WO2017181920A1 - 一种重组人粒细胞集落刺激因子的制备方法 - Google Patents

一种重组人粒细胞集落刺激因子的制备方法 Download PDF

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WO2017181920A1
WO2017181920A1 PCT/CN2017/080737 CN2017080737W WO2017181920A1 WO 2017181920 A1 WO2017181920 A1 WO 2017181920A1 CN 2017080737 W CN2017080737 W CN 2017080737W WO 2017181920 A1 WO2017181920 A1 WO 2017181920A1
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stimulating factor
granulocyte colony
human granulocyte
expression vector
recombinant human
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French (fr)
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喻馗
王宏伟
田静
曾翔
孔祥林
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江苏恒瑞医药股份有限公司
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Priority to CN201780004772.4A priority Critical patent/CN108368512B/zh
Publication of WO2017181920A1 publication Critical patent/WO2017181920A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • 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 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.
  • various types of cells in the body eg, fibroblasts, endothelial cells
  • T cells 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.
  • human granulocyte colony-stimulating factor is mainly used for leukopenia caused by cancer, chemotherapy and other reasons, and is an important auxiliary drug in the process of tumor release and chemotherapy.
  • human granulocyte colony-stimulating factor cDNA was successfully cloned.
  • 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 of 207 and 204 amino acids, each with a 30 amino acid leader sequence.
  • the mature protein molecules are 177 and 174 amino acids, respectively.
  • the former is at the N-position of the mature molecule. Except for the insertion of 3 amino acids, the remaining sequences are 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.
  • human granulocyte colony stimulating factor is recombinantly expressed using mammalian cells.
  • 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.
  • 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.
  • 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, Optimization of culture medium, optimization of culture conditions and other means to achieve high expression of engineering bacteria; (3) optimization of renaturation conditions, change the purification process.
  • 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.
  • 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.
  • the Rosetta host strain derived from BL21 can be used to enhance the expression of eukaryotic proteins with rare codons of E. coli.
  • 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 present invention.
  • the method can significantly increase the expression level of the target protein, ie, recombinant human granulocyte stimulating factor, and solve the problem of rare codons appearing directly in the prokaryotic expression system using human cDNA sequences.
  • the invention provides a preparation method of recombinant human granulocyte colony-stimulating factor, and the protein sequence of the recombinant human granulocyte colony-stimulating factor is shown in SEQ ID NO. 3, and the human gene corresponding to the recombinant human granulocyte colony-stimulating factor corresponding to the protein sequence
  • the sequence is shown in SEQ ID NO. 1, which is characterized by comprising the step of synthesizing a cDNA sequence which is mutated from a human cDNA sequence, a construction step of the recombinant expression vector, and a recombinant expression vector for inducing expression in a host cell. A step of.
  • 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. , CpG islands, RNA labyrinths, repeats, and restriction enzyme sites that may interfere with cloning, optimize recombinant human granulocyte colony-stimulating factor gene sequences.
  • the cleavage sites required for cloning and recombination are introduced at both ends, respectively.
  • 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 improved (see Figures 2A and 2B), and the GC content was also improved ( See Figure 3A and Figure 3B).
  • the method for preparing the recombinant human granulocyte colony stimulating factor provided by the present invention further comprises the step of constructing the recombinant expression vector.
  • the recombinant expression vector involved in the methods provided herein is selected from the group consisting of a temperature expression vector (e.g., pBV220) and a substrate-inducing vector (e.g., pET9a), preferably a substrate-inducible expression vector, most preferably the pET series.
  • a temperature expression vector e.g., pBV220
  • a substrate-inducing vector e.g., pET9a
  • a substrate-inducible expression vector most preferably the 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).
  • the method provided by the present invention includes the following steps:
  • the synthesized cDNA sequence is ligated to an expression vector to construct a recombinant expression vector.
  • 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 (e.g., pBV220) and a substrate-inducing vector (e.g., pET9a), preferably a substrate-inducible expression vector, most preferably the pET series.
  • a temperature expression vector e.g., pBV220
  • a substrate-inducing vector e.g., pET9a
  • a substrate-inducible expression vector most preferably 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 a recombinant human granulocyte colony as represented by the protein sequence SEQ ID NO. After the stimulating factor, the method further comprises the step of coupling the recombinant human granulocyte colony stimulating factor with the water-soluble polymer, and the specific operation method can refer to the patent CN101172161B.
  • the present invention also provides a water-soluble polymer modified conjugate of recombinant human granulocyte colony-stimulating factor, the water-soluble polymer being selected from the group consisting of polyethylene glycol, polypropylene glycol, polylactic acid, etc., preferably polyethylene glycol
  • the molecular weight of the alcohol, polyethylene glycol is selected from 2KD to 100KD, preferably from 5KD to 100KD.
  • n is selected from an integer of from 50 to 2500, preferably from an integer of from 400 to 500, and G is a protein sequence of the sequence of SEQ ID NO.
  • Figure 3A and Figure 3B Comparison of the sequence content of SEQ ID NO. 1 ( Figure 3A) with the sequence SEQ ID NO. 2 ( Figure 3B).
  • Figure 4 Shake flask-induced expression of recombinant expression strains, lane 1: pET9a-GCSF-1/BL21 (DE3), lane 2: pET9a-GCSF-2/BL21 (DE3), lane 3: pET9a-GCSF-2/ BL21 (DE3), Lane 4: pBV220-GCSF-3/DH5a, Lane 5: pBV220-GCSF-4/DH5a, Lane 6: pBV220-GCSF-4/DH5a, Lane 7: Empty Carrier Sample, Lane 8: Empty Bacterial sample, lane M: Protein Marker.
  • Figure 5 Recombinant expression strain 5L fermentor induced expression yield assay, wherein 1: fermentation sample was diluted 8 times, 2: fermentation sample was diluted 16 times.
  • Example 1 Construction of recombinant human granulocyte colony-stimulating factor substrate-inducible expression strain
  • 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-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.
  • 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 recombinant cloning plasmid (pUC57-GCSF-1, pUC57-GCSF-2) and the expression vector pET9a were digested with NdeI and BamHI endonuclease (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.
  • the ligation product was transformed into E. coli BL21 (DE3) competent cells, and LB plates containing a final concentration of 50 ⁇ g/ml Kana were plated to screen for recombinants.
  • the 5L scale fermentation is carried out in the following 4 steps:
  • glycerol bacteria 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 the bacteria.
  • the OD 600 is about 2.5.
  • 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 O11g/L, KH 2 PO 4 2.7g/L, sterilized at 121 ° C for 30 min.
  • the culture temperature was set to At 37 ° C, the aeration rate was maintained at 6 L/min, the pH was controlled at about 7.0 with ammonia water, and the solution was cultured at 37 ° C.
  • the dissolved oxygen was controlled by stirring and feeding at 30% to 110%.
  • 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
  • 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 is shown below, in SEQ ID An EcoRI restriction site was added to the 5' end of the NO.2, and a BamHI restriction site was added to the 3' end.
  • the recombinant cloning plasmid (pUC57-GCSF-3, pUC57-GCSF-4) and the expression vector pBV220 were digested with EcoRI and BamHI endonuclease (Fermentas), respectively.
  • 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.
  • the ligation product was transformed into E. coli DH5a competent cells, and LB plates containing a final concentration of 100 ⁇ g/ml Amp were applied to screen for recombinants.
  • the 5L scale fermentation is carried out in the following 4 steps:
  • 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 O11g/L, KH 2 PO 4 2.7g/L, sterilized at 121 ° C for 30 min.
  • the culture temperature was set to At 30 ° C, the aeration rate was maintained at 6 L/min, the pH was controlled at about 7.0 with ammonia water, and the solution was cultured at 30 ° C.
  • the dissolved oxygen was controlled by stirring and feeding at 30% to 110%.
  • 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
  • Example 4 Screening of recombinant human granulocyte colony-stimulating factor cDNA sequence
  • the recombinant human granulocyte colony stimulating factor gene sequence was synthesized by Nanjing Kingsray Biotech Co., Ltd.
  • 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.
  • 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 recombinant cloning plasmid (pUC57-GCSF-5, pUC57-GCSF-6) and the expression vector pET9a were digested with NdeI and BamHI endonuclease (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.
  • the ligation product was transformed into E. coli BL21 (DE3) competent cells, and LB plates containing a final concentration of 50 ⁇ g/ml Kana were applied to screen for recombinants.

Abstract

提供了一种重组人粒细胞集落刺激因子的制备方法，其中采用的cDNA是在人源序列基础上根据大肠杆菌密码子偏好性修改而得到的，其在大肠杆菌工程菌中表达量高。

Description

一种重组人粒细胞集落刺激因子的制备方法 技术领域
本发明涉及一种重组人粒细胞集落刺激因子的制备方法，属于生物化学与分子生物学领域。
背景技术
人粒细胞集落刺激因子(hGCSF)是一种对刺激造血细胞包括成熟中性粒细胞，巨噬细胞和树状细胞的生长、分化和存活起重要作用的细胞因子。当受到微生物或者炎症细胞因子的刺激时，体内各种类型的细胞(例如：成纤维细胞，内皮细胞)都会产生人粒细胞集落刺激因子，人粒细胞集落刺激因子的活化在先天免疫应答中起重要作用。当受到特殊的抗原刺激时，T细胞也会分泌人粒细胞集落刺激因子。由于对造血细胞的促进作用，人粒细胞集落刺激因子常被用作对免疫低下个体治疗的生物药。hGCSF的活化失控与诸如关节炎和多发性硬化等自发免疫症状相关。自身抗体对hGCSF生物学活性的中和引起另一中自身免疫疾病---特发型肺泡蛋白沉积症，并且hGCSF可以治疗这种症状。目前，人粒细胞集落刺激因子主要应用于癌症放、化疗等原因引起的白细胞减少症，是肿瘤放、化疗过程中重要的辅助药物。
1986年人粒细胞集落刺激因子cDNA克隆成功，人粒细胞集落刺激因子基因全长2.5kb，包括5个外显子和4个内含子，人粒细胞集落刺激因子基因位于17号染色体。人类有两种不同的hGCSFcDNA，分别编码含207和204个氨基酸的前体蛋白，均有30个氨基酸的先导序列，成熟蛋白分子分别为177和174个氨基酸，前者除了在成熟分子N端35位处插入了3个氨基酸外，其余的序列与174氨基酸分子相同。174氨基酸分子的生物学活性比177氨基酸分子的生物学活性高20倍。
通常而言，人粒细胞集落刺激因子使用哺乳动物细胞进行重组表达。但是，为了分离和纯化hGCSF，培养细胞并从培养上清液中分离GCSF蛋白，该方法存在GCSF产量低的缺点，因此不适于大量生产。已知糖基化hGCSF的糖链对于hGCSF的活性而言不是必需的，并且使用哺乳动物细胞产生糖基化hGCSF需要昂贵的材料和设备，因此，这样的方法从经济角度上来说不可行。1991年，第一个通过大肠杆菌表达系统生产的重组人粒细胞集落刺激因子药物Filgrastim(Neupogen，r-metHuGCSF，Amgen Inc)在美国上市，此后国内外有多家公司的大肠杆菌表达系统生产的rhGCSF药物上市。
目前报道的关于提高重组人粒细胞集落刺激因子产量的主要方法有以下几种(1)通过改变表达体系来提高表达量；(2)改变发酵工艺，如高密度发酵、 优化培养基、优化培养条件等手段实现工程菌的高表达；(3)优化复性条件，更改纯化工艺。但是这些方法只是生产工艺方面的优化，并没有在分子水平上提高人粒细胞集落刺激因子的重组表达产量。
如上文所述，糖基化hGCSF的糖链对于hGCSF的活性而言不是必需的，人粒细胞集落刺激因子可使用更加经济和方便的大肠杆菌表达系统进行生产，中国专利CN1156575C、CN1108303A、CN1088107C、CN101591660A等都是直接采用人源cDNA序列在大肠杆菌表达系统中重组表达人粒细胞集落刺激因子，而大肠杆菌表达系统中真核蛋白的表达会出现稀有密码子问题。尽管可以使用从BL21衍生而来的Rosetta宿主菌增强带有大肠杆菌稀有密码子的真核蛋白的表达。但是Rosetta宿主菌的筛选标记为氯霉素抗性，在药物生产中该抗生素的使用具有较大的限制。如何提高重组人粒细胞集落刺激因子的产量，最大限度的降低成本，是个巨大的挑战。
发明内容
本发明提供一种重组人粒细胞集落刺激因子的制备方法，该方法采用不同于重组人粒细胞集落刺激因子人源序列SEQ ID NO.1的cDNA在原核表达系统中表达，通过本发明提供的方法可使目的蛋白即重组人粒细胞刺激因子的表达量显著提高，解决了直接采用人源cDNA序列在原核生物表达体系中出现的稀有密码子问题。
本发明提供了一种重组人粒细胞集落刺激因子的制备方法，重组人粒细胞集落刺激因子的蛋白质序列见SEQ ID NO.3，该蛋白序列对应的重组人粒细胞集落刺激因子的人源基因序列见SEQ ID NO.1，该方法的特征在于包含合成cDNA序列的步骤，所述cDNA由人源cDNA序列突变而成，还包括重组表达载体的构建步骤以及重组表达载体在宿主细胞中诱导表达的步骤。
本发明中合成cDNA序列的步骤，采用全基因合成法，根据大肠杆菌密码子偏好性，同时优化序列GC含量，mRNA二级结构，消除剪接位点，polyA位点，Chi位点和RBS位点，CpG岛，RNA不稳定模体，重复序列以及可能干扰克隆的限制性酶切位点，优化重组人粒细胞集落刺激因子基因序列。为了便于载体构建，在两端分别引入克隆重组所需的的酶切位点。
在一个优选的实施方案中，本发明提供的方法中合成的cDNA序列见SEQ ID NO.2。
本发明提供的重组人粒细胞集落刺激因子的cDNA序列SEQ ID NO.2的合成是将重组人粒细胞集落刺激因子的人源基因序列中的34个碱基替换得到，序列中所有的大肠杆菌低频密码子均被高频密码子所替换。合成的cDNA序列SEQ ID NO.2在大肠杆菌中的密码子适应性增加(见图1A和图1B)，密码子使用频率得到提升(见图2A和图2B)，此外GC含量也有所改善(见图3A和图3B)。
本发明提供的重组人粒细胞集落刺激因子的制备方法还包括重组表达载体的构建的步骤。
本发明提供的方法中涉及的重组表达载体选自温度表达载体(例如pBV220)和底物诱导载体(例如pET9a)，优选底物诱导表达载体，最优选pET系列。
本发明提供的重组人粒细胞集落刺激因子的制备方法还包括重组表达载体在宿主细胞中诱导表达的步骤。
本发明提供的方法中涉及的宿主细胞为大肠杆菌，优选大肠杆菌Escherichia coli DH5a、BL21(DE3)、Rosetta(DE3)、Origami(DE3)、JM109、HSM174(DE3)。
具体来讲，本发明提供的方法包含以下步骤：
(1)采用全基因合成法合成重组人粒细胞集落刺激因子基因；
(2)合成的cDNA序列与表达载体连接，构建重组表达载体。
(3)构建的表达载体在大肠杆菌中进行高效表达。
通过本发明提供的cDNA序列的重组表达产量可以达到人源cDNA的2倍以上。
本发明还提供了一种根据上述全基因合成法合成的可编码重组人粒细胞集落刺激因子的cDNA序列，见SEQ ID NO.2。
本发明还提供了一种重组人粒细胞集落刺激因子基因的重组表达载体，该表达载体含有SEQ ID NO.2。
本发明提供的重组表达载体选自温度表达载体(例如pBV220)和底物诱导载体(例如pET9a)，优选底物诱导表达载体，最优选pET系列。
本发明还提供了一种可表达重组人粒细胞集落刺激因子的宿主细胞，该宿主细胞含有上述的重组表达载体。
本发明提供的宿主细胞为大肠杆菌，优选大肠杆菌Escherichia coli DH5a、BL21(DE3)、Rosetta(DE3)、Origami(DE3)、JM109、HSM174(DE3)。
本发明还提供了一种制备重组人粒细胞集落刺激因子的水溶性聚合物修饰的偶联物的方法，该方法采用前述步骤得到如蛋白质序列SEQ ID NO.3所示的重组人粒细胞集落刺激因子后，进一步包含重组人粒细胞集落刺激因子与水溶性聚合物偶联的步骤，具体操作方法可参考专利CN101172161B。
本发明还提供了一种重组人粒细胞集落刺激因子的水溶性聚合物修饰的偶联物，所述的水溶性聚合物选自聚乙二醇、聚丙二醇、聚乳酸等，优选聚乙二醇，聚乙二醇的分子量选自2KD-100KD，优选5KD-100KD。
本发明提供的重组人粒细胞集落刺激因子的水溶性聚合物修饰物的偶联物结构如式(Ⅰ)所示：
Figure PCTCN2017080737-appb-000001
m选自50-2500的整数，优选自400-500的整数，G为序列SEQ ID NO.3所示的蛋白序列。
附图说明
图1A和图1B：序列SEQ ID NO.1(图1A)与序列SEQ ID NO.2(图1B)密码子适应指数(CAI)对比。
图2A和图2B：序列SEQ ID NO.1(图2A)与序列SEQ ID NO.2(图2B)密码子使用频率(FOP)对比。
图3A和图3B：序列SEQ ID NO.1(图3A)与序列SEQ ID NO.2(图3B)GC含量对比。
图4：重组表达菌株的摇瓶诱导表达，其中泳道1：pET9a-GCSF-1/BL21(DE3)，泳道2：pET9a-GCSF-2/BL21(DE3)，泳道3：pET9a-GCSF-2/BL21(DE3)，泳道4：pBV220-GCSF-3/DH5a，泳道5：pBV220-GCSF-4/DH5a，泳道6：pBV220-GCSF-4/DH5a，泳道7：空载体菌样品，泳道8：空菌样品，泳道M：蛋白标记(Protein Marker)。
图5：重组表达菌株5L发酵罐诱导表达产量测定，其中1：发酵样品稀释8倍，2：发酵样品稀释16倍。
具体实施方式
以下结合实施例用于进一步描述本发明，但这些实施例并非限制本发明的范围。
实施例1:重组人粒细胞集落刺激因子底物诱导表达菌株的构建
1、基因合成
重组人粒细胞集落刺激因子基因序列由南京金斯瑞生物公司合成。
重组人粒细胞集落刺激因子人源序列SEQ ID NO.1：
Figure PCTCN2017080737-appb-000002
Figure PCTCN2017080737-appb-000003
合成的重组人粒细胞集落刺激因子基因序列GCSF-1如下所示，在SEQ ID NO.1 5’端加了一个Nde I酶切位点，3’端添加了一个BamHI酶切位点。
Figure PCTCN2017080737-appb-000004
重组人粒细胞集落刺激因子cDNA序列SEQ ID NO.2：
Figure PCTCN2017080737-appb-000005
合成的重组人粒细胞集落刺激因子基因序列GCSF-2如下所示，在SEQ ID NO.2 5’端加了一个Nde I酶切位点，3’端添加了一个BamHI酶切位点。
Figure PCTCN2017080737-appb-000006
Figure PCTCN2017080737-appb-000007
对应的蛋白序列如SEQ ID NO.3所示
Figure PCTCN2017080737-appb-000008
2、重组表达菌株pET9a-GCSF-1/BL21(DE3)、pET9a-GCSF-2/BL21(DE3)的构建
采用NdeI和BamHI内切酶(Fermentas)分别对重组克隆质粒(pUC57-GCSF-1、pUC57-GCSF-2)和表达载体pET9a进行双酶切消化。采用快速胶回收试剂盒(Promega)对目的基因片段(GCSF-1、GCSF-2)和表达载体pET9a酶切产物进行胶回收，用T4连接酶(New England Biolabs)对目的基因片段(GCSF-1、GCSF-2)和表达载体pET9a回收片段进行DNA连接。最后将连接产物转化大肠杆菌BL21(DE3)感受态细胞，涂布含终浓度50μg/mlKana的LB平板筛选重组子。
挑取阳性克隆，抽提质粒并由南京金斯瑞公司测序验证，得到pET9a-GCSF-1/BL21(DE3)、pET9a-GCSF-2/BL21(DE3)表达菌株。
3、重组表达菌株pET9a-GCSF-1/BL21(DE3)、pET9a-GCSF-2/BL21(DE3)的摇瓶诱导表达
将测序验证的阳性克隆接种至含2mlLB(50μg/mlKana)培养基的12ml菌种培养管中，37℃恒温摇床225rpm振荡培养至OD600＝4左右(16-18小时)。转接入50ml LB(50μg/mlKana)培养基中，37℃恒温摇床220rpm振荡培养约1.5h至OD600＝0.8左右。在培养基中加入约1mM IPTG，37℃恒温摇床220rpm振荡培养4-5小时，表达结束后，设置离心力为5000g离心10分钟取沉淀，SDS-PAGE电泳分析见图4。
4、重组表达菌株pET9a-GCSF-1/BL21(DE3)、pET9a-GCSF-2/BL21(DE3)5L发酵罐诱导表达
5L规模发酵分如下4步进行：
1.取2ml甘油菌接种至装有200mlLB培养基(酵母粉5g/L，大豆蛋白胨10g/L，NaCl5g/L)的1L摇瓶中，37℃恒温摇床220rpm振荡培养约9h，培养 至菌种OD600＝2.5左右。
2.将培养好的种子液接种至5L发酵罐中进行发酵，使用3L LB培养基(其组分为：胰蛋白胨10g/L，酵母粉5g/L，NaCl 5g/L，泡敌0.03％，Na2HPO4·12H2O11g/L，KH2PO4 2.7g/L，121℃灭菌30min。葡萄糖5g/L，MgSO4 0.3g/L，115℃灭菌30min)，培养温度设定为37℃，通气量保持6L/min，用氨水控制pH在7.0左右，37℃培养，通过搅拌和补料控制溶氧在30％～110％。
3.OD600达到25左右时，加入约1mM IPTG，诱导时间4h。
4.产量测定
表1
  菌株 诱导后2h 诱导后4h 诱导后6h
优化前 pET9a-GCSF-1/BL21(DE3) 1.23(g/L) 1.51(g/L) 1.74(g/L)
优化后 pET9a-GCSF-2/BL21(DE3) 2.36(g/L) 3.04(g/L) 3.49(g/L)
由表1中结果可知：采用SEQ ID NO.2基因序列构建的工程菌的目的蛋白的表达量是采用人源的重组人粒细胞集落刺激因子基因序列SEQ ID NO.1构建的工程菌目的蛋白的表达量的2倍以上。
实施例2:重组人粒细胞集落刺激因子温度诱导表达菌株的构建
1.基因合成
重组人粒细胞集落刺激因子基因序列由南京金斯瑞生物公司合成。
合成的重组人粒细胞集落刺激因子基因序列GCSF-3如下所示，在SEQ ID NO.15’端加了一个EcoRI酶切位点，3’端添加了一个BamHI酶切位点。
Figure PCTCN2017080737-appb-000009
合成的重组人粒细胞集落刺激因子基因序列GCSF-4如下所示，在SEQ ID  NO.2 5’端加了一个EcoRI酶切位点，3’端添加了一个BamHI酶切位点。
Figure PCTCN2017080737-appb-000010
对应的蛋白序列如SEQ ID NO.3所示
Figure PCTCN2017080737-appb-000011
2.重组表达菌株pBV220-GCSF-3/DH5a、pBV220-GCSF-4/DH5a的构建
采用EcoRI和BamHI内切酶(Fermentas)分别对重组克隆质粒(pUC57-GCSF-3、pUC57-GCSF-4)和表达载体pBV220进行双酶切消化。采用快速胶回收试剂盒(Promega)对目的基因片段(GCSF-3、GCSF-4)和表达载体pBV220酶切产物进行胶回收，用T4连接酶(New England Biolabs)对目的基因片段(GCSF-3、GCSF-4)和表达载体pBV220回收片段进行DNA连接。最后将连接产物转化大肠杆菌DH5a感受态细胞，涂布含终浓度100μg/mlAmp的LB平板筛选重组子。
挑取阳性克隆，抽提质粒并由南京金斯瑞公司测序验证，得到pBV220-GCSF-3/DH5a、pBV220-GCSF-4/DH5a表达菌株。
3.重组表达菌株pBV220-GCSF-3/DH5a、pBV220-GCSF-4/DH5a的摇瓶诱导表达
将测序验证的阳性克隆接种至含2mlLB(100μg/mlAmp)培养基的12ml菌种培养管中，30℃恒温摇床225rpm振荡培养至OD600＝4左右(16-18小时)。转接入50ml LB(100μg/mlAmp)培养基中，30℃恒温摇床220rpm振荡培养约1.5h至OD600＝0.8左右。升温至42℃恒温摇床220rpm振荡培养4-5小时，表达结束后，设置离心力为5000g离心10分钟取沉淀，SDS-PAGE电泳分析。
4.重组表达菌株pBV220-GCSF-3/DH5a、pBV220-GCSF-3/DH5a 5L发酵罐诱导表 达
5L规模发酵分如下4步进行：
1.取2ml甘油菌接种至装有200mlLB培养基(酵母粉5g/L，大豆蛋白胨10g/L，NaCl 5g/L)的1L摇瓶中，30℃恒温摇床220rpm振荡培养约9h，培养至菌种OD600＝2.5左右。
2.将培养好的种子液接种至5L发酵罐中进行发酵，使用3L LB培养基(其组分为：胰蛋白胨10g/L，酵母粉5g/L，NaCl 5g/L，泡敌0.03％，Na2HPO4·12H2O11g/L，KH2PO4 2.7g/L，121℃灭菌30min。葡萄糖5g/L，MgSO4 0.3g/L，115℃灭菌30min)，培养温度设定为30℃，通气量保持6L/min，用氨水控制pH在7.0左右，30℃培养，通过搅拌和补料控制溶氧在30％～110％。
3.OD600达到25左右时，升温至42℃，诱导时间4h。
4.产量测定
表2
  菌株 诱导后2h 诱导后4h 诱导后6h
优化前 pBV220-GCSF-3/DH5a 0.53(g/L) 0.81(g/L) 1.24(g/L)
优化后 pBV220-GCSF-4/DH5a 1.56(g/L) 2.18(g/L) 2.52(g/L)
由表2中结果可知：采用SEQ ID NO.2基因序列构建的工程菌的目的蛋白的表达量是采用人源的重组人粒细胞集落刺激因子基因序列SEQ ID NO.1构建的工程菌目的蛋白的表达量的2倍以上。
实施例3:重组表达菌株5L发酵罐诱导表达产量的测定
1、取对照品30μl，用纯化水稀释配制0.20g/L对照品溶液，进一步用纯化水稀释成浓度梯度。取1ml混合均匀的发酵液，12000rpm离心5分钟，弃上清，沉淀中加入1ml纯化水混匀。取52μl稀释样品，加入20μl
Figure PCTCN2017080737-appb-000012
LDS Sample Buffer(4×)和8μl
Figure PCTCN2017080737-appb-000013
Reducing Agent(10×)，混匀，70℃水浴10分钟，12000rpm离心1分钟备用。
2、选择
Figure PCTCN2017080737-appb-000014
4-12％Bis-Tris Gel与
Figure PCTCN2017080737-appb-000015
MOPS SDS Running Buffer(20×)与200V恒压电泳约50分钟至溴酚蓝迁移至胶底，停止电泳。电泳完毕，取出胶片，置约100ml纯化水中，摇床振荡5min，重复三次，弃去纯化水。加入约100ml染色液，摇床振荡染色1h直到出现清晰的蛋白条带；弃去染色液，加入约100ml纯化水，摇床振荡脱色过夜至凝胶背景透明。
3、将电泳后的凝胶置凝胶成像仪中，拍照分析见图5。
实施例4:重组人粒细胞集落刺激因子cDNA序列筛选
1、基因合成
重组人粒细胞集落刺激因子基因序列由南京金斯瑞生物公司合成。
重组人粒细胞集落刺激因子cDNA序列SEQ ID NO.4：
Figure PCTCN2017080737-appb-000016
合成的重组人粒细胞集落刺激因子基因序列GCSF-5如下所示，在SEQ ID NO.4 5’端加了一个Nde I酶切位点，3’端添加了一个BamHI酶切位点。
Figure PCTCN2017080737-appb-000017
重组人粒细胞集落刺激因子cDNA序列SEQ ID NO.5：
Figure PCTCN2017080737-appb-000018
合成的重组人粒细胞集落刺激因子基因序列GCSF-6如下所示，在SEQ ID NO.5 5’端加了一个Nde I酶切位点，3’端添加了一个BamHI酶切位点。
Figure PCTCN2017080737-appb-000019
对应的蛋白序列如SEQ ID NO.3所示
2、重组表达菌株pET9a-GCSF-5/BL21(DE3)、pET9a-GCSF-6/BL21(DE3)的构建
采用NdeI和BamHI内切酶(Fermentas)分别对重组克隆质粒(pUC57-GCSF-5、pUC57-GCSF-6)和表达载体pET9a进行双酶切消化。采用快速胶回收试剂盒(Promega)对目的基因片段(GCSF-5、GCSF-6)和表达载体pET9a酶切产物进行胶回收，用T4连接酶(New England Biolabs)对目的基因片段(GCSF-5、GCSF-6)和表达载体pET9a回收片段进行DNA连接。最后将连接产物转化大肠杆菌BL21(DE3)感受态细胞，涂布含终浓度50μg/ml Kana的LB平板筛选重组子。
挑取阳性克隆，抽提质粒并由南京金斯瑞公司测序验证，得到pET9a-GCSF-5/BL21(DE3)、pET9a-GCSF-6/BL21(DE3)表达菌株。
3、重组表达菌株pET9a-GCSF-1/BL21(DE3)、pET9a-GCSF-2/BL21(DE3)、pET9a-GCSF-5/BL21(DE3)、pET9a-GCSF-6/BL21(DE3)的摇瓶诱导表达
将测序验证的阳性克隆接种至含2mlLB(50μg/ml Kana)培养基的12ml菌种培养管中，37℃恒温摇床225rpm振荡培养至OD600＝4左右(16-18小时)。转接入50ml LB(50μg/ml Kana)培养基中，37℃恒温摇床220rpm振荡培养约1.5h至OD600＝0.8左右。在培养基中加入约1mM IPTG，37℃恒温摇床220rpm振荡培养4-5小时，表达结束后，设置离心力为5000g离心10分钟取沉淀，SDS-PAGE电泳分析，按实施例3所示方法进行定量，结果见下表：
表3：
Figure PCTCN2017080737-appb-000020

Claims (13)

  1. 一种重组人粒细胞集落刺激因子的制备方法，所述的重组人粒细胞集落刺激因子的蛋白质序列见SEQ ID NO.3，其特征在于所述的制备方法包括合成cDNA序列的步骤，所述cDNA由是人源cDNA突变而成，还包括重组表达载体的构建步骤以及重组表达载体在宿主细胞中诱导表达的步骤。
  2. 根据权利要求1所述的制备方法，其特征在于所述的cDNA序列的合成依据选自：密码子偏好性、GC含量、CpG二核苷酸含量、mRNA二级结构、剪接位点、polyA位点、Chi位点、核糖体结合位点、CpG岛、RNA不稳定模体、重复序列、干扰克隆的限制性酶切位点。
  3. 根据权利要求1所述的制备方法，其特征在于所述cDNA的序列为SEQ ID NO.2。
  4. 根据权利要求1所述的制备方法，其特征在于所述重组表达载体选自温度表达载体和底物诱导表达载体，优选底物诱导表达载体，最优选pET系列。
  5. 根据权利要求1所述的制备方法，其特征在于所述的宿主细胞为大肠杆菌，优选大肠杆菌Escherichia coliDH5a、BL21(DE3)、Rosetta(DE3)、Origami(DE3)、JM109、HSM174(DE3)。
  6. 一种可编码重组人粒细胞集落刺激因子的cDNA序列，其特征在于所述序列为SEQ ID NO.2。
  7. 一种重组人粒细胞集落刺激因子基因的重组表达载体，其特征在于所述表达载体含有权利6所述的cDNA序列。
  8. 根据权利要求7所述的重组表达载体，其特征在于所述表达载体选自温度表达载体和底物诱导载体，优选底物诱导表达载体，最优选pET系列。
  9. 一种可表达重组人粒细胞集落刺激因子的宿主细胞，所述的宿主细胞含有权利要求7、8任一项所述的表达载体。
  10. 根据权利要求8所述的宿主细胞，其特征在于所述的宿主细胞为大肠杆 菌，优选为大肠杆菌，优选Escherichia coli DH5a、BL21(DE3)、Rosetta(DE3)、Origami(DE3)、JM109、HSM174(DE3)。
  11. 一种重组人粒细胞集落刺激因子的水溶性聚合物修饰的偶联物的制备方法：其特征在于所述方法包括权利要求1-5任一项所述的重组人粒细胞集落刺激因子的制备方法，还包括重组人粒细胞集落刺激因子与水溶性聚合物偶联的步骤。
  12. 一种根据权利要求11所述的制备方法制得的重组人粒细胞集落刺激因子的水溶性聚合物修饰的偶联物，其特征在于所述水溶性聚合物为聚乙二醇。
  13. 一种权利要求12所述的重组人粒细胞集落刺激因子的水溶性聚合物修饰的偶联物，其结构如式Ⅰ所示
    Figure PCTCN2017080737-appb-100001
    m选自50-2500的整数，优选自400-500的整数，G为SEQ ID NO.3所示的蛋白序列。
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