WO2019184372A1 - 高效表达重组人神经生长因子的基因组合 - Google Patents

高效表达重组人神经生长因子的基因组合 Download PDF

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WO2019184372A1
WO2019184372A1 PCT/CN2018/114594 CN2018114594W WO2019184372A1 WO 2019184372 A1 WO2019184372 A1 WO 2019184372A1 CN 2018114594 W CN2018114594 W CN 2018114594W WO 2019184372 A1 WO2019184372 A1 WO 2019184372A1
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gene
rhngf
expression
seq
prongf
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陈海
孙洪亮
张怡
王跃生
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江苏中新医药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a gene combination for efficiently expressing recombinant human nerve growth factor (rhNGF) having natural activity in a eukaryotic expression system.
  • rhNGF human nerve growth factor
  • NGF neurotrophic nutrition and axonal growth, and it plays an important role in the regulation of the development, differentiation, growth, regeneration and functional properties of central and peripheral neurons.
  • NGF is synthesized in vivo in pro- form (proNGF), including "gene B", leader peptide and mature NGF.
  • proNGF pro- form
  • Gene B contributes to the secretion of the protein; a two-part conserved region in the leader peptide is required for proNGF expression, enzymatic hydrolysis to form a biologically active protein, and secretion of mature NGF, and it also contributes to the correct folding of the protein.
  • proNGF contains a potential N-glycosylation site, and glycosylation of the leader peptide in proNGF contributes to its endoplasmic reticulum.
  • ProNGF forms a biologically active mature NGF after hydrolysis of a specific site by furin or prohormone convertase.
  • Mature NGF has 118 amino acids and is a double-stranded dimer structure. There are 6 cysteine residues in the single chain, which can form 3 pairs of intrachain disulfide bonds (Cys 58 -Cys 108 , Cys 68 -Cys 110 , Cys 15 -Cys 80 ). The correct formation of disulfide bonds is NGF has the necessary conditions for activity.
  • mNGF rat nerve growth factor
  • hNGF human nerve growth factor
  • the rhNGF eye drops developed by Domoutheastern of Italy were approved by the European Medicines Agency for the treatment of moderate to severe neurotrophic keratitis on July 6, 2017.
  • the active ingredient rhNGF uses the E. coli expression system as proNGF.
  • Formal expression due to the lack of effective post-translational modification function of the system, is not conducive to the correct folding of mature NGF protein, the expression product is inclusion body, the subsequent process requires in vitro renaturation, protease excision of the leader peptide to obtain the target protein, resulting in complex production process , low yield of products, high production costs and other shortcomings.
  • rhNGF The efficient expression of rhNGF in eukaryotic expression systems is a technical problem that needs to be solved. Because the high expression vector is the main factor for achieving high yield of rhNGF. The expression level of rhNGF can be increased by using appropriate expression control sequences and reasonable structural arrangement during the construction of the expression vector.
  • the object of the present invention is to optimize a gene expression regulatory element of recombinant human nerve growth factor, and to provide a gene combination for efficiently expressing rhNGF.
  • the present inventors have found that the expression level of rhNGF in a eukaryotic expression system can be remarkably improved by selecting an appropriate regulatory element to be ligated to a human nerve growth factor precursor gene (hereinafter also referred to as a proNGF gene).
  • a proNGF gene a human nerve growth factor precursor gene
  • the present invention provides a gene combination for efficiently expressing rhNGF, which is: "gene B - gene A";
  • the gene A which has a nucleotide sequence as shown in SEQ ID No. 7, is referred to as a proNGF gene, and comprises a leader peptide gene and mature hNGF, and an amino acid encoding the sequence of SEQ ID No. 8.
  • the gene B has a nucleotide sequence as shown in SEQ ID No. 3 (Pre) or SEQ ID No. 5 (abbreviated as Luc), which encodes the sequence shown in SEQ ID No. 4 or SEQ ID No. 6. Amino acids.
  • the invention provides a second gene combination for efficiently expressing rhNGF, and further comprises an element having a gene C, and the connection sequence is: "gene C-gene B-gene A";
  • the gene C has a nucleotide sequence as shown in SEQ ID No. 1 (referred to as glo) or SEQ ID No. 2 (abbreviated as aden).
  • the gene combination is constructed into an expression vector.
  • the expression vector is a eukaryotic expression vector and can be introduced into a host cell by transient transfection or stable transfection.
  • the host cell is a mammalian cell.
  • the mammalian cells are Chinese hamster ovary (CHO) cells, human embryonic kidney 293 cells, COS cells or HeLa cells.
  • the present invention performs the following research work:
  • the "gene C” glo (as shown in SEQ ID No. 1) and aden (as shown in SEQ ID No. 2) are respectively constructed into the expression vector of 2 by restriction enzymes to obtain a "gene. C” - "Gene B” - a prokaryotic expression vector of the proNGF gene combination, and the effect of "gene C” on the transient expression of rhNGF was evaluated by the method described in 2 using the vector described above as a control.
  • the cell pool with high yield and good cell growth was selected to be monoclonal by limiting dilution method, and the engineered cell strain with high expression of rhNGF was obtained by screening.
  • the growth curve, cell viability and rhNGF expression level of the engineered cell lines were measured in a bioreactor.
  • the biological activity of rhNGF was determined by TF-1 cell/MTS colorimetry.
  • Figure 1 SfiI digestion of the eukaryotic expression vector p-Pre/Nat/Luc-pro-rhNGF(SfiI)-1 containing the "gene B"-proNGF gene combination, wherein:
  • Figure 2 AvrII, BstZ17I double digestion assay of eukaryotic expression vector p-Pre/Nat/Luc-pro-rhNGF(SfiI)-1 containing "gene B"-proNGF gene combination, wherein:
  • Figure 3 Left and right diagrams are the results of two experiments with the effect of two "gene B"-proNGF gene combinations on the transient expression of rhNGF.
  • Figure 4 Left and right images are the results of two experiments of "gene C” - "gene B” - the effect of proNGF gene combination on the transient expression of rhNGF.
  • Figure 5 Schematic diagram of rhNGF eukaryotic expression vector containing "gene C” - "gene B” - proNGF gene combination;
  • Figure 6 SDS-PAGE analysis of Capto-S purified from 6 cell batch culture supernatants.
  • Figure 7 Trends in growth curve, cell viability, and rhNGF expression levels of engineered cell lines cultured in bioreactors.
  • Figure 8 RhNGF-stimulated TF-1 cell proliferation activity curve as determined by TF-1 cell/MTS colorimetry.
  • SEQ ID NO. 1 The nucleotide sequence of "Gene C" glo.
  • SEQ ID NO. 2 Nucleotide sequence of "Gene C” aden.
  • SEQ ID NO. 3 Nucleotide sequence of "Gene B” Pre.
  • SEQ ID NO. 4 Amino acid sequence of "Gene B” Pre.
  • SEQ ID NO. 5 Nucleotide sequence of "gene B” Luc.
  • SEQ ID NO. 6 Amino acid sequence of "gene B" Luc.
  • SEQ ID NO. 7 Nucleotide sequence of proNGF.
  • SEQ ID NO. 8 Amino acid sequence of proNGF.
  • SEQ ID NO. 9 Nucleotide sequence of "Gene B” Nat.
  • SEQ ID NO. 10 Amino acid sequence of "gene B” Nat.
  • the amino acid sequence of rhNGF was searched from the protein sequence database UniProtKB to obtain the sequence of ID P01138.
  • the amino acid sequence of proNGF consisted of a 103 amino acid leader peptide and a 120 amino acid mature hNGF, and the C-terminal two amino acids (RA) to NGF.
  • the biological activity has no effect, and the natural NGF protein also deletes the two amino acids, so the proNGF amino acid sequence of the present invention consists of a 103 amino acid leader peptide and 118 amino acid hNGF, as shown in SEQ ID NO. Sre Biotechnology Co., Ltd. reverse-translated the proNGF amino acid sequence into a DNA sequence as shown in SEQ ID NO. 7 and synthesized according to the characteristics of CHO cell expression.
  • CHO cells can be used as host cells, eukaryotic expression vector contains two insertion points, can express two genes at the same time, can also be removed as needed
  • the second expression unit constructs a single gene expression vector containing a dihydrofolate reductase selection marker and a puromycin resistance gene, which can be used for simultaneous pressure screening of MTX and puromycin to improve the quality of the screening.
  • the “gene B” Pre and “gene B” Luc were selected to direct the secretion expression of rhNGF, which was compared with the natural "gene B” Nat of hNGF.
  • Gene B the gene sequence is shown in SEQ ID NO. 3, synthesized together with the proNGF gene sequence by Kingsray Biotechnology Co., Ltd., the sequence is referred to as Pre-pro-rhNGF, and the 5' and 3' ends are respectively added with AvrII.
  • the BstZ17I restriction site was constructed on plasmid pUC57 to form plasmid pUC57-Pre-pro-rhNGF.
  • the gene Pre-pro-rhNGF was obtained by restriction endonuclease AvrII and BstZ17I double-digested plasmid pUC57-Pre-pro-rhNGF, and the Pre-pro-rhNGF gene fragment of about 770 bp was obtained by double digestion, and the gel recovery kit was used. After purification, it is ready for use.
  • "Gene B" Luc, Nat the gene sequence is shown in SEQ ID NO. 5, SEQ ID NO. 9, and is added to the 5' end of the proNGF gene by PCR using a primer using pUC57-Pre-pro-rhNGF as a template.
  • Luc-pro-rhNGF and Nat-pro-rhNGF Two gene fragments of about 740 bp, referred to as Luc-pro-rhNGF and Nat-pro-rhNGF, were obtained, and the PCR product was purified by a common DNA product purification kit, and the concentration was measured by an ultraviolet spectrophotometer.
  • the purified gene Luc-pro-rhNGF and Nat-pro-rhNGF were digested by restriction endonucleases AvrII and BstZ17I, and purified for use.
  • the restriction vector was digested with restriction endonucleas AvrII and BstZ17I, and the digested product was purified by DNA.
  • the kit was purified.
  • the gene trans-pro-rhNGF, Luc-pro-rhNGF and Nat-pro-rhNGF digested with AvrII and BstZ17I were ligated to the same double-digested expression vector by T4 DNA ligase, and the linker was chemically transformed into Top. 10 competent cells.
  • the single colony grown by transformation was screened for positive clone by PCR. If the target gene was successfully ligated to the expression vector, the PCR product size was about 1000 bp; the empty vector PCR product was about 260 bp.
  • the positive clones p-Pre-pro-rhNGF-8, p-Nat-pro-rhNGF-1, and p-Luc-pro-rhNGF-5 were selected for sequencing, and the three vectors were analyzed by sequence alignment.
  • the B"-proNGF gene combination sequence is identical to the theoretical sequence.
  • the second expression unit (CMV/EF1 expression cassette) in the eukaryotic expression vector should be deleted, and the first expression unit is retained, and the vector is transformed into a vector expressing only one unit.
  • the correct vectors p-Pre-pro-rhNGF-8, p-Nat-pro-rhNGF-1, p-Luc-pro-rhNGF-5 were sequenced using restriction endonuclease SfiI.
  • the digested product was purified by DNA product purification kit, and the SfiI-digested vector was self-ligated by T4 DNA ligase, and the linker was chemically transformed into Top 10 competent cells, and the grown single colony was passed through the bacterial PCR. Positive clones were screened, and the vector PCR product of the CMV/EF1 expression cassette was successfully removed without banding, otherwise the PCR product was a 535 bp band.
  • the single-colon extraction plasmid p-Pre-pro-rhNGF(SfiI)-1, p-Nat-pro-rhNGF(SfiI)-1, p-Luc- was selected.
  • Pro-rhNGF(SfiI)-1 was detected by SfiI single digestion and AvrII and BstZ17I double digestion.
  • the positive clones were cut without SfiI single-stranded, as shown in Figure 1.
  • AvrII and BstZ17I were digested.
  • a strip of about 740 bp in size is shown in Figure 2.
  • the medium was FortiCHO, supplemented with 8 mM glutamine as complete medium. Culture conditions: orbital shaker (orbital diameter 2.5 cm), rotation speed 130 rpm, carbon dioxide concentration 8%, temperature 37 °C. CHO cells were passaged at 1.5-2.5 x 10 6 /mL, and the density after passage was 3-5 ⁇ 10 5 /mL.
  • Two reagents were added in a 1.5 mL microcentrifuge tube at a ratio of 1.67 ⁇ L of FreeStyle Max transfection reagent per 10 6 cells and 48.33 ⁇ L of optiPRO SFM, and gently mixed. Immediately mix the diluted Max solution with the DNA solution and leave it at room temperature for 10 minutes, up to 20 minutes. The DNA:MAX mixed solution was added dropwise to the cell suspension and immediately placed in a shaker culture.
  • the DNA sequences of "gene C" glo and aden were synthesized by Kingsray Biotechnology Co., Ltd. and constructed on the same plasmid pUC57, which are 150 bp and 296 bp in size respectively.
  • the ends of the sequence contain AvrII restriction sites, and the corresponding plasmids are called pUC57-glo-aden.
  • Glo and aden were obtained by restriction endonuclease AvrII single digestion of plasmid pUC57-glo-aden, purified by gel recovery kit and used.
  • Gene C was added to the vector p-Pre-pro-rhNGF (SfiI)-1, which has a higher level of transient expression of rhNGF as described in 3.
  • the restriction enzyme was digested with restriction endonuclease AvrII, and the digested product was purified by a common DNA product purification kit, and the "gene C” fragment glo and aden which were digested with AvrII were ligated to the vector p-Pre by T4 DNA ligase.
  • the linker was chemically transformed into Top 10 competent cells.
  • the single colonies grown by transformation were subjected to PCR screening to obtain positive clones, which were named p-glo-Pre-pro-rhNGF (SfiI) and p-aden-Pre-pro-rhNGF (SfiI), respectively.
  • the recombinant gene was sequenced, and the "gene C" glo and aden sequences were identical to the designed sequence by sequence alignment analysis, thereby obtaining a eukaryotic expression vector containing the "gene C"-"gene B"-proNGF gene combination.
  • Linearized p-glo-Pre-pro-rhNGF (SfiI) and p-aden-Pre-pro-rhNGF (SfiI) expression vectors were transiently transfected into CHO cells, cultured for 48 hours, samples were taken, and rhNGF in supernatant was detected by ELISA. The content was evaluated for expression efficiency and was performed twice in an independent experiment as shown in FIG.
  • the cells were divided into two portions. One portion was added with 10 ⁇ g/mL puromycin and 100 nM MTX; the other portion was added with 20 ⁇ g/mL puromycin and 200 nM MTX. When the cell viability was restored to 85% or more, each cell was further divided into two portions, and 30 ⁇ g/mL puromycin/500 nM MTX and 50 ⁇ g/mL puromycin/1000 nM MTX were added, respectively, and the screening was continued. The criterion for screening end is that the cell viability is greater than 90%. A total of 6 cell pools were obtained from the two rounds of screening. After analyzing the specific yield, a cell pool with a higher specific yield and good cell growth was selected for the monoclonal.
  • the cloning medium was supplemented with 6 mM glutamine for FortiCHO. Dilute the cells to be cloned to 2-5 cells/mL. The cell suspension was added to a 96-well plate with an 8-channel pipette at 200 ⁇ L per well. The cells were placed in a carbon dioxide incubator and cultured at 37 ° C under 5% carbon dioxide. According to the speed of cloning, after 11-14 days of culture, 20 ⁇ L of the monoclonal-producing wells were used to analyze the concentration of rhNGF by ELISA.
  • the high-expression clones were transferred from 96-well plates to 48-well plates, 200 ⁇ L of fresh medium was added, MTX and puromycin were added to screen the drug to the concentration of the monoclonal pre-cell pool, and after passage, the cells were passaged to a 12-well plate, etc.
  • the cells in the 12-well plate were basically subcultured. The cells were removed into a centrifuge tube, and the supernatant was removed by centrifugation. The cells were washed once with PBS, resuspended in 1 mL of fresh medium, and added to a 6-well plate to obtain 30 ⁇ L of the cell suspension.
  • a 6-well plate was placed in an incubator for 2-4 hours, 100 ⁇ L of the culture solution was taken out, and the supernatant was centrifuged, and 1 mL of fresh medium and a screening drug were added to each well of a 6-well plate to continue the culture.
  • the culture supernatant was analyzed for rhNGF concentration by ELISA.
  • the specific yield of the cells was calculated using the formula, and the specific yield was equal to rhNGF concentration/cell density/incubation time.
  • the clones were subjected to a second round of screening according to specific yield.
  • the cells obtained by the screening were subjected to the passaging stability test, and 6 cells which performed well in the stability test were selected for batch culture, and the rhNGF in the batch culture supernatant was preliminarily purified by Capto S column, and subjected to SDS-PAGE.
  • the results are shown in Fig. 6. It indicates that the content of proNGF protein in rhNGF expressed by 13C5 cells is low in batch culture. Since proNGF protein is a product-related impurity, the lower the content, the better, thus selecting 13C5 cells as engineering cells.
  • Strain is the content of proNGF protein in rhNGF expressed by 13C5 cells is low in batch culture. Since proNGF protein is a product-related impurity, the lower the content, the better, thus selecting 13C5 cells as engineering cells.
  • the growth curve, cell viability and rhNGF expression level of engineering cell lines were determined in the bioreactor of Example 3.
  • the fed-batch (fed) culture was used as the production culture mode of the engineering cell line, and the culture scale was enlarged from 30 mL of the shake flask to 2.5 L, and further enlarged to a 28 L-scale mechanically stirred bioreactor capable of in-situ sterilization, stirring paddle It is a single inclined blade paddle, and the working stirring speed is 125 rpm, and the bubble is ventilated.
  • the dissolved oxygen control first controls the air flow, and after reaching the maximum gas flow set value, the oxygen is introduced, the air flow is simultaneously reduced, and the total flow rate is kept constant.
  • the pH is maintained at 7.2 by controlling the CO 2 flow rate at the beginning of the culture. As the cell grows, the pH will decrease first, then rebound and rise.
  • the cell enters the plateau from the exponential growth phase on the fifth day, and the viable cell density remains stable for the remaining 6-10 days.
  • the highest cell density is 1.2 ⁇ 10 7 /mL, and the cell viability is maintained at More than 90%, the concentration of rhNGF continued to increase rapidly during the culture, and reached 78 mg/L at the end of the culture.
  • the TF-1 cell/MTS colorimetric method is a classical method for the determination of the biological activity of nerve growth factor, which is included in the three editions of the Chinese Pharmacopoeia 2015.
  • the biological activity of rhNGF is determined by this method, and the international standard product (item number) :93/556, NIBSC), mNGF for injection (trade name: Su Peptide, Shutai Shen (Beijing) Biopharmaceutical Co., Ltd.) for comparison.
  • TF-1 cells Human erythrocyte leukemia cells (TF-1 cells, domesticated NGF-dependent, sourced from the Chinese Food and Drug Administration Recombinant Protein Chamber) with good growth medium (1640+10%FBS+1%P/S)
  • the cells were inserted into a 96-well plate at a volume of 5000 cells per well at a volume of 100 ⁇ L per well; then 100 ⁇ L of the NGF to be tested in a 3-fold gradient of the basal medium was added to each well (rhNGF, international standard (Std), threonide).
  • rhNGF is equivalent to the international standard (Std) activity in stimulating the proliferation activity of TF-1 cells (EC 50 is 5.30 ng/mL, 5.26 ng/mL, respectively), and stronger than Supisheng (Sutaisheng). Activity (EC 50 is 14.82 ng/mL).

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Abstract

提供一种高效表达重组人神经生长因子（rhNGF）的基因组合，以及该基因组合在制备rhNGF中的应用。

Description

高效表达重组人神经生长因子的基因组合 技术领域：
本发明涉及生物技术领域，特别涉及一种在真核表达系统中高效表达具有天然活性的重组人神经生长因子(rhNGF)的基因组合。
背景技术：
NGF具有神经元营养和促轴突生长双重生物学功能，它对中枢及周围神经元的发育、分化、生长、再生和功能特性的表达均具有重要的调控作用。
NGF在体内以前体形式(proNGF)合成，包括“基因B”、前导肽和成熟NGF。“基因B”有助于蛋白的分泌；前导肽中有两部分保守区域为proNGF表达、酶解形成具有生物活性的蛋白以及成熟NGF的分泌所必需，且其还有助于蛋白的正确折叠。proNGF包含潜在的N糖基化位点，proNGF中前导肽的糖基化有助于其出内质网。proNGF经弗林蛋白酶或激素酶原转化酶在特定部位水解后形成具有生物学活性的成熟NGF。成熟NGF有118个氨基酸，呈双链二聚体结构。单链内共有6个半胱氨酸残基，可对应形成3对链内二硫键(Cys 58-Cys 108，Cys 68-Cys 110，Cys 15-Cys 80)，二硫键的正确形成是NGF具有活性的必要条件。
我国是世界上第一个也是唯一一个批准注射用mNGF(鼠神经生长因子)上市的国家。mNGF与人神经生长因子(hNGF)在蛋白质氨基酸序列上有10％差异，作为异源蛋白质长期使用有潜在的安全性问题，且存在鼠源病毒污染风险。
20世纪90年代起，国外多家制药公司和研发机构相继开始研究重组人神经生长因子，但迄今尚未有开发成功的rhNGF产品上市。其主要原因是NGF表达水平低、表达产物生物学活性差以及hNGF与mNGF的同源性问题。
例如，意大利Dompé公司研发的rhNGF滴眼液于2017年7月6日获欧洲药品管理局批准用于中度至重度神经营养性角膜炎的治疗，其中的活性成分rhNGF采用大肠杆菌表达系统以proNGF形式进行表达，由于该系统缺乏有效的翻译后修饰功能，不利于成熟NGF蛋白的正确折叠，表达产物为包涵体，后续工艺需要体外复性、蛋白酶切除前导肽才能获得目标蛋白，导致生产工艺复杂、产物得率低、生产成本高等缺点。
在真核表达系统中高效表达rhNGF是目前需要解决的技术难题。因为高效表达载体是 实现rhNGF高产率的主要因素。在表达载体构建过程中采用合适的表达调控序列和合理的结构排列，可以提高rhNGF的表达水平。
发明内容
本发明的目的是优化重组人神经生长因子的基因表达调控元件，提供高效表达rhNGF的基因组合。
本发明人发现，通过选择适当的调控元件与人神经生长因子前体基因(以下亦简称为proNGF基因)进行连接，可以显著提高在真核表达系统中rhNGF的表达水平。
本发明提供了一种高效表达rhNGF的基因组合，所述组合为：“基因B—基因A”；
所述基因A，其核苷酸序列如SEQ ID No.7所示，称为proNGF基因，包含前导肽基因和成熟hNGF，编码SEQ ID No.8所示序列的氨基酸；
所述基因B，其核苷酸序列如SEQ ID No.3(简称Pre)，或SEQ ID No.5(简称Luc)所示，它们编码SEQ ID No.4或SEQ ID No.6所示序列的氨基酸。
本发明提供了第二种高效表达rhNGF的基因组合，还包括有基因C的元件，连接顺序为：“基因C—基因B—基因A”；
所述基因C，其核苷酸序列如SEQ ID No.1(简称glo)，或SEQ ID No.2(简称aden)所示。
经实验证实，本发明提供的基因组合可以提高rhNGF的分泌效率，进而提高rhNGF的表达水平。
所述基因组合构建至表达载体。
所述表达载体为真核表达载体，可以通过瞬时转染或稳定转染的方式导入宿主细胞。
所述宿主细胞为哺乳动物细胞。所述哺乳动物细胞为中国仓鼠卵巢(CHO)细胞、人胚肾293细胞、COS细胞或Hela细胞。
具体地，本发明进行了如下研究工作：
1、从蛋白质序列数据库UniProtKB中查询hNGF的氨基酸序列，获得ID为P01138的proNGF序列，如SEQ ID No.8所示，由金斯瑞生物科技有限公司根据CHO细胞表达的特点将proNGF氨基酸序列逆翻译为DNA序列并合成，如SEQ ID No.7所示。
2、在proNGF的5’端分别添加“基因B”Pre、Luc，如SEQ ID No.3、SEQ ID No.5所示，获得不同“基因B”—proNGF基因组合，将其插入到真核表达载体中，通过瞬时转染导入CHO细胞，经培养后离心取上清，利用ELISA测定上清中rhNGF的含量，以proNGF的天然“基因B”为对照，基因序列如SEQ ID No.9(简称Nat)所示，编码的氨基酸序 列如SEQ ID No.10所示，比较不同“基因B”引导下的rhNGF表达水平。
3、利用限制性内切酶将“基因C”glo(如SEQ ID No.1所示)、aden(如SEQ ID No.2所示)分别构建至2所述表达载体中，获得含有“基因C”—“基因B”—proNGF基因组合的真核表达载体，以2所述载体为对照，通过2所述方法评估“基因C”对rhNGF瞬时表达的影响。
4、将3所述表达载体转染CHO细胞中，同时加入嘌呤霉素(puromycin)和甲氨蝶呤(MTX)进行两轮加压筛选以获得细胞池。
5、选择比产率高、细胞生长良好的细胞池利用有限稀释法进行单克隆，筛选获得高效表达rhNGF的工程细胞株。
6、生物反应器中测定工程细胞株的生长曲线、细胞活率及rhNGF表达水平的变化趋势。
7、采用TF-1细胞/MTS比色法测定rhNGF的生物学活性。
经实验证实，本发明提供的“基因B—proNGF”基因组合，相较于NGF天然基因组合，在真核表达系统中，rhNGF瞬时表达水平显著性提高(见实施例1)；
在所述“基因B—proNGF基因”组合5’端添加“基因C”可以进一步显著性提高rhNGF的瞬时表达水平(见实施例1)；基于所述“基因C—基因B—proNGF”基因组合构建的工程细胞株在生物反应器中培养，上清中rhNGF的表达量达78mg/L(见实施例2、3)，生物学活性与国际标准品相当，强于注射用mNGF(见实施例4)。
附图说明
图1：含有“基因B”—proNGF基因组合的真核表达载体p-Pre/Nat/Luc–pro-rhNGF(SfiI)－1的SfiI酶切检测，其中：
M：DL5000 DNA Marker；
1：p-Pre-pro-rhNGF-8，SfiI酶切；
2：p-Pre-pro-rhNGF(SfiI)-1，SfiI酶切；
3：p-Nat-pro-rhNGF(SfiI)-1，SfiI酶切；
4：p-Luc-pro-rhNGF(SfiI)-1，SfiI酶切。
图2：含有“基因B”—proNGF基因组合的真核表达载体p-Pre/Nat/Luc–pro-rhNGF(SfiI)－1的AvrII、BstZ17I双酶切检测，其中：
M：DL5000 DNA Marker；
1：p-Pre-pro-rhNGF(SfiI)-1，AvrII、BstZ17I双酶切；
2：p-Nat-pro-rhNGF(SfiI)-1，AvrII、BstZ17I双酶切；
3：p-Luc-pro-rhNGF(SfiI)-1，AvrII、BstZ17I双酶切。
图3：左右二图分别是两次“基因B”—proNGF基因组合对rhNGF瞬时表达的影响实验的结果。
图4：左右二图分别是两次“基因C”—“基因B”—proNGF基因组合对rhNGF瞬时表达的影响实验的结果。
图5：含“基因C”—“基因B”—proNGF基因组合的rhNGF真核表达载体简图；
“基因C”、“基因B”、前体序列(Pro)和成熟NGF序列(rhNGF)构成完整的重组基因组合。
图6：6株细胞批次培养上清Capto-S纯化后SDS-PAGE分析。
图7：生物反应器中培养的工程细胞株的生长曲线、细胞活率及rhNGF表达水平的变化趋势。
图8：TF-1细胞/MTS比色法测定的rhNGF刺激TF-1细胞增殖活性曲线。
序列表信息
SEQ ID NO.1：“基因C”glo的核苷酸序列。
SEQ ID NO.2：“基因C”aden的核苷酸序列。
SEQ ID NO.3：“基因B”Pre的核苷酸序列。
SEQ ID NO.4：“基因B”Pre的氨基酸序列。
SEQ ID NO.5：“基因B”Luc的核苷酸序列。
SEQ ID NO.6：“基因B”Luc的氨基酸序列。
SEQ ID NO.7：proNGF的核苷酸序列。
SEQ ID NO.8：proNGF的氨基酸序列。
SEQ ID NO.9：“基因B”Nat的核苷酸序列。
SEQ ID NO.10：“基因B”Nat的氨基酸序列。
具体实施方式
以下实施例仅用于举例说明本发明的方法和装置，并不限定本发明的范围。
实施例1表达rhNGF的基因组合的确定
1、proNGF基因的获得
从蛋白质序列数据库UniProtKB中查询rhNGF的氨基酸序列，获得ID为P01138的序列，proNGF的氨基酸序列由103个氨基酸的前导肽和120个氨基酸的成熟hNGF组成，C 末端两个氨基酸(RA)对NGF的生物活性无影响，且天然NGF蛋白也会缺失这两个氨基酸，所以本发明的proNGF氨基酸序列由103个氨基酸的前导肽和118个氨基酸的hNGF组成，如SEQ ID NO.8所示，由金斯瑞生物科技有限公司根据CHO细胞表达的特点将proNGF氨基酸序列优化逆翻译为如SEQ ID NO.7所示的DNA序列并合成。
2、表达系统的选择
选择哺乳动物细胞表达系统进行rhNGF的表达，包含CHO细胞和真核表达载体，CHO细胞可以作为宿主细胞，真核表达载体包含两个插入点，可以同时表达两个基因，也可以根据需要移除第二个表达单元，构建单基因表达载体，该载体含有二氢叶酸还原酶选择标记和嘌呤霉素抗性基因，可用于MTX和puromycin的同时加压筛选，提高筛选的质量。
3、“基因B”元件的筛选
选择“基因B”Pre、“基因B”Luc引导rhNGF的分泌表达，与hNGF的天然“基因B”Nat进行比较。
3.1“基因B”—proNGF基因组合的获得
“基因B”Pre，基因序列如SEQ ID NO.3所示，与proNGF基因序列一起由金斯瑞生物科技有限公司合成，获得序列简称Pre-pro-rhNGF，5’和3’端分别添加AvrII、BstZ17I酶切位点，构建于质粒pUC57上，形成质粒pUC57-Pre-pro-rhNGF。基因Pre-pro-rhNGF通过限制性内切酶AvrII、BstZ17I双酶切质粒pUC57-Pre-pro-rhNGF获得，通过双酶切获得大小770bp左右的Pre-pro-rhNGF基因片段，利用胶回收试剂盒纯化后待用。“基因B”Luc、Nat，基因序列如SEQ ID NO.5、SEQ ID NO.9所示，通过引物利用PCR的方法以pUC57-Pre-pro-rhNGF为模板添加至proNGF基因的5’端，获得2条740bp左右的基因片段，分别简称Luc-pro-rhNGF、Nat-pro-rhNGF，利用普通DNA产物纯化试剂盒纯化PCR产物，紫外分光光度计测定浓度。对纯化后的基因Luc-pro-rhNGF、Nat-pro-rhNGF通过限制性内切酶AvrII、BstZ17I进行双酶切，纯化后待用。
3.2含有“基因B”—proNGF基因组合的真核表达载体的构建
为将含不同“基因B”的proNGF基因构建至真核表达载体的EF2/CMV杂合启动子下游，利用限制性内切酶AvrII、BstZ17I对表达载体进行双酶切，酶切产物经DNA纯化试剂盒进行纯化。
利用T4 DNA连接酶将AvrII、BstZ17I双酶切后的基因Pre-pro-rhNGF、Luc-pro-rhNGF、Nat-pro-rhNGF连接至相同双酶切后的表达载体上，连接子化学转化至Top 10感受态细胞。转化长出的单菌落通过菌体PCR筛选阳性克隆，若目的基因成功连 接至表达载体上，则PCR产物大小为1000bp左右；空载体的PCR产物为260bp左右。
挑选PCR筛选的阳性克隆p-Pre-pro-rhNGF-8、p-Nat-pro-rhNGF-1、p-Luc-pro-rhNGF-5进行测序，经序列比对分析，三个载体的“基因B”—proNGF基因组合序列与理论序列一致。
为表达rhNGF，应删去真核表达载体中第二个表达单元(CMV/EF1表达框)，保留第一个表达单元，变构为只表达一个单元的载体。为移除CMV/EF1表达框，利用限制性内切酶SfiI对测序正确的载体p-Pre-pro-rhNGF-8、p-Nat-pro-rhNGF-1、p-Luc-pro-rhNGF-5进行酶切，酶切产物经DNA产物纯化试剂盒纯化后，通过T4DNA连接酶对SfiI酶切的载体进行自连，连接子化学转化至Top 10感受态细胞，长出的单菌落通过菌体PCR筛选阳性克隆，成功移除CMV/EF1表达框的载体PCR产物无条带，否则PCR产物为535bp的条带。
根据菌体PCR的筛选结果，选择PCR产物无条带的单菌落抽提质粒p-Pre-pro-rhNGF(SfiI)-1，p-Nat-pro-rhNGF(SfiI)-1，p-Luc-pro-rhNGF(SfiI)-1，分别通过SfiI单酶切和AvrII、BstZ17I双酶切检测，阳性克隆的SfiI单酶切无条带切下，如图1所示，AvrII、BstZ17I双酶切切下大小740bp左右的条带，如图2所示。
将酶切鉴定正确的质粒p-Pre/Nat/Luc-pro-rhNGF(SfiI)-1进行测序，经序列比对分析，三个表达载体插入的“基因B”—proNGF基因组合序列与设计序列一致。
3.3“基因B”—proNGF基因组合对rhNGF表达的影响
采用瞬时转染的方法测试各种“基因B”—proNGF基因组合表达rhNGF的效率。
(1)CHO细胞培养条件
培养基FortiCHO，补加8mM谷氨酰胺后为完全培养基。培养条件：轨道摇床(轨道直径2.5cm)，转速130rpm，二氧化碳浓度8％，温度37℃。CHO细胞在达到1.5-2.5×10 6/mL时传代，传代后密度3-5×10 5/mL。
(2)细胞转染方法
在转染前一天传代细胞，密度为5-6×10 5/mL。在转染前用完全培养基调整细胞密度到1×10 6/mL。根据实验目的选择合适的转染体积。在1.5mL微离心管中，按每10 6待转细胞1.67μg的比例分别加入线性化后的表达载体p-Pre/Nat/Luc-pro-rhNGF(SfiI)-1，加入optiPRO SFM使终体积为每10 6待转细胞50μL，温和混均。在另一个1.5mL微离心管中按每10 6细胞1.67μL FreeStyle Max转染试剂和48.33μL optiPRO SFM的比例加入两种试剂，温和混均。立即把稀释后的Max溶液同DNA溶液混合，室温放置10min，最长不 要超过20min。逐滴把DNA：MAX混合溶液加入到细胞悬液中，立即放入摇床培养。
(3)rhNGF表达水平的测定
瞬时转染后，细胞培养48小时收样，ELISA测量不同“基因B”引导下rhNGF的表达水平，独立实验进行两次，如图3所示。结果表明“基因B”Pre、Luc-proNGF基因组合与NGF天然基因组合Nat-proNGF相比，rhNGF表达水平显著提高，所以Pre、Luc“基因B”与proNGF基因组合是较优的选择。
4、“基因C”元件的选择
在目的基因5’端添加“基因C”，可以增强mRNA的稳定性，进而增加目的蛋白的表达。选择“基因C”glo和aden，序列如SEQ ID No.1、SEQ ID No.2所示，与“基因B”、proNGF基因进行组合，考察其对rhNGF表达的影响。
4.1“基因C”的获得
“基因C”glo和aden的DNA序列由金斯瑞生物科技有限公司合成，构建于同一质粒pUC57上，大小分别为150bp和296bp，序列两端都含有AvrII酶切位点，对应的质粒称为pUC57-glo-aden。glo和aden通过限制性内切酶AvrII单酶切质粒pUC57-glo-aden获得，利用胶回收试剂盒纯化后待用。
4.2含有“基因C”—“基因B”—proNGF基因组合的真核表达载体的构建
选择在3中所述瞬时表达rhNGF水平较高的载体p-Pre-pro-rhNGF(SfiI)－1上添加“基因C”。通过限制性内切酶AvrII对其进行酶切，酶切产物经普通DNA产物纯化试剂盒进行纯化，利用T4DNA连接酶将AvrII酶切后的“基因C”片段glo、aden连接至载体p-Pre-pro-rhNGF(SfiI)上，连接子化学转化至Top 10感受态细胞。转化长出的单菌落经菌体PCR筛选获得阳性克隆，分别命名为p-glo-Pre-pro-rhNGF(SfiI)和p-aden-Pre-pro-rhNGF(SfiI)，对这两个载体内的重组基因进行测序，经序列比对分析，“基因C”glo、aden序列与设计序列一致，由此获得含有“基因C”—“基因B”—proNGF基因组合的真核表达载体。
4.3“基因C”—“基因B”—proNGF基因组合对rhNGF表达的影响
同样采用瞬时转染的方法测试“基因C”—“基因B”—proNGF基因组合表达rhNGF的效率。CHO细胞培养条件、细胞转染方法同3.3中所述。
线性化的p-glo-Pre-pro-rhNGF(SfiI)和p-aden-Pre-pro-rhNGF(SfiI)表达载体瞬时转染CHO细胞，培养48小时后收样，通过ELISA检测上清中rhNGF含量评估表达效率，独立实验进行两次，如图4所示。结果证实在“基因B”—proNGF基因组合5’端添加“基 因C”可以进一步显著提高rhNGF的表达水平，两个“基因C”之间无明显差异，由此确定“基因C”—“基因B”—proNGF基因组合是表达rhNGF更优的选择，对应的真核表达载体中插入的基因元件组合依次为“基因C”、“基因B”、proNGF基因，如图5所示。
实施例2工程细胞株的建立
1、CHO细胞培养条件、细胞转染方法见3.3所述。
2、稳定筛选
转染48小时后，把细胞分成两份。一份加入10μg/mL puromycin和100nM MTX；另一份加入20μg/mL puromycin和200nM MTX。待细胞活率恢复到85％以上时，每份细胞再分成两份，分别加入30μg/mL puromycin/500nM MTX和50μg/mL puromycin/1000nM MTX，继续筛选。筛选结束的准则是细胞活率大于90％。两轮筛选共得到6个细胞池，分析比产率后，选择比产率高、细胞生长良好的细胞池用于单克隆。
3、有限稀释法单克隆和克隆筛选
克隆培养基为FortiCHO补加6mM谷氨酰胺。稀释待克隆的细胞到2-5个细胞/mL。用8通道移液器加细胞悬液到96孔板中，每孔200μL。把细胞放入二氧化碳培养箱中在37℃、5％二氧化碳下培养。根据成克隆的速度，培养11-14天后，从生成单克隆的孔中取20μL用ELISA的方法分析rhNGF的浓度。选取表达量高的克隆从96孔板中移到48孔板中，加入200μL新鲜培养基，加入MTX和puromycin筛选药物到单克隆前细胞池筛选的浓度，长满后传代到12孔板，等12孔板中细胞基本达到传代密度，取出细胞到离心管中，离心除去上清，用PBS洗一遍细胞后，重悬浮在1mL新鲜培养基中，加入到6孔板中，取30μL细胞悬液用于分析细胞密度，把6孔板放入培养箱培养2-4小时，取出100μL培养液离心收获上清，向6孔板每孔中加入1mL新鲜培养基和筛选药物，继续培养。培养上清用ELISA分析rhNGF浓度。采用公式计算细胞的比产率，比产率等于rhNGF浓度/细胞密度/孵育时间。按照比产率对克隆进行第二轮筛选。
对筛选获得的细胞进行传代稳定性试验，选取稳定性试验中表现良好的6株细胞进行批次培养，用Capto S层析柱初步纯化批次培养上清中的rhNGF，进行SDS-PAGE检测，结果如图6所示，表明在批次培养中13C5细胞表达的rhNGF中proNGF蛋白的含量较低，由于proNGF蛋白是产品相关的杂质，其含量越低越好，由此选择13C5细胞作为工程细胞株。
实施例3生物反应器中测定工程细胞株的生长曲线、细胞活率及rhNGF表达水平的变化趋势
采用流加(补料)培养作为工程细胞株的生产培养模式，将培养规模从摇瓶的30mL放大到2.5L，再进一步放大到28L规模可原位灭菌的机械搅拌生物反应器，搅拌桨为单斜叶桨，工作搅拌转速125rpm，大泡通气。溶氧控制先级联控制空气流量，达到最大气体流量设定值后，开始通入氧气，同步减少空气流量，保持总流量不变。pH在培养的初期通过控制CO 2流量维持在7.2，随细胞生长，pH会先降低，后反弹上升，待反弹至7.2时，用稀盐酸控制在设定值(pH7.2)，直到培养结束。细胞培养过程中定时取样监测细胞密度、活率以及rhNGF的浓度，汇总结果如图7所示。
从结果可以看出，培养至第5天细胞由指数生长期进入平台期，在余下6-10天的活细胞密度大致保持稳定，最高细胞密度达1.2×10 7/mL，细胞活率维持在90％以上，培养过程中rhNGF浓度持续快速升高，培养结束时达78mg/L。
实施例4 TF-1细胞/MTS比色法测定rhNGF的生物学活性
TF-1细胞/MTS比色法是《中国药典》2015年版三部收录的用于测定神经生长因子生物学活性的经典方法，采用此方法测定rhNGF的生物学活性，同时与国际标准品(货号：93/556，NIBSC)、注射用mNGF(商品名：苏肽生，舒泰神(北京)生物制药股份有限公司)进行比较。
将生长状态良好的人红细胞白血病细胞(TF-1细胞，已驯化的NGF依赖型，来源为中国食品药品检定研究院重组蛋白室)用基础培养基(1640+10％FBS+1％P/S)以5000个细胞每孔的量接入96孔板，每孔体积100μL；然后每孔分别加入100μL以基础培养基3倍梯度稀释的待测NGF(rhNGF、国际标准品(Std)、苏肽生(Sutaisheng))溶液，浓度设置为100、33、11、3.3、1.1、0.33、0.11、0.033ng/mL，每浓度两个复孔；混匀后放入37℃，5％CO 2培养箱中培养72h；每孔加入20μL MTS，37℃，混匀孵育3h；于酶标仪492nm处检测各孔的OD值；以Graphpad 6.0软件拟合各组的吸光值-浓度关系曲线(选择四参数非线性回归方程拟合)；计算出各样品刺激TF-1细胞增殖的EC 50值，结果如图8所示。
从结果可以看出，在刺激TF-1细胞增殖活性上，rhNGF与国际标准品(Std)活性相当(EC 50分别为5.30ng/mL,5.26ng/mL)，而强于苏肽生(Sutaisheng)的活性(EC 50为14.82ng/mL)。

Claims (6)

  1. 一种高效表达rhNGF的基因组合，所述组合为：“基因B—基因A”；
    所述基因A，其核苷酸序列如SEQ ID No.7所示；
    所述基因B，其核苷酸序列如SEQ ID No.3或SEQ ID No.5所示。
  2. 权利要求1所述的基因组合在制备rhNGF中的应用。
  3. 权利要求1所述的基因组合，还包括有基因C的元件，连接顺序为：
    “基因C—基因B—基因A”；
    所述基因C，其核苷酸序列如SEQ ID No.1或SEQ ID No.2所示。
  4. 权利要求3所述的基因组合在制备rhNGF中的应用。
  5. 核苷酸序列如SEQ ID No.7所示的人神经生长因子前体基因。
  6. SEQ ID No.3或SEQ ID No.5所示核苷酸序列的基因在制备rhNGF中的应用。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5272063A (en) * 1988-11-22 1993-12-21 Syntex (U.S.A.) Inc. Process of making human nerve growth factor
CN103880943A (zh) * 2014-01-20 2014-06-25 厦门北大之路生物工程有限公司 一种rhNGF成熟肽的制备方法
CN104203264A (zh) * 2011-12-19 2014-12-10 瓦克化学有限公司 新型proNGF突变体及其在生产β-NGF中的用途
CN108456681A (zh) * 2018-03-26 2018-08-28 江苏中新医药有限公司 高效表达重组人神经生长因子的基因组合

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102586190B (zh) * 2011-01-11 2014-01-01 北京华安科创生物技术有限公司 高效表达重组人神经生长因子的cho细胞株及其构建方法
CN103074374B (zh) * 2013-01-16 2014-04-02 薛博夫 人β-NGF的重组表达载体及含该载体的重组细胞株
CN106810611A (zh) * 2015-11-30 2017-06-09 中国科学院深圳先进技术研究院 抗cMet和CD3特异性双靶向抗体及其制备方法和应用、含该双靶向抗体表达盒的微环DNA及应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5272063A (en) * 1988-11-22 1993-12-21 Syntex (U.S.A.) Inc. Process of making human nerve growth factor
CN104203264A (zh) * 2011-12-19 2014-12-10 瓦克化学有限公司 新型proNGF突变体及其在生产β-NGF中的用途
CN103880943A (zh) * 2014-01-20 2014-06-25 厦门北大之路生物工程有限公司 一种rhNGF成熟肽的制备方法
CN108456681A (zh) * 2018-03-26 2018-08-28 江苏中新医药有限公司 高效表达重组人神经生长因子的基因组合

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 8 March 2018 (2018-03-08), TAKANO, S. ET AL.: "Beta-Nerve Growth Factor Precursor [Homo Sapiens", XP055641356, retrieved from NCBI Database accession no. NP-002497.2 *
SHEN, LI ET AL.: "Progress on Research and Clinical Application of Nerve Growth Factor", PROGRESS IN MICROBIOLOGY AND IMMUNOLOGY, vol. 43, no. 6, 31 December 2015 (2015-12-31), ISSN: 1005-5673 *

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