WO2013013626A1

WO2013013626A1 - Deletion mutant of recombinant human nerve growth factor, preparation method and use thereof

Info

Publication number: WO2013013626A1
Application number: PCT/CN2012/079188
Authority: WO
Inventors: 陈薇; 侯利华; 宋小红; 于婷; 付玲; 于蕊; 房婷
Original assignee: 中国人民解放军军事医学科学院生物工程研究所
Priority date: 2011-07-28
Filing date: 2012-07-26
Publication date: 2013-01-31
Also published as: CN102898514A; CN102898514B

Abstract

Disclosed is a deletion mutant of a recombinant human nerve growth factor. The mutant is a peptide chain of the complete human nerve growth factor, with 3 amino-acids at C-terminal deleted, wherein the amino-acid sequence of the mutant is as shown in sequence list SEQ ID No.1, or the amino-acid sequence of the mutant is an amino-acid sequence that shares over 95% homology with that which is shown in SEQ ID No.1 and has the biological activity of a nerve growth factor. The mutant in the invention retains the C-terminal integrity in rhNGF by the strategy of expressing rhNGF with 3 amino-acids deleted at C-terminal (rhNGF-D3), so the expression level of rhNGF-D3 protein in eukaryotic cells is increased five- to ten-fold by this modification compared to rhNGF, with the same bioactivity and uniform protein C-terminal.

Description

重组人神经生长因子缺失突变体及其制备方法和用途技术领域 Recombinant human nerve growth factor deletion mutant, preparation method and use thereof

本发明涉及一种突变体，具体的说是一种在真核表达***中高效表达并保持相似的生物学活性的重组人神经生长因子缺失突变体。 The present invention relates to a mutant, in particular to a recombinant human nerve growth factor deletion mutant that efficiently expresses and maintains similar biological activity in a eukaryotic expression system.

背景技术 Background technique

神经生长因子（Nerve Growth Factor, NGF) 是 Levi- Montlcini于 1953 年在小鼠肉瘤细胞内发现的第一个神经营养因子， NGF 能够维持和促进交感神经及来自神经嵴的感觉神经细胞的存活、分化和成熟以及执行功能，是参与损伤神经再生和功能修复的重要因素。 Nerve Growth Factor (NGF) is the first neurotrophic factor discovered by Levi-Montlcini in mouse sarcoma cells in 1953. NGF can maintain and promote the survival of sympathetic nerves and sensory nerve cells from neural crest. Differentiation and maturation and executive function are important factors involved in injured nerve regeneration and functional repair.

NGF存在于多种物种中，在雄性小鼠颌下腺、牛***、蛇毒、豚鼠前列腺和人胎盘中组织中含量丰富。其中小鼠 NGF与人 NGF氨基酸序列同源性达到 90%。利用生物技术提取自小鼠颌下腺的鼠 NGF及提取自人胎盘组织的人 NGF 注射液目前已在中国上市，临床上主要用于治疗视神经损伤等，例如中毒性周围神经损伤、外伤性周围神经损伤、面神经炎等疾病。考虑到鼠 NGF 应用于人体的种属差异性及小鼠作为原材料所具有的潜在的致病因子风险及人胎盘组织原材料的限制，发展基因工程技术制备重组人 NGF ( rhNGF, recombinant human NGF) 来取代提取的鼠 NGF和人 NGF具有很好的应用前景。 NGF is present in a variety of species and is abundant in tissues in the submandibular gland, bovine seminal plasma, snake venom, guinea pig prostate gland, and human placenta in male mice. The amino acid sequence of mouse NGF and human NGF reached 90%. Human NGF injection extracted from mouse submandibular gland and human NGF injection extracted from human placenta tissue has been marketed in China. It is mainly used for the treatment of optic nerve injury, such as toxic peripheral nerve injury and traumatic peripheral nerve injury. , facial neuritis and other diseases. Considering the species diversity of murine NGF applied to humans and the potential risk factors of human raw materials for placenta and the limitations of human placental tissue raw materials, genetic engineering technology was developed to prepare recombinant human NGF (rhGF). It has a good application prospect instead of the extracted mouse NGF and human NGF.

人体内成熟的 NGF以同源二聚体的形式存在，每条肽链包含 120个氨基酸。人 NGF基因位于 1号染色体短臂上，完整的 NGF外显子由 241个氨基酸组成，通常称为 pr印 ro NGF前体，在内质网中 pr印 ro NGF前体的信号肽被切割下来，形成 pro NGF前体（223个氨基酸）， pro NGF 前体在内质网中以同源二聚体形式存在，然后转移至高尔基体中，前体部分经 Furin酶切，形成成熟的 NGF二聚体（单体含有 120个氨基酸），被转运至细胞外，同时也有部分未经切割的 pro NGF前体分泌至细胞外。 The mature NGF in the human body exists as a homodimer, and each peptide chain contains 120 amino acids. The human NGF gene is located on the short arm of chromosome 1, and the complete NGF exon consists of 241 amino acids, commonly referred to as the pr-po NGF precursor. The signal peptide of the pr-labeled N-N precursor is cleaved in the endoplasmic reticulum. , pro NGF precursor (223 amino acids) is formed, pro NGF precursor exists as a homodimer in the endoplasmic reticulum, and then transferred to the Golgi apparatus, and the precursor part is digested by Furin to form mature NGF II. The polymer (the monomer contains 120 amino acids) is transported extracellularly, and a portion of the uncleaved pro NGF precursor is also secreted outside the cell.

提取自小鼠颌下腺的鼠 NGF通常在 N端、 C端有一些氨基酸缺失，例如 N 端缺失 8个氨基酸、 C端缺失 2个或 3个氨基酸，这几种缺失突变体同时存在于鼠 NGF中，有研究表明 N端或 C端缺失的 NGF并不影响其生物学活性。在利用真核细胞制备 rhNGF时，同样发现经常存在 C端缺失 2到 3个氨基酸的情况，会形成同源或者异源二聚体，例如 120/120、 118/118、 117/117、 120/118、 117/118等，这就给 rhNGF的质量控制造成了一定的困扰。 Mouse NGF extracted from mouse submandibular gland usually has some amino acid deletions at the N-terminus and C-terminus, such as deletion of 8 amino acids at the N-terminus and deletion of 2 or 3 amino acids at the C-terminus. These deletion mutants are also present in murine NGF. Studies have shown that NGF deleted at the N-terminus or C-terminus does not affect its biological activity. When rhNGF is prepared by using eukaryotic cells, it is also found that there are often two or three amino acid deletions at the C-terminus, which will result in homologous or heterodimers, such as 120/120, 118/118, 117/117, 120/118, 117/118, etc., which caused some troubles for the quality control of rhNGF.

发明内容 Summary of the invention

本发明的目的在于提供一种在真核细胞中高效表达的重组人神经生长因子缺失突变体。 It is an object of the present invention to provide a recombinant human nerve growth factor deletion mutant which is highly expressed in eukaryotic cells.

本发明的具体技术方案如下： The specific technical solutions of the present invention are as follows:

一种重组人神经生长因子缺失突变体，所述突变体是完整人神经生长因子肽链 C端缺失 3个氨基酸；所述突变体氨基酸序列为： A recombinant human nerve growth factor deletion mutant, wherein the mutant is a complete human neural growth factor peptide chain C-terminal deletion of 3 amino acids; the mutant amino acid sequence is:

( 1 ) 如序列表 SEQ ID No. 1所示。或 (1) as shown in the sequence listing SEQ ID No. 1. Or

( 2 ) 所述突变体的氨基酸序列为与 SEQ ID No. 1 中所示的氨基酸序列具有 95%以上同源性且具有神经生长因子生物学活性的氨基酸序列。编码上述重组人神经生长因子缺失突变体的基因。 (2) The amino acid sequence of the mutant is an amino acid sequence having 95% or more homology with the amino acid sequence shown in SEQ ID No. 1, and having a biological activity of a nerve growth factor. A gene encoding the above recombinant human nerve growth factor deletion mutant.

所述基因如 SEQ ID No. 2所示。 The gene is shown as SEQ ID No. 2.

一种制备重组人神经生长因子缺失突变体基因的方法，所述步骤如下：根据人 NGF 靈序列 ( Genbank : NM— 002506 ) 设计引物，分离人外周血白蛋白，提取其总 RNA, 反转录为 cDNA, 以此 cDNA为模板，扩增人 NGF基因及缺失突变体基因。 A method for preparing a recombinant human nerve growth factor deletion mutant gene, the steps are as follows: designing a primer according to a human NGF sequence (Genbank: NM-002506), isolating human peripheral blood albumin, extracting total RNA, and reverse transcription For cDNA, this cDNA was used as a template to amplify the human NGF gene and the deletion mutant gene.

一种表达载体，所述载体含有上述方法制备得到的基因。 An expression vector comprising the gene prepared by the above method.

所述表达载体为真核表达载体。真核细胞可以使用酵母细胞、昆虫细胞、植物细胞、哺乳动物细胞等，基因导入细胞的方法可以使用稳定转染或瞬时转染。 The expression vector is a eukaryotic expression vector. The eukaryotic cells can use yeast cells, insect cells, plant cells, mammalian cells, etc., and the method of gene introduction into cells can be carried out by stable transfection or transient transfection.

一种含有上述表达载体的宿主细胞。 A host cell comprising the above expression vector.

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细胞、人胚肾 293细胞、 COS细胞或 Hela细胞。 The host cell is a mammalian cell. The mammalian cells are Chinese hamster ovary cells, human embryonic kidney 293 cells, COS cells or Hela cells.

一种表达上述载体的方法，是将含有重组人神经生长因子缺失突变体基因的表达载体转化至宿主细胞，培养所得重组细胞表达得到重组人神经生长因子缺失突变体。 A method for expressing the above vector is to transform an expression vector containing a recombinant human nerve growth factor deletion mutant gene into a host cell, and the cultured recombinant cell is expressed to obtain a recombinant human nerve growth factor deletion mutant.

本发明的有益效果： The beneficial effects of the invention:

本发明所述的突变体采取表达 C末端缺失 3个氨基酸 rhNGF( rhNGF-D3 ) 的策略来保持 rhNGF的 C末端完整性，并且通过此改造，相比较于 rhNGF, 在真核细胞中 rhNGF-D3蛋白表达水平提高 5〜10倍，生物活性保持不变，蛋白 C端均一。本发明的可实施性和突出实质性特点以及积极效果可通过以下实例得以体现，但不限制其范围。 The mutant of the present invention adopts a strategy of expressing a C-terminal deletion of three amino acids rhNGF (rhNGF-D3) to maintain the C-terminal integrity of rhNGF, and by this modification, compared to rhNGF, rhNGF-D3 in eukaryotic cells The protein expression level is increased by 5 to 10 times, the biological activity remains unchanged, and the protein C end is uniform. The applicability and salient features and positive effects of the present invention can be embodied by the following examples without limiting the scope thereof.

附图说明 DRAWINGS

图 1为编码人 NGF及其突变体的薩序列； rhNGF-D3突变体序列与人 NGF 序列相比缺乏 3' 端的 9bp (下划线标记的序列）； Figure 1 shows the Sag sequence encoding human NGF and its mutant; the rhNGF-D3 mutant sequence lacks the 3' end 9 bp (underlined sequence) compared to the human NGF sequence;

图 2为 rhNGF-D3氨基酸序列； Figure 2 is the amino acid sequence of rhNGF-D3;

图 3为表达 rhNGF或 rhNGF-D3的载体结构图； Figure 3 is a diagram showing the structure of a vector expressing rhNGF or rhNGF-D3;

图 4为 1F1G8和 2F5重组细胞系在 10LWAVE生物反应器中的细胞生长曲线； 1F1G8表达 rhNGF, 2F5细胞表达 rhNGF-D3;细胞培养采取流加培养模式，共培养 12天； Figure 4 shows the cell growth curve of 1F1G8 and 2F5 recombinant cell lines in a 10LWAVE bioreactor; 1F1G8 expresses rhNGF, 2F5 cells express rhNGF-D3; cell culture adopts fed-batch culture mode, and co-culture for 12 days;

图 5为 rhNGF和 rhNGF_D3蛋白表达水平； Figure 5 shows the expression levels of rhNGF and rhNGF_D3 proteins;

图 6为 rhNGF蛋白纯化结果； Figure 6 shows the purification results of rhNGF protein;

图 7为 rhNGF-D3蛋白纯化结果； Figure 7 shows the results of purification of rhNGF-D3 protein;

图 8为 rhNGF-D3蛋白 N端氨基酸序列测定结果； Figure 8 shows the results of the N-terminal amino acid sequence of rhNGF-D3 protein;

图 9为 rhNGF-D3蛋白分子量测定结果； Figure 9 shows the results of molecular weight determination of rhNGF-D3 protein;

图 10为瞬时转染情况下 rhNGF和 rhNGF-D3蛋白表达水平分析。 Figure 10 shows the expression levels of rhNGF and rhNGF-D3 proteins in the case of transient transfection.

具体实施方式 detailed description

实施例 1 重组人神经生长因子缺失突变体（rhNGF-D3) 基因克隆及载体构建根据已公布的人 NGF DNA序列 (Genbank: 匪― 002506) 设计引物，分离人外周血白蛋白，利用 TriZol (购自 Invitrogen) ，提取其总 RNA, 反转录为 cDNA, 反转录体系为模板 RNA 2 μ g， 5 反应液4 1， dNTP混合液（ lOmM, each) 1μ 1， RNase inhibitor (20U/ 1) ，随机引物 2μ 1， M- MLV反转录酶（200υ/μ 1) 1μ 1, 加水补足总体积至 20μ 1。室温放置 lO min后， 42°C 反应 l h。以此 cDNA为模板，扩增人 NGF基因及缺失突变体基因，扩增上游引物为 " 5 ' -atgaa ttcca ccatg tccat gttgt tctac actc t ga~3 ' ，，，下游引物分别为 " 5' -atccc gggtt atcag gctct tctca cagcc ttcct get- 3'Example 1 Recombinant Human Nerve Growth Factor Deletion Mutant (rhNGF-D3) Gene Cloning and Vector Construction Primers were designed according to the published human NGF DNA sequence (Genbank: 匪-002506) to isolate human peripheral blood albumin, using TriZol (purchased From Invitrogen, extract total RNA, reverse transcription into cDNA, reverse transcription system as template RNA 2 μg, 5 reaction solution 4 1, dNTP mixture (10 mM, each) 1μ 1, RNase inhibitor (20U/ 1) , random primer 2μ 1, M- MLV reverse transcriptase (200 υ / μ 1) 1μ 1, add water to make up the total volume to 20μ 1 . After lO min at room temperature, react at 42 ° C for 1 h. Using this cDNA as a template, the human NGF gene and the deletion mutant gene were amplified, and the upstream primer was amplified as " 5 ' -atgaa ttcca ccatg tccat gttgt tctac actc t ga~3 ' , , and the downstream primers were respectively " 5 ' -atccc Gggtt atcag gctct tctca cagcc ttcct get- 3'

(用于全长 NGF基因扩增），，禾口 "5， -atccc gggtt atcac acagc cttcc tgctg agcacac-3' (用于 NGF缺失突变体基因扩增） " 。起始密码子处设有 Kozak 序列（CCACCATG), PCR扩增条件为 94°C- 3min; 94°C- 30s， 58°C- 30s， 72°C- lmin, 30个循环； 72°C- 7 mine 得到人 NGF基因全长 726 bp , 包括人 NGF的 pr印 ro序列部分， NGF缺失突变体基因全长 717 bp, 序列如 SEQ ID No. 2所示。如图 1所示，在人 NGF全长基因的 3 ' 端缺失 AGAAGAGCCT (代表 C末端的 3个氨基酸）。以此基因表达的人神经生长因子突变体蛋白氨基酸序列（117个氨基酸 SEQ ID No. 1所示）如图 2所示，编码 pr印 ro NGF-D3的基因***到表达载体中，信号肽（图中下划线标记的序列）被切割后，形成 pro NGF-D3前体，再经 furin酶酶切后，酶切位点如图中箭头所示，形成成熟的 rhNGF-D3。成熟的 rhNGF-D3蛋白含有 117个氨基酸，缺乏完整人 NGF蛋白 C端的 R、 R、 A三个氨基酸。 (for full-length NGF gene amplification), and "5, -atccc gggtt atcac acagc cttcc tgctg agcacac-3' (for NGF deletion mutant gene amplification)". The Kozak sequence (CCACCATG) was set at the start codon, and the PCR amplification conditions were 94 ° C - 3 min; 94 ° C - 30 s, 58 ° C - 30 s, 72 ° C - l min, 30 cycles; 72 ° C - 7 mine The human NGF gene was 726 bp in length, including the pr-ro sequence of human NGF, and the NGF deletion mutant gene was 717 bp in length, and the sequence is shown in SEQ ID No. 2. As shown in Figure 1, AGAAGAGCCT (representing three amino acids at the C-terminus) was deleted at the 3' end of the human NGF full-length gene. The human nerve growth factor mutant protein amino acid sequence expressed by this gene (117 amino acids shown in SEQ ID No. 1) is shown in Figure 2. The gene encoding pr-ro NGF-D3 is inserted into an expression vector, and the signal peptide ( The underlined sequence in the figure) was cleaved to form a pro NGF-D3 precursor, which was digested with furin, and the restriction site was shown by the arrow in the figure to form mature rhNGF-D3. The mature rhNGF-D3 protein contains 117 amino acids and lacks the three amino acids R, R and A of the C-terminus of the intact human NGF protein.

重组人神经生长因子缺失突变体（rhNGF-D3 ) 载体构建： Construction of recombinant human nerve growth factor deletion mutant (rhNGF-D3) vector:

将上述获得的 PCR产物人 NGF DNA 序列及 SEQ ID No. 2所示的 NGF缺失突变体基因序列经 EcoRI和 Sma l (购自 NEB ) 双酶切后，与***到经同样双酶切的 pCI-neo载体（Invitrogen公司）连接，连接条件为 16°C，过夜。将连接产物转化感受态细菌 DH 5 (1，感受态菌铺于带有氨苄青霉素的 LB琼脂平板上，利用 PCR鉴定挑选出阳性克隆， pCI-neo NGF 及 pCI-neo NGF-D3质粒经 DNA测序鉴定正确，质粒结构如图 3所示。 The PCR product human NGF DNA sequence obtained above and the NGF deletion mutant gene sequence shown in SEQ ID No. 2 were digested with EcoRI and Sma1 (purchased from NEB), and inserted into the same double-digested pCI. -neo vector (Invitrogen) was ligated at 16 ° C overnight. The ligation product was transformed into competent bacteria DH 5 (1, competent bacteria were plated on LB agar plates with ampicillin, positive clones were selected by PCR, and pCI-neo NGF and pCI-neo NGF-D3 plasmids were sequenced by DNA. The identification is correct, and the plasmid structure is shown in Figure 3.

实施例 2 Example 2

将制备好的 pCI-neo NGF 及 pCI-neo NGF-D3质粒经电转化至 CHO S细胞（购自 Invitrogen公司），电转仪为 Gene pulser Xcell (BIO- RAD公司），电转条件为 160V， 150ms。培养基为丽 EM加入 10% 胎牛血清，电转后，用培养基将电转杯内的细胞洗出，铺于 35mm细胞培养皿内。第三天，培养基内加入 600 μ g/ml G418 ( Sigma公司）加压筛选抗性细胞，经抗性筛选存活的单克隆细胞团，转移至 96孔板内，利用 dot-blot检测细胞上清中重组蛋白表达水平，挑选高表达的细胞克隆进入到悬浮培养。 The prepared pCI-neo NGF and pCI-neo NGF-D3 plasmids were electrotransformed into CHO S cells (purchased from Invitrogen), and the electrorotation device was a Gene pulser Xcell (BIO-RAD), and the electroporation conditions were 160 V, 150 ms. The medium was supplemented with 10% fetal bovine serum. After electroporation, the cells in the electric rotor were washed out with a medium and placed in a 35 mm cell culture dish. On the third day, 600 μg/ml G418 (Sigma) was added to the culture medium to screen for resistant cells, and the surviving monoclonal cell mass was screened by resistance, transferred to a 96-well plate, and detected by dot-blot. The level of recombinant protein expression in the Qing Dynasty was selected and the highly expressed cell clones were selected for suspension culture.

宿主细胞优选为哺乳动物细胞，本实施例中使用的是中国仓鼠卵巢（CH0) 细胞，人胚肾 293细胞、 COS细胞、 Hela细胞等也可应用。 The host cell is preferably a mammalian cell. In the present example, Chinese hamster ovary (CH0) cells, human embryonic kidney 293 cells, COS cells, Hela cells and the like are also applicable.

实施例 3 Example 3

选择有较好表达水平的细胞株转移至 40ml摇瓶（Corning公司）中，挑选适应无血清悬浮培养的细胞株，所用培养基为 SFM4 (Hyclone公司），培养条件为 37°C，比较细胞生长曲线、重组蛋白表达水平（定量 ELISA检测， BD公司， dy265 ) ，最终确定重组细胞系 1F1G8 (表达全长重组人神经生长因子（rhNGF) ) 和细胞系 2F5 (表达重组人神经生长因子 C末端缺失 3个氨基酸的突变体（rhNGF-D3 ) ) 作为下一步研究的重组细胞系。 Cell lines with good expression levels were selected and transferred to a 40 ml shake flask (Corning). Cell lines adapted to serum-free suspension culture were selected. The medium used was SFM4 (Hyclone), and the culture conditions were 37 ° C. Curve, recombinant protein expression level (quantitative ELISA assay, BD, dy265), final identification of recombinant cell line 1F1G8 (expressing full-length recombinant human nerve growth factor (rhNGF)) and cell line 2F5 (representing recombinant human nerve growth factor C-terminal deletion) 3 amino groups The acid mutant (rhNGF-D3) was used as a recombinant cell line for further study.

对细胞系 1F1G8和 2F5在 10L WAVE生物反应器（Satorious公司）中进行了小规模的批式流加细胞培养工艺摸索，两株细胞系保持相似的生长曲线，如图 4所示，但 rhNGF-D3蛋白的表达水平比 rhNGF高大约 10倍以上，如图 5所示。 The cell lines 1F1G8 and 2F5 were subjected to a small-scale batch-flow and cell culture process in a 10L WAVE bioreactor (Satorious). The two cell lines maintained a similar growth curve, as shown in Figure 4, but rhNGF- The expression level of D3 protein is about 10 times higher than that of rhNGF, as shown in FIG.

实施例 4 Example 4

将实施例 3 获得的细胞液进行离心，超滤浓缩换液后，首先上样于 Sepharose Fastf low柱（GE公司），利用加入 1 mol/1 NaCl 的缓冲液洗脱后，陆续上样于 Phenyl Fastf low ( low sub, GE公司）、 Phenyl HP ( GE公司）和 Superdex 75分子筛凝胶柱（GE公司），纯化仪为 AKTA Purifier, 得到纯度大于 90%的重组蛋白，如图 6和图 7所示。 The cell liquid obtained in Example 3 was centrifuged, and after ultrafiltration and concentration, the sample was first applied to a Sepharose Fastf low column (GE), and eluted with a buffer of 1 mol/1 NaCl, and then successively loaded on Phenyl. Fastf low (low sub, GE), Phenyl HP (GE) and Superdex 75 molecular sieve gel column (GE), the purification instrument is AKTA Purifier, and the recombinant protein with purity greater than 90% is obtained, as shown in Figure 6 and Figure 7. Show.

实施例 5 Example 5

NGF测活的方法使用 TF-1细胞， TF-1细胞表面具体有 NGF高亲和力受体 TrkA, NGF能够通过剂量依赖关系促进 TF-1细胞的增殖，通过 MTT法来检测 NGF刺激细胞增殖的能力。 rhNGF活性标准品是来自于英国 NIBSC 的重组人 NGF活性国际标准品，其比活性为 1 X 10⁶ AU/mg，所测的 rhNGF和 rhNGF-D3 比活性分别为 9. 6 X 10⁵和 9. 8 X 10⁵ AU/mg。可以看出，两种蛋白生物比活性相当。 The method of NGF measurement uses TF-1 cells. The surface of TF-1 cells specifically has NGF high affinity receptor TrkA. NGF can promote the proliferation of TF-1 cells in a dose-dependent manner, and the ability of NGF to stimulate cell proliferation can be detected by MTT assay. . The rhNGF activity standard is an international standard for recombinant human NGF activity from NIBSC in the United Kingdom, and its specific activity is 1 X 10 ⁶ AU/mg, and the specific activities of rhNGF and rhNGF-D3 are 9. 6 X 10 ⁵ and 9 respectively. . 8 X 10 ⁵ AU/mg. It can be seen that the two protein organisms have comparable specific activities.

实施例 6 Example 6

对 rhNGF-D3蛋白进行 N端测定和分子量质谱测定，委托军事医学科学院仪器测定分析中心测定。 N端 5个氨基酸为 SSSHP, 与理论计算相同，如图 8 所示。 rhNGF-D3蛋白理论分子量为 13110 Da，实际测定分子量为 13102 Da, 如图 9所示，相差 8 Da, 在误差范围之内。由于该蛋白测定 C端氨基酸序列较为困难，未能检测出来。但根据质谱测定分子量的结果分析，蛋白中只有单一的分子量存在，说明形成了 C端均一的蛋白。 The N-terminus and molecular weight mass spectrometry of rhNGF-D3 protein were determined by the Instrumental Analysis and Analysis Center of the Academy of Military Medical Sciences. The N-terminal 5 amino acids are SSSHP, which is the same as the theoretical calculation, as shown in Figure 8. The theoretical molecular weight of rhNGF-D3 protein is 13110 Da, and the actual molecular weight is 13102 Da. As shown in Fig. 9, the difference is 8 Da, which is within the error range. Since the protein was difficult to detect the C-terminal amino acid sequence, it was not detected. However, according to the results of mass spectrometry determination of molecular weight, only a single molecular weight exists in the protein, indicating that a protein having a uniform C terminus is formed.

实施例 7 Example 7

为了进一步明确人 NGF C端缺失 3个氨基酸对重组蛋白表达水平的影响，说明我们以上获得的 rhNGF和 rhNGF-D3的表达水平差异不是由于不同细胞克隆的表达水平差异造成的，我们进行了两种蛋白的瞬时表达水平的检测。表达***为 FreeStyle™ MAX 293 Expression System ( Invitrogen公司），将 pCI-neo NGF 及 pCIieo NGF-D3质粒转染至 293F细胞，蛋白表达在 40 ml 摇瓶中进行，使用无血清悬浮培养，每种蛋白进行三个重复，利用 ELISA检测上清中的 NGF表达量，计算平均值及标准差。重组蛋白表达水平见图 10。 In order to further clarify the effect of the deletion of 3 amino acids at the C-terminus of human NGF on the expression level of recombinant protein, it is indicated that the difference in expression levels of rhNGF and rhNGF-D3 obtained above is not due to the difference in expression levels of different cell clones. Detection of transient expression levels of proteins. The expression system was FreeStyleTM MAX 293 Expression System (Invitrogen), and pCI-neo NGF and pCIieo NGF-D3 plasmids were transfected into 293F cells with protein expression at 40 ml. In a shake flask, serum-free suspension culture was used, and each protein was subjected to three replicates. The expression of NGF in the supernatant was measured by ELISA, and the mean value and standard deviation were calculated. The recombinant protein expression levels are shown in Figure 10.

从图中可以看出， rhNGF-D3的表达水平明显高于 rhNGF,相对于 rhNGF, rhNGF-D3的表达水平提高了 5〜10倍。说明基因的变化对重组蛋白表达水平起着决定性的作用。 It can be seen from the figure that the expression level of rhNGF-D3 is significantly higher than that of rhNGF, and the expression level of rhNGF-D3 is increased by 5 to 10 times compared with rhNGF. This indicates that changes in genes play a decisive role in the expression level of recombinant proteins.

Claims

权利要求 Rights request

1. 一种重组人神经生长因子缺失突变体，其特征在于，所述突变体是完整人神经生长因子肽链 C端缺失 3个氨基酸；所述突变体氨基酸序列为：A recombinant human nerve growth factor deletion mutant, characterized in that the mutant is a complete human nerve growth factor peptide chain C-terminal deletion of three amino acids; the mutant amino acid sequence is:

( 1 ) 如序列表 SEQ ID No. 1所示；或 (1) as shown in the sequence listing SEQ ID No. 1; or

( 2 ) 所述突变体的氨基酸序列为与 SEQ ID No. 1中所示的氨基酸序列具有 95%以上同源性且具有神经生长因子生物学活性的氨基酸序列。 (2) The amino acid sequence of the mutant is an amino acid sequence having 95% or more homology with the amino acid sequence shown in SEQ ID No. 1, and having a biological activity of a nerve growth factor.

2. 编码权利要求 1或 2所述重组人神经生长因子缺失突变体的基因。 2. A gene encoding the recombinant human nerve growth factor deletion mutant of claim 1 or 2.

3. 根据权利要求 3 所述的编码重组人神经生长因子缺失突变体的基因，其特征在于，所述基因如 SEQ ID No. 2所示。 The gene encoding a recombinant human nerve growth factor deletion mutant according to claim 3, wherein the gene is represented by SEQ ID No. 2.

4. 一种制备权利要求 2或 3所述重组人神经生长因子缺失突变体基因的方法，其特征在于，所述步骤如下：根据人 NGF DNA序列（Genbank : 匪— 002506 ) 设计引物，分离人外周血白蛋白，提取其总 RNA, 反转录为 cDNA, 以此 cDNA 为模板，扩增人 NGF基因及缺失突变体基因。 A method for producing the recombinant human nerve growth factor deletion mutant gene according to claim 2 or 3, wherein the step is as follows: designing a primer according to a human NGF DNA sequence (Genbank: 匪-002506), separating the human Peripheral blood albumin, which extracts total RNA and reverse transcribes into cDNA, and uses this cDNA as a template to amplify the human NGF gene and the deletion mutant gene.

5. 一种表达载体，其特征在于，所述载体含有权利要求 4制备得到的基因。 An expression vector comprising the gene prepared according to claim 4.

6. 根据权利要求 5所述的表达载体，其特征在于，所述表达载体为真核表达载体。 The expression vector according to claim 5, wherein the expression vector is an eukaryotic expression vector.

7. 一种表达权利要求 5或 6所述载体的方法，其特征在于，将含有重组人神经生长因子缺失突变体基因的表达载体转化至宿主细胞，培养所得重组细胞表达得到重组人神经生长因子缺失突变体。 A method for expressing the vector according to claim 5 or 6, wherein the expression vector containing the recombinant human nerve growth factor deletion mutant gene is transformed into a host cell, and the recombinant cell cultured is expressed to obtain recombinant human nerve growth factor. Missing mutants.

8. 含有权利要求 5或 6所述表达载体的宿主细胞。 8. A host cell comprising the expression vector of claim 5 or 6.

9. 根据权利要求 8所述的宿主细胞，其特征在于，所述宿主细胞为哺乳动物细胞。 The host cell according to claim 8, wherein the host cell is a mammalian cell.

10. 根据权利要求 9所述的宿主细胞，其特征在于，所述哺乳动物细胞为中国仓鼠卵巢细胞、人胚肾 293细胞、 COS细胞或 Hela细胞。 The host cell according to claim 9, wherein the mammalian cell is a Chinese hamster ovary cell, a human embryonic kidney 293 cell, a COS cell or a Hela cell.