control group, and was restored to the reference range of normal mice (0.8X 10)⁹～6.8×10⁹L), the rhG-CSF-Mut/RTBD1 fusion protein of the invention is proved to be successful in treating leukopenia.

The invention discloses a human granulocyte colony stimulating factor mutant recombinant fusion protein, a preparation method and application thereof, and a person skilled in the art can realize the recombinant fusion protein by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.

Sequence listing

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