CN107699590A - 一种制备重组人α‑L‑艾杜糖醛酸酶的方法 - Google Patents
一种制备重组人α‑L‑艾杜糖醛酸酶的方法 Download PDFInfo
- Publication number
- CN107699590A CN107699590A CN201710952574.2A CN201710952574A CN107699590A CN 107699590 A CN107699590 A CN 107699590A CN 201710952574 A CN201710952574 A CN 201710952574A CN 107699590 A CN107699590 A CN 107699590A
- Authority
- CN
- China
- Prior art keywords
- iduronidase
- rhidua
- idua
- human
- pcmv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims description 16
- 101001019502 Homo sapiens Alpha-L-iduronidase Proteins 0.000 claims abstract description 47
- 102000056929 human IDUA Human genes 0.000 claims abstract description 23
- 238000011282 treatment Methods 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 239000013613 expression plasmid Substances 0.000 claims abstract description 15
- 239000013604 expression vector Substances 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 230000010473 stable expression Effects 0.000 claims abstract description 3
- 239000012634 fragment Substances 0.000 claims description 10
- 239000013612 plasmid Substances 0.000 claims description 10
- 238000001976 enzyme digestion Methods 0.000 claims description 6
- 238000004255 ion exchange chromatography Methods 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 6
- 241000699802 Cricetulus griseus Species 0.000 claims description 5
- 210000001672 ovary Anatomy 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 5
- 208000002678 Mucopolysaccharidoses Diseases 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 206010028093 mucopolysaccharidosis Diseases 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 27
- 102100035028 Alpha-L-iduronidase Human genes 0.000 abstract description 22
- 208000028781 Mucopolysaccharidosis type 1 Diseases 0.000 abstract description 18
- 206010056886 Mucopolysaccharidosis I Diseases 0.000 abstract description 14
- 229920002683 Glycosaminoglycan Polymers 0.000 abstract description 10
- 102000004627 Iduronidase Human genes 0.000 abstract description 8
- 108010003381 Iduronidase Proteins 0.000 abstract description 8
- 230000004071 biological effect Effects 0.000 abstract description 6
- 239000000758 substrate Substances 0.000 abstract description 5
- 230000015556 catabolic process Effects 0.000 abstract description 4
- 238000006731 degradation reaction Methods 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 238000006911 enzymatic reaction Methods 0.000 abstract description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 abstract description 2
- 208000035475 disorder Diseases 0.000 abstract description 2
- 230000004060 metabolic process Effects 0.000 abstract description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 238000004520 electroporation Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000005571 anion exchange chromatography Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000002641 enzyme replacement therapy Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 230000008499 blood brain barrier function Effects 0.000 description 2
- 210000001218 blood-brain barrier Anatomy 0.000 description 2
- 238000005277 cation exchange chromatography Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 208000030159 metabolic disease Diseases 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000009168 stem cell therapy Methods 0.000 description 2
- 238000009580 stem-cell therapy Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 108010064942 Angiopep-2 Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 206010041660 Splenomegaly Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 230000001149 cognitive effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000016245 inborn errors of metabolism Diseases 0.000 description 1
- 208000015978 inherited metabolic disease Diseases 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004199 lung function Effects 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01076—L-Iduronidase (3.2.1.76)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及一种人α‑L‑艾杜糖醛酸酶表达载体,它是pCMV‑IDUA表达质粒。以及一种重组人α‑L‑艾杜糖醛酸酶的制备方法。本发明通过克隆表达编码人α‑L‑艾杜糖醛酸酶的基因,转染中国仓鼠卵巢细胞系DG44,并在该细胞中实现了α‑L‑艾杜糖醛酸酶的稳定表达。最终获得的α‑L‑艾杜糖醛酸酶具有催化底物α‑Liduronide酶促反应的生物活性,适合用于治疗因α‑L‑艾杜糖醛酸酶缺乏致糖胺聚糖降解代谢障碍引发的粘多糖贮积症I型(MPS‑Ⅰ)。
Description
技术领域
本发明属于基因工程技术领域,涉及人α-L-艾杜糖醛酸酶基因重组载体,用该载体转化的宿主重组表达人α-L-艾杜糖醛酸酶的方法及应用。
背景技术
粘多糖贮积症(Mucopolysaccharidosis,MPS)由酸性粘多糖降解酶缺乏,致糖胺聚糖降解代谢障碍引起,根据不同酶缺陷(大约10余种),临床上又分为8型,其中MPS-Ⅰ是我国发病率相对较高的类型。溶酶体α-L-艾杜糖醛酸酶(α-L-iduronidase,IDUA)缺陷致糖胺聚糖(glycosaminoglycans,GAGs)降解代谢障碍引发MPS-Ⅰ,多在婴儿或儿童期发生,是一种致残、致死性遗传性代谢疾病。重组人α-L-艾杜糖醛酸酶(recombinantα-L-iduronidase,rhIDUA)作为酶替代治疗(ERT)药物在临床治疗显示患者均能安全度过童年期,且伴随明显的认知功能恢复和某些症状的改善。
除ERT治疗手段外,造血干细胞治疗也是十分有效的手段。ERT以及造血干细胞治疗目前在中国仍属于空白领域,虽然,中国专利(CN201180051220.1)公开了一种反义寡核苷酸,用于调节IDUA表达来达到治疗MPS-Ⅰ型粘多糖贮积症的目的,另有中国专利(201380041239.7)公开了一种IDUA与血管肽素-2的重组融合蛋白,通过连接血管素肽靶向血脑屏障,有效改善中枢溶酶体中的葡糖氨基葡聚糖(粘多糖)的贮积。重组IDUA在欧美上市并用于MPS-Ⅰ治疗已超过十年,但我国该领域的治疗至今仍是空白。尽管重组IDUA尚不能透过血脑屏障,但其改善MPS-Ⅰ患者肺功能,尤其改善肝、脾肿大的疗效十分显著,在治疗MPS-Ⅰ并延长患者生命周期仍具有十分重要的意义。
目前虽然国外大型医疗企业有生产重组人α-L-艾杜糖醛酸酶的治疗试剂,但是该试剂价格较高,对于临床患者的治疗用药较为不利。由于治疗药物的缺失,使得国内对于该领域的治疗研究缺失严重,不利于基因治疗技术的发展,特别是对于MPS-Ⅰ型婴幼儿的治疗技术发展缓慢。
发明内容
本发明的目的在于克服现有技术中缺少关于重组表达人α-L-艾杜糖醛酸酶(rhIDUA)研究的不足,提供一种重组表达人IDUA的方法,其包含编码IDUA的核苷酸序列、表达载体以及宿主细胞DG44细胞。
同时,本发明也公开了重组IDUA的生物活性以及用于MPS-Ⅰ型粘多糖贮积症治疗的用途。
为了实现上述发明目的,本发明提供了以下技术方案:
一种人α-L-艾杜糖醛酸酶(rhIDUA)表达载体,它是pCMV-IDUA表达质粒。
一种pCMV-IDUA表达质粒构建方法,用BglII和PacI对pCMV质粒进行双酶切后回收大片段,再用BglII和PacI对pUC-IDUA质粒进行双酶切回收目的基因片段,酶切后将产生相同黏性末端的载体片段与目的基因进行连接。
通过双酶切回收获取目的基因片段,然后进行目的基因转化结合,其中rhIDUA的碱基序列如SEQ ID NO:1所示,通过如此方式获得表达质粒载体,实现制备所需要的表达载体,它是表达质粒,性质稳定,转化效率高。
一种制备人α-L-艾杜糖醛酸酶(rhIDUA)的方法,包括用上述pCMV-IDUA表达质粒转化宿主细胞,培养转化体,获得人α-L-艾杜糖醛酸酶(rhIDUA)。
进一步,所述宿主细胞是中国仓鼠卵巢DG44(CHO DG44)细胞、人胚胎肾细胞HEK293细胞。优选中国仓鼠卵巢DG44悬浮细胞系,具有更好的表达稳定性和表达水平。
重组人α-L-艾杜糖醛酸酶(rhIDUA),将所述表达载体转化宿主细胞,培养转化体,从培养物获得重组人α-L-艾杜糖醛酸酶(rhIDUA)。
进一步,将重组质粒pCMV-IDUA经电转法转染至CHO DG44细胞中,得到转化体。
优选地,将电转后细胞混合液转移至含SFM4培养基的6孔板中,置于37℃孵箱中培养,48h后加入10nM MTX进行加压筛选,待细胞活率恢复至90%以上,MTX增加至100nM;细胞加入MTX至500nM,待活率恢复至90%以上后,将细胞转入摇瓶培养,在培养第3天和第5天分别补加4g/L葡萄糖,继续培养至第7天。取培养液在4℃条件下,以5000rpm离心15min,收集上清经0.45μm滤膜过滤后进行纯化,得到重组人α-L-艾杜糖醛酸酶。
进一步,将所得rhIDUA的纯化采用离子交换层析纯化。优选地,采用阴离子交换层析或阳离子交换层析。
一种用中国仓鼠卵巢DG44(CHO DG44)细胞重组表达人α-L-艾杜糖醛酸酶(rhIDUA)的方法,其特征在于所述方法包括:
a.rhIDUA的碱基序列如SEQ ID NO:1所示;
b.构建pCMV-IDUA表达质粒;
c.在CHO DG44细胞的稳定表达;
d.离子交换层析纯化rhIDUA。
通过将rhIDUA和pCMV连接构建pCMV-IDUA表达质粒,转化CHO DG44细胞,筛选出稳定表达的目标遗传系,进行培养制备得到大量rhIDUA,然后经过离子交换层析纯化得到纯净产物rhIDUA。如此制备的rhIDUA可以应用于MPS-Ⅰ型婴幼儿的治疗,对于促进基因治疗技术的发展具有重要意义。
上述方法制备得到的rhIDUA,用于制备粘多糖贮积症MPS-Ⅰ型的治疗的生物制剂。所述rhIDUA具有催化底物α-Liduronide酶促反应的生物活性,可用于粘多糖贮积症MPS-Ⅰ型的治疗。
与现有技术相比,本发明的有益效果:
1.本发明将IDUA结合pCMV构建表达质粒,通过转化宿主细胞可以实现体外重组制备α-L-艾杜糖醛酸酶,为治疗MPS-Ⅰ型粘多糖贮积症的研究提供所需的基因治疗制剂原料,对于相应的研究促进具有重要意义。
2.本发明利用pCMV-IDUA表达质粒转化宿主细胞,培养转化体,培养所得培养液进行离子交换层析处理,可以获取大量高纯度的重组人α-L-艾杜糖醛酸酶(rhIDUA),具有纯净度高,应用效果好的特点。
3.本发明通过克隆表达编码人α-L-艾杜糖醛酸酶的基因,转染中国仓鼠卵巢细胞系DG44,并在该细胞中实现了α-L-艾杜糖醛酸酶的稳定表达。最终获得的α-L-艾杜糖醛酸酶具有催化底物α-Liduronide酶促反应的生物活性,适合用于治疗因α-L-艾杜糖醛酸酶缺乏致糖胺聚糖降解代谢障碍引发的粘多糖贮积症I型(MPS-Ⅰ)
附图说明:
图1是pCMV-IDUA-1,2,3酶切鉴定结果。
图2是重组IDUA蛋白的SEC-HPLC纯度分析。
具体实施方式
下面结合试验例及具体实施方式对本发明作进一步的详细描述。但不应将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明内容所实现的技术均属于本发明的范围。
<实施例1>
rhIDUA表达质粒构建
针对CHO种属进行IDUA编码区基因序列(GenBank:M74715.1)的优化和合成,将合成的基因序列重组至质粒载体pUC19中,获得包含目的基因的质粒为pUC-IDUA。用BglII和PacI对pCMV质粒进行双酶切后回收大片段,再用BglII和PacI对pUC-IDUA质粒进行双酶切回收目的基因片段,酶切后将产生相同黏性末端的载体片段与目的基因进行连接。将连接产物转化至Top10大肠杆菌感受态细胞中,涂布于2YT平板培养基上,37℃静止过夜。挑取单菌落小量培养后提取质粒酶切鉴定。
rhIDUA的序列如下:
SEQ ID No 1
GCCACCATGAGGCCTCTGAGGCCCCGTGCTGCTCTGCTCGCCCTCCTCGCTTCCCTGCTGGCCGCTCCTCCTGTGGCTCCCGCCGAAGCTCCTCACCTGGTGCAGGTGGACGCTGCTAGGGCTCTGTGGCCCCTGAGGCGGTTTTGGCGGAGCACCGGCTTCTGTCCTCCTCTCCCTCACAGCCAGGCCGATCAGTATGTGCTGTCCTGGGACCAGCAACTGAATCTGGCCTACGTGGGAGCCGTGCCCCACAGGGGCATTAAGCAGGTGAGGACCCACTGGCTGCTGGAGCTGGTCACCACAAGGGGATCCACCGGCAGGGGCCTCAGCTATAACTTCACCCATCTCGACGGCTACCTGGACCTCCTGCGGGAAAACCAGCTGCTCCCTGGCTTCGAGCTGATGGGCAGCGCCTCCGGCCACTTTACAGACTTCGAGGATAAGCAGCAGGTGTTCGAATGGAAGGACCTGGTCAGCTCCCTCGCTCGGAGGTACATTGGCAGGTACGGACTGGCCCACGTGTCCAAGTGGAACTTTGAGACCTGGAACGAGCCTGACCACCACGACTTCGACAACGTGTCCATGACCATGCAGGGCTTCCTGAACTACTACGACGCCTGCAGCGAGGGCCTGAGGGCCGCCTCCCCCGCCCTGAGGCTCGGCGGCCCTGGCGACTCCTTTCACACACCTCCCCGTAGCCCCCTGAGCTGGGGACTGCTGCGGCATTGCCATGACGGCACCAACTTCTTCACAGGAGAGGCCGGCGTGCGGCTGGATTATATCTCCCTGCACCGGAAGGGAGCTCGGAGCTCCATCAGCATTCTGGAGCAGGAGAAAGTGGTGGCTCAGCAGATCCGGCAACTGTTCCCCAAATTCGCTGACACCCCCATCTACAACGATGAGGCTGACCCTCTCGTGGGCTGGAGCCTCCCTCAGCCTTGGCGGGCTGACGTCACCTACGCCGCTATGGTCGTGAAGGTCATCGCCCAGCATCAAAACCTGCTCCTCGCTAACACAACATCCGCTTTCCCCTACGCCCTGCTGTCCAACGACAACGCCTTTCTCAGCTACCACCCCCATCCTTTTGCCCAGAGGACACTCACCGCCCGGTTTCAGGTGAACAACACCAGGCCTCCTCACGTGCAGCTGCTGCGGAAGCCTGTCCTCACCGCTATGGGCCTCCTGGCCCTGCTGGATGAGGAGCAGCTCTGGGCTGAGGTCTCCCAGGCTGGAACCGTGCTGGATTCCAATCATACCGTGGGAGTGCTCGCTTCCGCTCATAGGCCCCAGGGCCCTGCTGATGCCTGGAGGGCTGCTGTCCTGATTTATGCTAGCGACGATACCCGGGCCCATCCTAATAGGTCCGTGGCTGTGACCCTGAGGCTCAGGGGAGTGCCTCCTGGACCTGGCCTGGTCTATGTGACCAGGTATCTGGACAACGGCCTGTGCTCCCCTGATGGCGAATGGCGGAGGCTGGGCAGGCCTGTCTTTCCCACAGCCGAGCAGTTCCGGCGGATGAGGGCTGCTGAAGACCCTGTGGCTGCTGCCCCCAGGCCCCTGCCTGCTGGAGGAAGGCTCACCCTGAGGCCTGCCCTGAGGCTGCCTTCCCTGCTGCTGGTCCATGTGTGCGCCAGGCCCGAGAAGCCTCCTGGACAGGTGACCCGGCTGAGGGCTCTGCCCCTGACACAGGGCCAGCTGGTCCTGGTGTGGAGCGACGAGCATGTCGGCTCCAAGTGCCTGTGGACATACGAAATCCAGTTCTCCCAGGACGGCAAGGCCTACACACCCGTGAGCAGGAAGCCTTCCACATTCAACCTCTTCGTGTTCTCCCCCGATACAGGCGCCGTCAGCGGAAGCTACAGGGTGAGGGCCCTCGATTATTGGGCCAGGCCTGGCCCCTTTAGCGATCCTGTGCCTTATCTGGAGGTGCCCGTGCCTCGGGGACCTCCCAGCCCCGGCAACCCCTGA
<实施例2>
rhIDUA在CHO DG44细胞中的表达
将实施例1鉴定正确的重组质粒pCMV-IDUA(如图1)经电转法转染至CHO DG44细胞中,将电转后细胞混合液转移至含SFM4培养基的6孔板中,置于37℃孵箱中培养,48h后加入MTX(10nM)进行加压筛选,待细胞活率恢复至90%以上,MTX增加至100nM;细胞加入MTX至500nM,待活率恢复至90%以上后,将细胞转入摇瓶培养,在培养第3天和第5天分别补加葡萄糖(4g/L),继续培养至第7天。取培养液在4℃条件下,以5000rpm离心15min,收集上清经0.45μm滤膜过滤后进行纯化。
<实施例3>
rhIDUA的纯化
采用离子交换层析技术对上述表达细胞的上清进行纯化,具体操作如下:
阴离子交换层析:将1mL HiTrapTM Q HP预装柱与AKTA purifier层析系统相连。流动相A为:20mM Tris-HCl,pH=8.0;流动相B为:20mM Tris-HCl,含1M氯化钠,pH=8.0;流速1.5mL/min,检测波长254nm和280nm。先用流动相A平衡层析柱5-10CV,对已收集处理的细胞上清液进行上样,目的蛋白结合于介质上,部分杂质穿透。上样结束后,采用流动相A清洗层析柱3-5CV,最后用流动相B对其目的蛋白进行梯度洗脱(0-50%B,20min),收集洗脱样品并将缓冲液置换至20mM柠檬酸(pH=6.0)中。层析柱用3-5CV的1M氯化钠再生,并用0.1M氢氧化钠进行清洗。
阳离子交换层析:将1ml HiTrapTM SP HP预装柱与AKTA purifier层析系统相连。流动相A为:20mM柠檬酸,pH=6.0;流动相B为:20mM柠檬酸,含1M氯化钠,pH=6.0;流速1.5mL/min,检测波长254nm和280nm。先用流动相A平衡层析柱5-10CV,将阴离子层析透析样品上样,目的蛋白结合于介质上,部分杂质穿透。上样结束后,采用流动相A进行冲洗层析柱3-5CV,线性梯度洗脱0-30%B,20min。
<实施例4>
rhIDUA的生物活性检测
采用公开报道的酶活性检测方法测试了rhIDUA的生物活性,结果如表1所示,rhIDUA催化底物α-Liduronide的相对酶活力为16203.27±251.782pmol/min/μg,这与文献资料报告IDUA的酶活力大于7500pmol/min/μg相符合,表明本研究中的rhIDUA具有良好的生物活性,可用于MPS-Ⅰ型粘多糖贮积症的治疗。
表1 rhIDUA催化荧光底物酶活力检测
SEQUENCE LISTING
<110> 成都中医药大学
<120> 一种制备重组人α-L-艾杜糖醛酸酶的方法
<130> 人工序列的描述:rhIDUA的序列
<160> 1
<210> 1
<211> 1968
<212> DNA
<213> 人工序列
<223> 人工序列的描述:rhIDUA的序列
<400> 1
gccaccatga ggcctctgag gccccgtgct gctctgctcg ccctcctcgc ttccctgctg 60
gccgctcctc ctgtggctcc cgccgaagct cctcacctgg tgcaggtgga cgctgctagg 120
gctctgtggc ccctgaggcg gttttggcgg agcaccggct tctgtcctcc tctccctcac 180
agccaggccg atcagtatgt gctgtcctgg gaccagcaac tgaatctggc ctacgtggga 240
gccgtgcccc acaggggcat taagcaggtg aggacccact ggctgctgga gctggtcacc 300
acaaggggat ccaccggcag gggcctcagc tataacttca cccatctcga cggctacctg 360
gacctcctgc gggaaaacca gctgctccct ggcttcgagc tgatgggcag cgcctccggc 420
cactttacag acttcgagga taagcagcag gtgttcgaat ggaaggacct ggtcagctcc 480
ctcgctcgga ggtacattgg caggtacgga ctggcccacg tgtccaagtg gaactttgag 540
acctggaacg agcctgacca ccacgacttc gacaacgtgt ccatgaccat gcagggcttc 600
ctgaactact acgacgcctg cagcgagggc ctgagggccg cctcccccgc cctgaggctc 660
ggcggccctg gcgactcctt tcacacacct ccccgtagcc ccctgagctg gggactgctg 720
cggcattgcc atgacggcac caacttcttc acaggagagg ccggcgtgcg gctggattat 780
atctccctgc accggaaggg agctcggagc tccatcagca ttctggagca ggagaaagtg 840
gtggctcagc agatccggca actgttcccc aaattcgctg acacccccat ctacaacgat 900
gaggctgacc ctctcgtggg ctggagcctc cctcagcctt ggcgggctga cgtcacctac 960
gccgctatgg tcgtgaaggt catcgcccag catcaaaacc tgctcctcgc taacacaaca 1020
tccgctttcc cctacgccct gctgtccaac gacaacgcct ttctcagcta ccacccccat 1080
ccttttgccc agaggacact caccgcccgg tttcaggtga acaacaccag gcctcctcac 1140
gtgcagctgc tgcggaagcc tgtcctcacc gctatgggcc tcctggccct gctggatgag 1200
gagcagctct gggctgaggt ctcccaggct ggaaccgtgc tggattccaa tcataccgtg 1260
ggagtgctcg cttccgctca taggccccag ggccctgctg atgcctggag ggctgctgtc 1320
ctgatttatg ctagcgacga tacccgggcc catcctaata ggtccgtggc tgtgaccctg 1380
aggctcaggg gagtgcctcc tggacctggc ctggtctatg tgaccaggta tctggacaac 1440
ggcctgtgct cccctgatgg cgaatggcgg aggctgggca ggcctgtctt tcccacagcc 1500
gagcagttcc ggcggatgag ggctgctgaa gaccctgtgg ctgctgcccc caggcccctg 1560
cctgctggag gaaggctcac cctgaggcct gccctgaggc tgccttccct gctgctggtc 1620
catgtgtgcg ccaggcccga gaagcctcct ggacaggtga cccggctgag ggctctgccc 1680
ctgacacagg gccagctggt cctggtgtgg agcgacgagc atgtcggctc caagtgcctg 1740
tggacatacg aaatccagtt ctcccaggac ggcaaggcct acacacccgt gagcaggaag 1800
ccttccacat tcaacctctt cgtgttctcc cccgatacag gcgccgtcag cggaagctac 1860
agggtgaggg ccctcgatta ttgggccagg cctggcccct ttagcgatcc tgtgccttat 1920
ctggaggtgc ccgtgcctcg gggacctccc agccccggca acccctga 1968
Claims (7)
1.一种人α-L-艾杜糖醛酸酶表达载体,其特征在于,它是pCMV-IDUA表达质粒。
2.一种pCMV-IDUA表达质粒构建方法,其特征在于,用BglII和PacI对pCMV质粒进行双酶切后回收大片段,再用BglII和PacI对pUC-IDUA质粒进行双酶切回收目的基因片段,酶切后将产生相同黏性末端的载体片段与目的基因进行连接。
3.制备人α-L-艾杜糖醛酸酶的方法,其特征在于,包括用上述pCMV-IDUA表达质粒转化宿主细胞,培养转化体,获得人α-L-艾杜糖醛酸酶。
4.权利要求3所述方法,其特征在于,所述宿主细胞是中国仓鼠卵巢DG44细胞。
5.重组人α-L-艾杜糖醛酸酶,将权利要求1所述表达载体转化宿主细胞,培养转化体,从培养物获得重组人α-L-艾杜糖醛酸酶。
6.一种用中国仓鼠卵巢DG44(CHO DG44)细胞重组表达人α-L-艾杜糖醛酸酶(rhIDUA)的方法,其特征在于所述方法包括:
a.rhIDUA的碱基序列如SEQ ID NO:1所示;
b.构建pCMV-IDUA表达质粒;
c.在CHO DG44细胞的稳定表达;
d.离子交换层析纯化rhIDUA。
7.如权利要求6方法制备得到的rhIDUA,用于制备粘多糖贮积症MPS-Ⅰ型的治疗的生物制剂。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710952574.2A CN107699590A (zh) | 2017-10-13 | 2017-10-13 | 一种制备重组人α‑L‑艾杜糖醛酸酶的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710952574.2A CN107699590A (zh) | 2017-10-13 | 2017-10-13 | 一种制备重组人α‑L‑艾杜糖醛酸酶的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN107699590A true CN107699590A (zh) | 2018-02-16 |
Family
ID=61184142
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710952574.2A Pending CN107699590A (zh) | 2017-10-13 | 2017-10-13 | 一种制备重组人α‑L‑艾杜糖醛酸酶的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107699590A (zh) |
Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6149909A (en) * | 1995-06-23 | 2000-11-21 | Women's And Children's Hospital | Synthetic α-L-iduronidase and genetic sequences encoding same |
CN1300323A (zh) * | 1998-05-13 | 2001-06-20 | 加州大学洛杉矶分校港口研究和教育院 | 重组α-L-艾杜糖苷酸酶,其生产和纯化的方法以及治疗其缺乏导致的疾病的方法 |
US6426208B1 (en) * | 1999-11-12 | 2002-07-30 | Harbor-Ucla Research And Education Institute | Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof |
US6569661B1 (en) * | 1999-11-12 | 2003-05-27 | Biomarin Pharmaceutical Inc. | Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof |
US6858206B2 (en) * | 1999-11-12 | 2005-02-22 | Emil D. Kakkis | Methods for treating diseases caused by deficiencies of recombinant alpha-L-iduronidase |
CN1694958A (zh) * | 2002-09-13 | 2005-11-09 | 昆士兰大学 | 以密码子翻译效率为基础的基因表达系统 |
US20090053219A1 (en) * | 2007-07-27 | 2009-02-26 | Armagen Technologies, Inc. | Methods and compositions for increasing alpha-l-iduronidase activity in the cns |
WO2011034951A2 (en) * | 2009-09-15 | 2011-03-24 | The Regents Of The University Of California | Assisted enzyme replacement therapy |
TW201305334A (zh) * | 2010-10-22 | 2013-02-01 | Opko Curna Llc | 藉由抑制α-L-艾杜糖醛酸酶(IDUA)之天然反義轉錄本以治療與IDUA相關之疾病 |
CN103354837A (zh) * | 2011-01-25 | 2013-10-16 | 日本化学研究株式会社 | 重组人艾杜糖醛酸2-硫酸酯酶的生产方法 |
US20150258129A1 (en) * | 1998-06-01 | 2015-09-17 | Mount Sinai School Of Medicine Of New York University | Method for Increasing the Activity of Lysosomal Enzymes |
CN105026554A (zh) * | 2013-03-15 | 2015-11-04 | 宾夕法尼亚大学托管会 | 用于治疗mps1的组合物和方法 |
WO2017136500A1 (en) * | 2016-02-03 | 2017-08-10 | The Trustees Of The University Of Pennsylvania | Gene therapy for treating mucopolysaccharidosis type i |
WO2017147123A1 (en) * | 2016-02-22 | 2017-08-31 | The University Of North Carolina At Chapel Hill | Aav-idua vector for treatment of mps i-associated blindness |
-
2017
- 2017-10-13 CN CN201710952574.2A patent/CN107699590A/zh active Pending
Patent Citations (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6149909A (en) * | 1995-06-23 | 2000-11-21 | Women's And Children's Hospital | Synthetic α-L-iduronidase and genetic sequences encoding same |
CN1300323A (zh) * | 1998-05-13 | 2001-06-20 | 加州大学洛杉矶分校港口研究和教育院 | 重组α-L-艾杜糖苷酸酶,其生产和纯化的方法以及治疗其缺乏导致的疾病的方法 |
US20150258129A1 (en) * | 1998-06-01 | 2015-09-17 | Mount Sinai School Of Medicine Of New York University | Method for Increasing the Activity of Lysosomal Enzymes |
US6426208B1 (en) * | 1999-11-12 | 2002-07-30 | Harbor-Ucla Research And Education Institute | Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof |
US6569661B1 (en) * | 1999-11-12 | 2003-05-27 | Biomarin Pharmaceutical Inc. | Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof |
US6858206B2 (en) * | 1999-11-12 | 2005-02-22 | Emil D. Kakkis | Methods for treating diseases caused by deficiencies of recombinant alpha-L-iduronidase |
CN101407796A (zh) * | 1999-11-12 | 2009-04-15 | 生物马林药物股份有限公司 | 重组α-L-艾杜糖苷酶,其生产和纯化的方法以及治疗其缺陷导致的疾病的方法 |
CN1694958A (zh) * | 2002-09-13 | 2005-11-09 | 昆士兰大学 | 以密码子翻译效率为基础的基因表达系统 |
US20090053219A1 (en) * | 2007-07-27 | 2009-02-26 | Armagen Technologies, Inc. | Methods and compositions for increasing alpha-l-iduronidase activity in the cns |
WO2011034951A2 (en) * | 2009-09-15 | 2011-03-24 | The Regents Of The University Of California | Assisted enzyme replacement therapy |
TW201305334A (zh) * | 2010-10-22 | 2013-02-01 | Opko Curna Llc | 藉由抑制α-L-艾杜糖醛酸酶(IDUA)之天然反義轉錄本以治療與IDUA相關之疾病 |
CN103354837A (zh) * | 2011-01-25 | 2013-10-16 | 日本化学研究株式会社 | 重组人艾杜糖醛酸2-硫酸酯酶的生产方法 |
CN105026554A (zh) * | 2013-03-15 | 2015-11-04 | 宾夕法尼亚大学托管会 | 用于治疗mps1的组合物和方法 |
WO2017136500A1 (en) * | 2016-02-03 | 2017-08-10 | The Trustees Of The University Of Pennsylvania | Gene therapy for treating mucopolysaccharidosis type i |
WO2017147123A1 (en) * | 2016-02-22 | 2017-08-31 | The University Of North Carolina At Chapel Hill | Aav-idua vector for treatment of mps i-associated blindness |
Non-Patent Citations (1)
Title |
---|
KAKKIS, ED; MATYNIA, A; JONAS, AJ; 等.: "《OVEREXPRESSION OF THE HUMAN LYSOSOMAL-ENZYME ALPHA-L-IDURONIDASE IN CHINESE-HAMSTER OVARY CELLS》", 《PROTEIN EXPRESSION AND PURIFICATION 》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2021083072A1 (zh) | 人胶原蛋白17型多肽、其生产方法和用途 | |
CN104160033B (zh) | 将靶向肽偶联于重组溶酶体酶上的方法 | |
CN116218864B (zh) | 一种重组Ⅲ型人源化胶原蛋白α1及其表达载体和应用 | |
CN100562576C (zh) | 一种分泌表达重组人成纤维细胞生长因子-21的生产方法 | |
CN102863539B (zh) | 融合串联抗菌肽及其制备方法 | |
US12319926B2 (en) | Recombinant humanized collagen type I alpha-1 (rhCol1A1), and expression vector and use thereof | |
CN111378027B (zh) | 一种索玛鲁肽前体的制备方法 | |
CN116284453A (zh) | 猪流行性腹泻病毒s蛋白突变体及其制备方法、应用 | |
CN112608933B (zh) | 一种重组蓝铜肽前体-寡肽的高纯度制备方法 | |
CN116096898A (zh) | 人类α半乳糖苷酶变体 | |
TWI638826B (zh) | Method for producing human epithelial cell proliferative factor | |
CN107699590A (zh) | 一种制备重组人α‑L‑艾杜糖醛酸酶的方法 | |
CN118909812A (zh) | 人源化iii型胶原蛋白工程菌、载体、制备方法及应用 | |
CN113735960A (zh) | 一种fgf重组蛋白治疗nash的应用 | |
CN117229381A (zh) | 一种重组人参降血糖肽及其制备方法和应用 | |
CN115232805B (zh) | 一种硫酸软骨素裂解酶及其重组菌株和应用 | |
CN102277371A (zh) | 脑钠肽的制备方法 | |
CN118147106A (zh) | 酶、复合酶、固定化酶及Tirzepatide的制备方法 | |
CN115040637A (zh) | Rcn2重组蛋白在制备预防和/或治疗骨质疏松症以及提升免疫力的药物中的应用 | |
CN111575314A (zh) | 尿激酶受体稳定突变体suPARcc在真核胞外蛋白表达中的应用 | |
CN114746548A (zh) | 用于生产来自日本曲霉的果糖基转移酶的核酸、载体、宿主细胞和方法 | |
CN112142848A (zh) | 一种重组人胰岛素及其纯化制备方法 | |
CN112322602B (zh) | 一种共表达磷脂酶促进蛋白在枯草芽孢杆菌中胞外表达的方法 | |
CN103911388B (zh) | 重组艾塞那肽的生产工艺 | |
CN119020325B (zh) | 一种驴源大肠杆菌噬菌体的解聚酶Dp14及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180216 |