TW201738263A - A preparation method of recombinant human granulocyte colony stimulating factor - Google Patents

Abstract

The invention relates to a preparation method of recombinant human granulocyte colony stimulating factor. Specifically, the cDNA sequence with the method of the invention is formed by the human sequence mutation, the method provided by the invention can make the expression of protein (recombinant human granulocyte colony stimulating factor) increased significantly.

Description

Preparation method of recombinant human granulocyte colony stimulating factor

The invention relates to a preparation method of recombinant human granulocyte colony stimulating factor, belonging to the fields of biochemistry and molecular biology.

Human granulocyte colony-stimulating factor (hGCSF) is a cytokine that plays an important role in stimulating the growth, differentiation and survival of hematopoietic cells including mature neutrophils, macrophages and dendritic cells. When stimulated by microorganisms or inflammatory cytokines, various types of cells in the body (eg, fibroblasts, endothelial cells) produce human granulocyte colony-stimulating factor, and activation of human granulocyte colony-stimulating factor plays an important role in the innate immune response. effect. When stimulated by a particular antigen, T cells also secrete human granulocyte colony-stimulating factor. Human granulocyte colony-stimulating factors are often used as biologic drugs for the treatment of immunocompromised individuals due to the promotion of hematopoietic cells. Uncontrolled activation of hGCSF is associated with spontaneous immune symptoms such as arthritis and multiple sclerosis. Neutralization of the biological activity of hGCSF by autoantibodies causes another autoimmune disease, a special alveolar proteinosis, and hGCSF can treat this condition. At present, human granulocyte colony-stimulating factor is mainly used for leukopenia caused by cancer, chemotherapy, etc. An important auxiliary drug in the course of chemotherapy.

In 1986, human granulocyte colony-stimulating factor cDNA was successfully selected. The human granulocyte colony-stimulating factor gene is 2.5 kb in length, including 5 exons and 4 introns. The human granulocyte colony-stimulating factor gene is located on chromosome 17. Humans have two different hGCSF cDNAs, each encoding a precursor protein containing 207 and 204 amino acids, each with a leader sequence of 30 amino acids, the mature protein molecules being 177 and 174 amino acids, respectively. The mature molecule has three amino acids inserted at the N-position of the N-terminus, and the remaining sequence is identical to the 174 amino acid molecule. The biological activity of the 174 amino acid molecule is 20 times higher than the biological activity of the 177 amino acid molecule.

Generally, human granulocyte colony stimulating factor is recombinantly expressed using mammalian cells. However, in order to isolate and purify hGCSF, the cells are cultured and the GCSF protein is isolated from the culture supernatant, which has the disadvantage of low GCSF yield and is therefore unsuitable for mass production. It is known that glycosylated glycosylation of hGCSF is not essential for the activity of hGCSF, and the use of mammalian cells to produce glycosylated hGCSF requires expensive materials and equipment, and thus such an approach is not economically feasible. In 1991, the first recombinant human granulocyte colony-stimulating factor drug Filgrastim (Neupogen, r-metHuGCSF, Amgen Inc) produced by the E. coli expression system was marketed in the United States. Since then, many companies have produced E. coli expression systems at home and abroad. The rhGCSF drug is marketed.

The main methods currently reported to increase the yield of recombinant human granulocyte colony-stimulating factor are as follows: (1) to increase the expression level by changing the expression system; (2) to change the fermentation process, such as high-density fermentation, optimized medium, Optimize culture conditions and other means to achieve high expression of engineering bacteria; (3) optimize renaturation conditions and modify the purification process. However, these methods are only optimization in the production process and do not increase the recombinant expression yield of human granulocyte colony-stimulating factor at the molecular level.

As described above, the glycosylation of glycosylated hGCSF is not essential for the activity of hGCSF, and human granulocyte colony-stimulating factor can be produced using a more economical and convenient E. coli expression system, Chinese patents CN1156575C, CN1108303A, CN1088107C, CN101591660A and the like all directly use human cDNA sequence to recombinantly express human granulocyte colony-stimulating factor in E. coli expression system, and the expression of eukaryotic protein in E. coli expression system may have rare codon problem. Although the Rosetta host strain derived from BL21 can be used to enhance the expression of eukaryotic proteins with rare codons of E. coli. However, the screening marker for the Rosetta host strain is chloramphenicol resistance, which has a large limitation in the production of the drug. How to increase the production of recombinant human granulocyte colony-stimulating factor and minimize the cost is a huge challenge.

The present invention provides a method for preparing a recombinant human granulocyte colony stimulating factor, which is expressed in a prokaryotic expression system using a cDNA different from the recombinant human granulocyte colony stimulating factor human sequence SEQ ID NO. 1, by the method provided by the present invention. The expression level of the target protein, ie recombinant human granulocyte stimulating factor, can be significantly increased, and the rare codon problem arising from the direct use of the human cDNA sequence in the prokaryotic expression system is solved.

The invention provides a system for recombinant human granulocyte colony stimulating factor The protein sequence of the recombinant human granulocyte colony-stimulating factor is SEQ ID NO. 3, and the human gene sequence of the recombinant human granulocyte colony-stimulating factor corresponding to the protein sequence is SEQ ID NO. 1, and the method is characterized by comprising A step of synthesizing a cDNA sequence which is mutated from a human cDNA sequence, and further comprises a step of constructing the recombinant expression vector and a step of inducing expression of the recombinant expression vector in the host cell.

The step of synthesizing the cDNA sequence in the present invention adopts the whole gene synthesis method, according to the codon preference of E. coli, simultaneously optimizes the sequence GC content, the mRNA secondary structure, eliminates the splice site, the polyA site, the Chi site and the RBS site. Point, CpG island, RNA labyrinth, repeat sequences and restriction enzyme sites that may interfere with selection, optimize the recombinant human granulocyte colony stimulating factor gene sequence. In order to facilitate vector construction, the restriction sites required for selection and recombination are introduced at both ends.

In a preferred embodiment, the cDNA sequence synthesized in the method provided by the present invention is shown in SEQ ID NO.

The cDNA sequence of the recombinant human granulocyte colony-stimulating factor provided by the present invention is synthesized by mutating 34 bases in the human gene sequence of the recombinant human granulocyte colony-stimulating factor, and all the Escherichia coli in the sequence Low frequency codons are replaced by high frequency codons. The codon adaptability of the synthesized cDNA sequence SEQ ID NO. 2 in E. coli increased (see Figure 1A and Figure 1B), the frequency of codon usage was increased (see Figures 2A and 2B), in addition to GC content There have also been improvements (see Figures 3A and 3B).

Preparation method of recombinant human granulocyte colony stimulating factor provided by the invention The method also includes the step of constructing a recombinant expression vector.

The recombinant expression vector involved in the method provided by the present invention is selected from the group consisting of a temperature expression vector (e.g., pBV220) and a substrate-inducing vector (e.g., pET9a), preferably a receptor-inducible expression vector, most preferably a pET series.

The method for preparing the recombinant human granulocyte colony stimulating factor provided by the present invention further comprises the step of inducing expression of the recombinant expression vector in the host cell.

The host cell involved in the method provided by the present invention is Escherichia coli, preferably Escherichia coli DH5a, BL21 (DE3), Rosetta (DE3), Origami (DE3), JM109, HSM174 (DE3).

Specifically, the method provided by the present invention includes the following steps:

(1) synthesizing recombinant human granulocyte colony-stimulating factor gene by whole-gene synthesis;

(2) The synthesized cDNA sequence is ligated to an expression vector to construct a recombinant expression vector.

(3) The constructed expression vector was highly expressed in E. coli.

The recombinant expression yield of the cDNA sequence provided by the present invention can be more than twice that of human cDNA.

The present invention also provides a cDNA sequence encoding a recombinant human granulocyte colony stimulating factor synthesized according to the above-described whole gene synthesis method, as shown in SEQ ID NO.

The present invention also provides a recombinant expression vector for a recombinant human granulocyte colony stimulating factor gene, which comprises SEQ ID NO.

The recombinant expression vector provided by the present invention is selected from the group consisting of a temperature expression vector (for example, pBV220) and a substrate-inducing vector (for example, pET9a), preferably induced by a substance. The expression vector is optimally the pET series.

The present invention also provides a host cell which expresses a recombinant human granulocyte colony stimulating factor, which comprises the above recombinant expression vector.

The host cell provided by the present invention is Escherichia coli, preferably Escherichia coli DH5a, BL21 (DE3), Rosetta (DE3), Origami (DE3), JM109, HSM174 (DE3).

The present invention also provides a method for preparing a water-soluble polymer modified conjugate of recombinant human granulocyte colony-stimulating factor, which comprises the step of obtaining recombinant human granulocyte colony-stimulation as represented by the protein sequence SEQ ID NO. After the factor, the step of coupling the recombinant human granulocyte colony stimulating factor with the water-soluble polymer is further included, and the specific operation method can refer to the patent CN101172161B.

The invention also provides a water-soluble polymer modified conjugate of recombinant human granulocyte colony stimulating factor, wherein the water-soluble polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, polylactic acid, etc., preferably polyethylene glycol. The molecular weight of the diol, polyethylene glycol is selected from 2KD to 100KD, preferably from 5KD to 100KD.

The conjugate structure of the water-soluble polymer modification of the recombinant human granulocyte colony-stimulating factor provided by the present invention is as shown in the formula (I):

m is selected from an integer of from 50 to 2500, preferably selected from an integer of from 400 to 500, and G is a protein sequence of the sequence of SEQ ID NO.

Figure 1A and Figure 1B: Sequence SEQ ID NO. 1 (Figure 1A) vs. Sequence SEQ ID NO. 2 (Figure 1 B) Codon Adaptation Index (CAI).

Figures 2A and 2B: Sequence SEQ ID NO. 1 (Figure 2A) is compared to sequence SEQ ID NO. 2 (Figure 2B) codon usage frequency (FOP).

Figures 3A and 3B: Sequence SEQ ID NO. 1 (Figure 3A) versus sequence SEQ ID NO. 2 (Figure 3B) GC content.

Figure 4: Shake flask-induced expression of recombinant expression strains, in which electrophoresis lane 1: pET9a-GCSF-1/BL21 (DE3), electrophoresis lane 2: pET9a-GCSF-2/BL21 (DE3), electrophoresis lane 3: pET9a- GCSF-2/BL21(DE3), electrophoresis channel 4: pBV220-GCSF-3/DH5a, electrophoresis channel 5: pBV220-GCSF-4/DH5a, electrophoresis channel 6: pBV220-GCSF-4/DH5a, electrophoresis channel 7: empty Carrier sample, electrophoresis channel 8: empty bacteria sample, electrophoresis channel M: protein marker (Protein Marker).

Figure 5: Recombinant expression strain 5L fermentor induced expression yield determination, wherein 1: fermentation sample diluted 8 times, 2: fermentation sample diluted 16 times.

The invention is further described in the following examples, but these examples are not intended to limit the scope of the invention.

Example 1: Construction of recombinant human granulocyte colony-stimulating factor receptor-inducible expression strain

1. Gene synthesis

The recombinant human granulocyte colony stimulating factor gene sequence was synthesized by Nanjing Kingsray Biotech Co., Ltd.

Recombinant human granulocyte colony stimulating factor human sequence SEQ ID NO.

The synthetic recombinant human granulocyte colony stimulating factor gene sequence GCSE-1 was shown below, and an Nde I restriction site was added to the 5' end of SEQ ID NO. 1, and a BamHI restriction site was added to the 3' end.

Recombinant human granulocyte colony stimulating factor cDNA sequence SEQ ID NO. 2:

The synthetic recombinant human granulocyte colony-stimulating factor gene sequence GCSF-2 was shown below, and an Nde I restriction site was added to the 5' end of SEQ ID NO. 2, and a BamHI restriction site was added to the 3' end.

The corresponding protein sequence is shown in SEQ ID NO.

2. Construction of recombinant expression strains pET9a-GCSF-1/BL21(DE3) and pET9a-GCSF-2/BL21(DE3)

Recombinant protoplasts (pUC57-GCSF-1, pUC57-GCSF-2) and expression vector pET9a were digested with NdeI and BamHI endonucleases (Fermentas), respectively. The target gene fragment (GCSF-1, GCSF-2) and the expression vector pET9a were subjected to gel recovery using a rapid gel recovery kit (Promega), and the target gene fragment (GCSF-1) was cloned with T4 ligase (New England Biolabs). , GCSF-2) and the expression vector pET9a recovered fragments for DNA ligation. Finally, the ligation product was transformed into E. coli BL21 (DE3) competent cells, and LB plates containing a final concentration of 50 μg/ml Kana were plated to screen for recombinants.

The positive selection strains were picked, and the plastids were extracted and verified by sequencing by Nanjing Kingsray Company to obtain pET9a-GCSF-1/BL21 (DE3) and pET9a-GCSF-2/BL21 (DE3) expression strains.

3. Induction of expression of recombinant expression strains pET9a-GCSF-1/BL21(DE3) and pET9a-GCSF-2/BL21(DE3)

The positive clones verified by sequencing were inoculated into a 12 ml culture tube containing 2 ml of LB (50 μg/ml Kana) medium, and shake cultured at 225 rpm on a 37 ° C constant temperature shaker to an OD ₆₀₀ = 4 (16-18 hours). Transfer into 50 ml LB (50 μg / ml Kana) medium, shake culture at 220 ° C for about 1.5 h at 37 ° C to OD ₆₀₀ = 0.8. About 1 mM IPTG was added to the culture medium, and the mixture was cultured at 37 ° C for 4 to 5 hours with shaking at 220 rpm. After the expression was completed, the sediment was centrifuged at 5000 g for 10 minutes to obtain a precipitate, and the SDS-PAGE electrophoresis analysis was shown in Fig. 4.

4. Induced expression of recombinant expression strains pET9a-GCSF-1/BL21(DE3), pET9a-GCSF-2/BL21(DE3)5L fermentor

The 5L scale fermentation is carried out in the following 4 steps:

1. Inoculate 2 ml of glycerol bacteria into a 1 L shake flask containing 200 ml of LB medium (yeast powder 5 g/L, soy peptone 10 g/L, NaCl 5 g/L), shake culture at 220 ° C for about 9 h at 37 ° C, and incubate to the strain. OD ₆₀₀ = 2.5 or so.

2. Inoculate the cultured seed solution into a 5L fermenter for fermentation, using 3L LB medium (the composition is: tryptone 10g / L, yeast powder 5g / L, NaCl 5g / L, bubble enemy 0.03%, Na ₂ HPO ₄ .12H ₂ O 11g / L, KH ₂ PO ₄ 2.7g / L, sterilized at 121 ° C for 30min. Glucose 5g / L, MgSO ₄ 0.3g / L, sterilized at 115 ° C for 30min), the culture temperature was set at 37 ° C, The aeration volume was maintained at 6 L/min, and the pH was controlled at about 7.0 with ammonia water. The culture was carried out at 37 ° C, and the dissolved oxygen was controlled by stirring and feeding at 30% to 110%.

3. When the OD ₆₀₀ reaches about 25, about 1 mM IPTG is added, and the induction time is 4 h.

4. Determination of yield

Table 1

From the results in Table 1, the expression level of the target protein of the engineered strain constructed using the SEQ ID NO. 2 gene sequence is the engineered protein of the engineered recombinant human granulocyte colony stimulating factor gene sequence SEQ ID NO. The expression level is more than 2 times.

Example 2: Construction of temperature-inducible expression strain of recombinant human granulocyte colony-stimulating factor

Gene synthesis

The recombinant human granulocyte colony stimulating factor gene sequence was synthesized by Nanjing Kingsray Biotech Co., Ltd.

The synthetic recombinant human granulocyte colony stimulating factor gene sequence GCSF-3 was shown below, and an EcoRI cleavage site was added to the SEQ ID NO. 15' end, and a BamHI cleavage site was added to the 3' end.

The synthetic recombinant human granulocyte colony stimulating factor gene sequence GCSF-4 was shown below, and an EcoRI cleavage site was added to the 5' end of SEQ ID NO. 2, and a BamHI restriction site was added to the 3' end.

The corresponding protein sequence is shown in SEQ ID NO.

2. Construction of recombinant expression strains pBV220-GCSF-3/DH5a, pBV220-GCSF-4/DH5a

Recombinant selection of recombinant plastids using EcoRI and BamHI endonuclease (Fermentas) (pUC57-

GCSF-3, pUC57-GCSF-4) and the expression vector pBV220 were subjected to double digestion. The target gene fragment (GCSF-3, GCSF-4) and the expression vector pBV220 were digested with a rapid gel recovery kit (Promega), and the target gene fragment (GCSF-3) was cloned with T4 ligase (New England Biolabs). , GCSF-4) and the expression vector pBV220 recovered fragments for DNA ligation. Finally, the ligation product was transformed into E. coli DH5a competent cells, and LB plates containing a final concentration of 100 μg/ml Amp were applied to screen for recombinants.

Pick positive colonies, extract the plastids and verify by sequencing by Nanjing Kingsray Company to obtain pBV220- GCSF-3/DH5a, pBV220-GCSF-4/DH5a expression strain.

3. Induction of expression of recombinant expression strains pBV220-GCSF-3/DH5a, pBV220-GCSF-4/DH5a by shake flask

The positive clones verified by sequencing were inoculated into a 12 ml culture tube containing 2 ml of LB (100 μg/ml Amp) medium, and shake cultured at 225 rpm on a constant temperature shaker at 30 ° C until an OD ₆₀₀ = 4 (16-18 hours). Transfer to 50 ml LB (100 μg / ml Amp) medium, shake culture at 220 ° C. on a constant temperature shaker at 30 ° C for about 1.5 h to about OD ₆₀₀ = 0.8. The temperature was raised to 42 ° C on a constant temperature shaker at 220 rpm for 4-5 hours with shaking. After the expression was completed, the precipitate was centrifuged at 5000 g for 10 minutes to precipitate, and analyzed by SDS-PAGE electrophoresis.

4. Induced expression of recombinant expression strains pBV220-GCSF-3/DH5a, pBV220-GCSF-3/DH5a 5L fermentor

The 5L scale fermentation is carried out in the following 4 steps:

1. 2 ml of glycerol bacteria were inoculated into a 1 L shake flask containing 200 ml of LB medium (yeast powder 5 g / L, soy peptone 10 g / L, NaCl 5 g / L), shaken at 220 ° C for 30 h at 30 ° C, and cultured to the strain. OD ₆₀₀ = 2.5 or so.

2. Inoculate the cultured seed solution into a 5L fermenter for fermentation, using 3L LB medium (the composition is: tryptone 10g / L, yeast powder 5g / L, NaCl 5g / L, bubble enemy 0.03%, Na ₂ HPO ₄ .12H ₂ O 11g / L, KH ₂ PO ₄ 2.7g / L, sterilized at 121 ° C for 30min. Glucose 5g / L, MgSO ₄ 0.3g / L, sterilized at 115 ° C for 30min), the culture temperature was set to 30 ° C, The aeration volume was maintained at 6 L/min, and the pH was controlled at about 7.0 with ammonia water. The culture was carried out at 30 ° C, and the dissolved oxygen was controlled by stirring and feeding at 30% to 110%.

3. When the OD ₆₀₀ reaches about 25, the temperature is raised to 42 ° C, and the induction time is 4 h.

4. Determination of yield

From the results in Table 2, the expression level of the target protein of the engineered strain constructed using the gene sequence of SEQ ID NO. 2 is the engineered protein of the engineered recombinant human granulocyte colony-stimulating factor gene sequence SEQ ID NO. The expression level is more than 2 times.

Example 3: Determination of induced expression yield of recombinant expression strain 5L fermentor

1. Take 30 μl of the reference substance, dilute with purified water to prepare a 0.20 g/L reference solution, and further dilute to a concentration gradient with purified water. 1 ml of the homogeneous fermentation broth was taken, centrifuged at 12,000 rpm for 5 minutes, the supernatant was discarded, and 1 ml of purified water was added to the precipitate to mix. 52 μl of the diluted sample was taken, 20 μl of NuPAGE® LDS Sample Buffer (4×) and 8 μl of NuPAGE® Reducing Agent (10×) were added, mixed, and subjected to a water bath at 70 ° C for 10 minutes, and centrifuged at 12,000 rpm for 1 minute.

2. Select NuPAGE® Novex® 4-12% Bis-Tris Gel and NuPAGE® MOPS SDS Running Buffer (20×) and 200V constant pressure electrophoresis for about 50 minutes until bromophenol blue migrates to the bottom of the gel and stop electrophoresis. Electrophoresis After completion, the film was taken out, placed in about 100 ml of purified water, shaken for 5 minutes on a shaker, and repeated three times, and the purified water was discarded. About 100 ml of the staining solution was added, and the mixture was shaken for 1 hour until a clear protein band appeared; the staining solution was discarded, about 100 ml of purified water was added, and the mixture was shaken and shaken overnight until the gel background was transparent.

3. Place the gel after electrophoresis in a gel imager. See Figure 5 for photo analysis.

Example 4: Screening of recombinant human granulocyte colony-stimulating factor cDNA sequence

1. Gene synthesis

The recombinant human granulocyte colony stimulating factor gene sequence was synthesized by Nanjing Kingsray Biotech Co., Ltd.

Recombinant human granulocyte colony stimulating factor cDNA sequence SEQ ID NO. 4:

The synthesized recombinant human granulocyte colony stimulating factor gene sequence GCSF-5 was shown below, and an Nde I restriction site was added to the 5' end of SEQ ID NO. 4, and a BamHI restriction site was added to the 3' end.

Recombinant human granulocyte colony stimulating factor cDNA sequence SEQ ID NO. 5:

The synthetic recombinant human granulocyte colony-stimulating factor gene sequence GCSF-6 was shown below, and an Nde I restriction site was added to the 5' end of SEQ ID NO. 5, and a BamHI restriction site was added to the 3' end.

The corresponding protein sequence is shown in SEQ ID NO.

2. Construction of recombinant expression strains pET9a-GCSF-5/BL21(DE3) and pET9a-GCSF-6/BL21(DE3)

Recombinant protoplasts (pUC57-GCSF-5, pUC57-GCSF-6) and expression vector pET9a were digested with NdeI and BamHI endonucleases (Fermentas), respectively. The target gene fragment (GCSF-5, GCSF-6) and the expression vector pET9a were subjected to gel recovery using a rapid gel recovery kit (Promega), and the target gene fragment (GCSF-5) was cloned with T4 ligase (New England Biolabs). , GCSF-6) and the expression vector pET9a recovered fragments for DNA ligation. Finally, the ligation product was transformed into E. coli BL21 (DE3) competent cells, and LB plates containing a final concentration of 50 μg/ml Kana were applied to screen for recombinants.

The positive selection strains were picked, and the plastids were extracted and verified by sequencing by Nanjing Kingsray Company to obtain pET9a-GCSF-5/BL21 (DE3) and pET9a-GCSF-6/BL21 (DE3) expression strains.

3. Shake of recombinant expression strains pET9a-GCSF-1/BL21(DE3), pET9a-GCSF-2/BL21(DE3), pET9a-GCSF-5/BL21(DE3), pET9a-GCSF-6/BL21(DE3) Bottle induced expression

The positive clones verified by sequencing were inoculated into a 12 ml culture tube containing 2 ml of LB (50 μg/ml Kana) medium, and shake cultured at 225 rpm on a 37 ° C constant temperature shaker to an OD ₆₀₀ = 4 (16-18 hours). Transfer to 50 ml LB (50 μg / ml Kana) medium, shake culture at 220 ° C for about 1.5 h at 37 ° C to OD ₆₀₀ = 0.8. Add about 1 mM IPTG to the culture medium, and incubate for 4-5 hours at 37 ° C with a constant temperature shaker at 220 rpm. After the expression is over, set the centrifugation force to 5000 g for 10 minutes to take the precipitate, and analyze by SDS-PAGE and quantify according to the method shown in Example 3. The results are shown in the table below:

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Claims (20)

A method for preparing a recombinant human granulocyte colony stimulating factor, wherein the protein sequence of the recombinant human granulocyte colony stimulating factor is SEQ ID NO. 3, the preparation method comprises the step of synthesizing a cDNA sequence which is mutated from a human cDNA Also included are the steps of constructing a recombinant expression vector and the step of inducing expression of the recombinant expression vector in a host cell. The preparation method according to the first aspect of the invention, wherein the synthesis of the cDNA sequence is selected from the group consisting of: codon preference, GC content, CpG dinucleotide content, mRNA secondary structure, splice site, polyA Site, Chi site, ribosome binding site, CpG island, RNA labyrinth, repeat sequence, restriction enzyme cleavage site for interference selection. The preparation method according to claim 1, wherein the sequence of the cDNA is SEQ ID NO. The preparation method according to the above aspect of the invention, wherein the recombinant expression vector is selected from the group consisting of a temperature expression vector and a substrate-induced expression vector. The preparation method according to the fourth aspect of the invention, wherein the recombinant expression vector is a substrate-induced expression vector. The preparation method according to claim 5, wherein the recombinant expression vector is a pET series. The preparation method according to claim 1, wherein the host cell is Escherichia coli. The preparation method according to claim 1, wherein the host cell is Escherichia coli DH5a, BL21 (DE3), Rosetta (DE3), Origami (DE3), JM109, and HSM174 (DE3). A cDNA sequence encoding a recombinant human granulocyte colony stimulating factor, which is SEQ ID NO. A recombinant expression vector for recombinant human granulocyte colony stimulating factor gene, which comprises the cDNA sequence of claim 9 of the patent application. The recombinant expression vector of claim 10, wherein the expression vector is selected from the group consisting of a temperature expression vector and a substrate induction vector. The recombinant expression vector of claim 11, wherein the expression vector is a substrate-induced expression vector. The recombinant expression vector of claim 12, wherein the expression vector is a pET series. A host cell which expresses a recombinant human granulocyte colony-stimulating factor, which comprises the expression vector of any one of claims 10 to 13. The host cell of claim 14, wherein the host cell is Escherichia coli. The host cell of claim 15, wherein the host cell is Escherichia coli DH5a, BL21 (DE3), Rosetta (DE3), Origami (DE3), JM109, HSM174 (DE3). A method for preparing a water-soluble polymer-modified conjugate of a recombinant human granulocyte colony-stimulating factor, the method comprising the method for preparing a recombinant human granulocyte colony stimulating factor according to any one of claims 1 to 8 Also included is a step of coupling a recombinant human granulocyte colony stimulating factor to a water soluble polymer. A water-soluble polymer-modified conjugate of recombinant human granulocyte colony-stimulating factor produced by the preparation method of claim 17, wherein the water-soluble polymer is polyethylene glycol. A water-soluble polymer modified conjugate of the recombinant human granulocyte colony stimulating factor according to claim 18, which has the structure shown in Formula I m is selected from an integer of 50 to 2500, and G is a protein sequence represented by SEQ ID NO. A water-soluble polymer modified conjugate of recombinant human granulocyte colony stimulating factor according to claim 19, wherein m is selected from an integer of from 400 to 500.