阅读说明：本技术 一种猫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 (Cat 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：一种猫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。该重组融合蛋白由猫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。制备方法为：猫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FeG-CSF-Mut/RTBD1融合蛋白表达；重组FeG-CSF-Mut/RTBD1融合蛋白纯化与复性。本发明将猫G-CSF突变体与蓖麻毒素B链截短肽连接获得长效粒细胞集落刺激因子,可增强粒细胞集落刺激因子的生物学活性,提高粒细胞集落刺激因子在体内血液中的蛋白稳定性,进而提高在机体内药物的半衰期。该重组融合蛋白具有治疗白细胞减少症的功能。(A cat granulocyte colony stimulating factor mutant recombinant fusion protein, a preparation method and application thereof, relating to the field of biological gene engineering. The recombinant fusion protein is formed by connecting a cat granulocyte colony stimulating factor mutant and ricin B chain truncated peptide through a flexible linker. The preparation method comprises the following steps: constructing a cat granulocyte colony stimulating factor mutation expression vector; constructing an escherichia coli recombinant expression vector; expressing recombinant FeG-CSF-Mut/RTBD1 fusion protein; purifying and renaturing the recombinant FeG-CSF-Mut/RTBD1 fusion protein. The cat G-CSF mutant is connected with ricin B chain truncated peptide to obtain the long-acting granulocyte colony stimulating factor, so that the biological activity of the granulocyte colony stimulating factor can be enhanced, the protein stability of the granulocyte colony stimulating factor in blood in vivo is improved, and the half-life period of a medicament in the body is further improved. The recombinant fusion protein has the function of treating leukopenia.)

1. The recombinant fusion protein is characterized in that the recombinant fusion protein is formed by connecting a cat 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 cat granulocyte colony stimulating factor mutant is prepared by the following steps: designing a site-directed mutagenesis primer, mutating aspartic acid at the 28 th position in a cat granulocyte colony-stimulating factor sequence into arginine and glutamine at the 121 th position into phenylalanine by taking recombinant plasmid pUC57-FeG-CSF as a template, and naming the mutated sequence as FeG-CSF-Mut;

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

SiteⅠ

Forward：5'-GTAAGGTCCAAGCTAGAGGCACAGCCCTGCAG-3'；

Reverse：5'-CTGCAGGGCTGTGCCTCTAGCTTGGACCTTAC-3'；

SiteⅡ

Forward：5'-GCAATTAATATCTGGCAGTTCATGGAGGATGTGGGTATGG-3'；

Reverse：5'CCATACCCACATCCTCCATGAACTGCCAGATATTAATTGC-3'。

3. the recombinant fusion protein of claim 2, wherein the mutated FeG-CSF-Mut sequence and the ricin B chain truncated peptide sequence are linked by a flexible linker to form a FeG-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 a cat granulocyte colony stimulating factor mutant recombinant fusion protein as claimed in any one of claims 1-4, comprising the steps of:

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

constructing an escherichia coli recombinant expression vector;

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

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

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

(1) performing homologous modeling on cat granulocyte colony stimulating factor molecules and cat granulocyte colony stimulating factor receptor molecules through a SWISS-MODEL online platform; docking cat granulocyte colony stimulating factor molecules and cat 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-FeG-CSF:

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

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

SiteⅠ

Forward：5'-GTAAGGTCCAAGCTAGAGGCACAGCCCTGCAG-3'；

Reverse：5'-CTGCAGGGCTGTGCCTCTAGCTTGGACCTTAC-3'；

SiteⅡ

Forward：5'-GCAATTAATATCTGGCAGTTCATGGAGGATGTGGGTATGG-3'；

Reverse：5'CCATACCCACATCCTCCATGAACTGCCAGATATTAATTGC-3'；

(4) the 28 th aspartic acid in the cat granulocyte colony-stimulating factor sequence is mutated into arginine, the 121 th glutamine is mutated into phenylalanine by taking the recombinant plasmid pUC57-FeG-CSF as a template, and the mutated sequence is named as FeG-CSF-Mut; the mutated FeG-CSF-Mut sequence and the ricin B chain truncated peptide sequence are connected by a flexible linker to form a FeG-CSF-Mut/RTBD1 sequence, the nucleotide sequence of the sequence is shown as SEQ ID NO. 1, the amino acid sequence of the sequence is shown as SEQ ID NO. 2, the sequence is inserted into a pMD18T vector, and a product with correct sequencing is named as a pMD18T-FeG-CSF-Mut/RTBD1 plasmid and serves as a FeG-CSF mutation expression vector.

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

carrying out double enzyme digestion on pMD18T-FeG-CSF-Mut/RTBD1 plasmid and 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 FeG-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.

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

inoculating correctly sequenced positive bacteria BL21(DE3)/pET30a-FeG-CSF-Mut/RTBD1 into 5mL LB culture medium containing Kan with the concentration of 100 mu g/mL according to the proportion of 1:100v/v, carrying out constant temperature shaking culture at 180rpm at 37 ℃ until OD600 is equal to 0.6, transferring the culture medium into 500mL of the same LB culture medium according to the proportion of 1:100v/v, supplementing Kan until the final concentration is 100 mu g/mL, and carrying out constant temperature shaking culture at 180rpm at 37 ℃ until OD600 is equal to 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 rFeG-CSF-Mut/RTBD1 is obtained by centrifugation at 8000rpm and 4 ℃.

9. The method for preparing the recombinant fusion protein of cat granulocyte colony-stimulating factor mutant as claimed in 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 the rFeG-CSF/RTBD1 inclusion body in a protein purification A liquid in a denaturing manner, loading a dissolving solution onto the column, and 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 the rFeG-CSF-Mut/RTBD1 solution purified by ion exchange chromatography to a column, mixing the protein purified solution C and the protein purified solution D by a gradient mixing module in a protein purification system, eluting the chelating Sepharose by imidazole with final concentration of 50mmol/L and 250mmol/L respectively, and collecting protein flow-through liquid, a mixed protein solution and a target protein solution respectively; performing SDS-PAGE analysis on the protein solution collected in each step, wherein the gel concentration is 12%;

(3) rFeG-CSF-Mut/RTBD1 protein renaturation: diluting the rFeG-CSF-Mut/RTBD1 protein solution purified by the two steps by using protein renaturation A liquid until the concentration of the rFeG-CSF-Mut/RTBD1 is 0.1mg/mL, filling the rFeG-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 hours, so as to completely remove urea and slowly and fully fold the rFeG-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 the rFeG-CSF-Mut/RTBD1 protein until the concentration is 1-1.5 mg/mL, and obtaining a recombinant FeG-CSF-Mut/RTBD1 fusion protein;

the composition of the protein purification solution A is as follows: 50mmol/LPB, 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/LTris, 8mol/LUrea, 0.5mol/LNaCl, 500mmol/LImidazole, pH 8.0;

the composition of the protein renaturation A solution is as follows: 50mmol/L of LTris, 8mol/L of LUrea, 10% m/V of sucrose, 10% v/V of glycerol, 5mmol/L of 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 a cat granulocyte colony stimulating factor mutant recombinant fusion protein as defined in any one of claims 1-4 in the manufacture of a medicament for the treatment of leukopenia.

Technical Field

The invention relates to the technical field of biological genetic engineering, in particular to a cat 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 cat granulocyte colony stimulating factor (rFeG-CSF-Mut/RTBD1) acts on the 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 macrophage cells and acid-phagocytic 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 has no toxicity when existing alone, and has the function of enhancing immune response of organism. RTB is a two-lobed molecule with the same folding topology composed of two different spherical regions, each comprising three subregions (α, β, γ), only 1 α and 2 β having significant galactose binding activity.

At present, researches on improving the activity of the recombinant fusion protein in treating leukopenia and prolonging the half life of the recombinant fusion protein by combining the cat G-CSF mutant and the ricin B chain truncated peptide into the recombinant fusion protein have not been reported.

Disclosure of Invention

The invention provides a cat 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 cat granulocyte colony stimulating factor mutant recombinant fusion protein which is formed by connecting a cat granulocyte colony stimulating factor mutant and ricin B chain truncated peptide through a flexible linker.

As a preferred embodiment, the cat granulocyte colony stimulating factor mutant is prepared by the following steps: designing a site-directed mutagenesis primer, mutating aspartic acid at the 28 th position in a cat granulocyte colony-stimulating factor sequence into arginine and glutamine at the 121 th position into phenylalanine by taking recombinant plasmid pUC57-FeG-CSF as a template, and naming the mutated sequence as FeG-CSF-Mut;

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

SiteⅠ

Forward：5'-GTAAGGTCCAAGCTAGAGGCACAGCCCTGCAG-3'；

Reverse：5'-CTGCAGGGCTGTGCCTCTAGCTTGGACCTTAC-3'；

SiteⅡ

Forward：5'-GCAATTAATATCTGGCAGTTCATGGAGGATGTGGGTATGG- 3'；

Reverse：5'CCATACCCACATCCTCCATGAACTGCCAGATATTAATTGC-3'。

as a preferred embodiment, the mutated FeG-CSF-Mut sequence and the ricin B chain truncated peptide sequence are connected by a flexible linker to form a FeG-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 discloses a preparation method of cat granulocyte colony stimulating factor mutant recombinant fusion protein, which comprises the following steps:

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

constructing an escherichia coli recombinant expression vector;

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

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

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

(1) performing homologous modeling on cat granulocyte colony stimulating factor molecules and cat granulocyte colony stimulating factor receptor molecules through a SWISS-MODEL online platform; docking cat granulocyte colony stimulating factor molecules and cat 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-FeG-CSF:

inquiring cat granulocyte colony stimulating factor gene sequence through NCBI Genbank database, carrying out whole gene synthesis on the obtained sequence, carrying out codon optimization by using escherichia coli codon preference during synthesis, and cloning into an escherichia coli cloning vector pUC57 to obtain an escherichia coli recombinant cloning vector pUC 57-FeG-CSF.

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

SiteⅠ

Forward：5'-GTAAGGTCCAAGCTAGAGGCACAGCCCTGCAG-3'；

Reverse：5'-CTGCAGGGCTGTGCCTCTAGCTTGGACCTTAC-3'；

SiteⅡ

Forward：5'-GCAATTAATATCTGGCAGTTCATGGAGGATGTGGGTATGG- 3'；

Reverse：5'CCATACCCACATCCTCCATGAACTGCCAGATATTAATTGC-3 '；

(4) using recombinant plasmid pUC57-FeG-CSF as a template, mutating aspartic acid at the 28 th position in a cat granulocyte colony-stimulating factor sequence into arginine, mutating glutamine at the 121 th position into phenylalanine, and naming the mutated sequence as FeG-CSF-Mut; the mutated FeG-CSF-Mut sequence and the ricin B chain truncated peptide sequence are connected by a flexible linker to form a FeG-CSF-Mut/RTBD1 sequence, the nucleotide sequence is shown as SEQ ID NO. 1, the amino acid sequence is shown as SEQ ID NO. 2, the mutated sequence is inserted into a pMD18T vector, and a product with correct sequencing is named as a pMD18T-FeG-CSF-Mut/RTBD1 plasmid and serves as a FeG-CSF mutation expression vector.

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

carrying out double enzyme digestion on pMD18T-FeG-CSF-Mut/RTBD1 plasmid and 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 FeG-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-FeG-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 culture medium into 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 rFeG-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 rFeG-CSF/RTBD1 inclusion bodies in a protein purification A liquid in a denaturing manner, loading a dissolved solution onto the column, and 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) metalChelating chromatography: pair of Ni-Supported proteins with protein purification solution B²⁺Balancing by using chemical Sepharose; loading the rFeG-CSF-Mut/RTBD1 solution purified by ion exchange chromatography to a column, mixing the protein purified solution C and the protein purified solution D by a gradient mixing module in a protein purification system, eluting the chelating Sepharose by imidazole with final concentration of 50mmol/L and 250mmol/L respectively, and collecting the protein flow-through solution, the hybrid protein solution and the target protein solution respectively; performing SDS-PAGE analysis on the protein solution collected in each step, wherein the gel concentration is 12%;

(3) rFeG-CSF-Mut/RTBD1 protein renaturation: diluting the rFeG-CSF-Mut/RTBD1 protein solution purified by the two steps by using protein renaturation A liquid until the concentration of the rFeG-CSF-Mut/RTBD1 is 0.1mg/mL, filling the rFeG-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 hours, so as to completely remove urea and slowly and fully fold the rFeG-CSF-Mut/RTBD1 protein; after the protein renaturation B liquid is exhausted, introducing a protein renaturation C liquid to remove residual cane sugar and glycerin components in the renaturation process, concentrating the rFeG-CSF-Mut/RTBD1 protein until the concentration is 1-1.5 mg/mL, and obtaining the recombinant FeG-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 application of cat granulocyte colony stimulating factor mutant recombinant fusion protein in preparing a medicament for treating leukopenia.

The invention has the beneficial effects that:

the cat granulocyte colony stimulating factor mutant recombinant fusion protein is formed by connecting a cat 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. The cat G-CSF mutant is connected with ricin B chain truncated peptide (RTBD1) to obtain the long-acting granulocyte colony stimulating factor, so that the biological activity of the granulocyte colony stimulating factor can be enhanced, the protein stability of the granulocyte colony stimulating factor in-vivo blood is improved, and the half-life period of the medicament in vivo is further improved. The invention reduces the cost of the granulocyte colony stimulating factor preparation, and has great potential and value in developing medicaments for treating leukopenia and veterinary clinical application.

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 embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

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

1. Construction of expression vector for cat granulocyte colony stimulating factor (FeG-CSF) mutation

(1) Performing homologous modeling on cat granulocyte colony stimulating factor molecules and cat granulocyte colony stimulating factor receptor molecules through a SWISS-MODEL online platform; docking cat granulocyte colony stimulating factor molecules and cat 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-FeG-CSF:

inquiring cat granulocyte colony stimulating factor gene sequence through NCBI Genbank database, carrying out whole gene synthesis on the obtained sequence, carrying out codon optimization by using escherichia coli codon preference during synthesis, and cloning into an escherichia coli cloning vector pUC57 to obtain an escherichia coli recombinant cloning vector pUC 57-FeG-CSF.

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

SiteⅠ

Forward：5'-GTAAGGTCCAAGCTAGAGGCACAGCCCTGCAG-3'；

Reverse：5'-CTGCAGGGCTGTGCCTCTAGCTTGGACCTTAC-3'；

SiteⅡ

Forward：5'-GCAATTAATATCTGGCAGTTCATGGAGGATGTGGGTATGG- 3'；

Reverse：5'CCATACCCACATCCTCCATGAACTGCCAGATATTAATTGC-3 '。

(4) the 28 th aspartic acid in the cat granulocyte colony-stimulating factor sequence is mutated into arginine and the 121 th glutamine is mutated into phenylalanine by taking the recombinant plasmid pUC57-FeG-CSF as a template so as to enhance the stability and the antiviral activity of the cat granulocyte colony-stimulating factor/RTBD 1 fusion protein, and the mutated sequence is named as FeG-CSF-Mut. The mutated FeG-CSF-Mut sequence and ricin B chain truncated peptide sequence (RTBD1 sequence, the nucleotide sequence is shown as SEQ ID NO:3 in the sequence table, and the amino acid sequence is shown as SEQ ID NO:4 in the sequence table) are passed through flexible linker (Gly)₄Ser)₃The connecting peptides are connected to form FeG-CSF-Mut/RTBD1 sequence, the nucleotide sequence is shown as SEQ ID NO. 1, the amino acid sequence is shown as SEQ ID NO. 2, the connecting peptides are inserted into the pMD18T vector, and after sequencing, the product with the correct sequencing is named as: pMD18T-FeG-CSF-Mut/RTBD1 plasmid as FeG-CSF mutation expression vector for downstream gene engineering operation. Wherein the adopted flexible linker is (Gly)₄Ser)₃A linker peptide.

2. Construction of recombinant expression vector for Escherichia coli

The pMD18T-FeG-CSF-Mut/RTBD1 plasmid and 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 FeG-CSF target fragment	4μL
Recovered pET30a vector fragment	1μL
		Reaction conditions	16 ℃ overnight

The ligation product was transformed into Trans10 competent cells. Converted and then coated to the endKan at a concentration of 100. mu.g/mL⁺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 FeG-CSF-Mut/RTBD1 fusion protein expression

Inoculating the positive bacterium BL21(DE3)/pET30a-FeG-CSF-Mut/RTBD1 with correct sequencing 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 equal to 0.6, then transferring into 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 equal to 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 pellets were obtained by centrifugation at 8000rpm and 4 ℃.

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

The cell pellet rFeG-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 is equilibrated by using protein purification A liquid (50mmol/L PB, 8mol/L Urea, pH 6.0), inclusion bodies of rFeG-CSF/RTBD1 are denatured and dissolved in the protein purification A liquid, the solution is loaded on the column, and a flow-through liquid in which the target protein is mainly present is 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 the rFeG-CSF-Mut/RTBD1 solution purified by ion exchange chromatography on a column, and mixing the protein purification solution C (50mmol/L Tris, 8mol/L Urea, 0.5mol/L NaCl, pH 8.0) and the 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, wherein the rFeG-CSF-Mut/RTBD1 solution is respectively used for mixingAnd (3) eluting the chelating Sepharose by using imidazole with final concentration of 50mmol/L and 250mmol/L, and respectively collecting protein flow-through liquid, mixed protein solution and target protein solution. The protein solution collected at each step was subjected to SDS-PAGE analysis at a gel concentration of 12%.

(3) rFeG-CSF-Mut/RTBD1 protein renaturation: the rFeG-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 rFeG-CSF-Mut/RTBD1 and loaded into a protein ultrafiltration system with a molecular weight cut-off of 3000 for dialytic renaturation, 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 hours, so as to completely remove urea and slowly and fully fold the rFeG-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 rFeG-CSF-Mut/RTBD1 protein until the concentration is 1-1.5 mg/mL, and obtaining the recombinant FeG-CSF-Mut/RTBD1 fusion protein.

EXAMPLE 2 pharmacodynamic assay of recombinant FeG-CSF-Mut/RTBD1 fusion protein

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 of the mice is taken as a blood routine to detect the number of White Blood Cells (WBC), and the success standard of the model adopted in the experiment is as follows: the number of leukocytes is lower than the lowest value of the reference range by 0.8X 10⁹And L. And (3) confirming that the preparation of the leukopenia mouse model is successful in the mice meeting the conditions. 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 rFeG-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 rFeG-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	rFeG-CSF-Mut/RTBD1

2. Routine blood test

The number of leukocytes in the blood of mice was measured using a blood routine analyzer. 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), demonstrating that the rFeG-CSF-Mut/RTBD1 fusion protein of the invention successfully treats leukopenia.

The invention discloses a cat granulocyte colony stimulating factor mutant recombinant fusion protein, a preparation method and application thereof, and a person skilled in the art can realize the fusion 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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