Method of preparing recombinant human alpha-L-iduronidase

An iduronidase and purpose technology, applied in the field of genetic engineering, can solve the problems of lack of treatment research, high price, unfavorable development of gene therapy technology, etc., and achieve the effect of good application effect and high purity

Inactive Publication Date: 2018-02-16
CHENGDU UNIV OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, although foreign large-scale medical companies have produced therapeutic reagents for recombinant human α-L-iduronidase, the price of this reagent is relatively high, which is unfavorable for the treatment of clinical patients.
Due to the lack of therapeutic drugs, the domestic treatment research in this field is seriously lacking, which is not conducive to the development of gene therapy technology, especially the slow development of treatment technology for MPS-I infants

Method used

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  • Method of preparing recombinant human alpha-L-iduronidase
  • Method of preparing recombinant human alpha-L-iduronidase
  • Method of preparing recombinant human alpha-L-iduronidase

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Experimental program
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Effect test

Embodiment 1

[0032] rhIDUA expression plasmid construction

[0033] The IDUA coding region gene sequence (GenBank: M74715.1) was optimized and synthesized for the CHO species, and the synthesized gene sequence was recombined into the plasmid vector pUC19 to obtain a plasmid containing the target gene as pUC-IDUA. Use BglII and PacI to double digest the pCMV plasmid and recover the large fragment, then use BglII and PacI to double digest the pUC-IDUA plasmid to recover the target gene fragment. After digestion, the vector fragment with the same sticky end and the target gene will be produced. to connect. The ligation product was transformed into Top10 Escherichia coli competent cells, spread on 2YT plate medium, and stood at 37°C overnight. Pick a single colony for small-scale culture and extract the plasmid for identification.

[0034] The sequence of rhIDUA is as follows:

[0035] SEQ ID No 1

[0036]GCCACCATGAGGCCTCTGAGGCCCCGTGCTGCTCTGCTCGCCCTCCTCGCTTCCCTGCTGGCCGCTCCTCCTGTGGCTCCCGCCG...

Embodiment 2

[0038] Expression of rhIDUA in CHO DG44 cells

[0039] The correct recombinant plasmid pCMV-IDUA (such as figure 1 ) were transfected into CHO DG44 cells by electroporation, and the cell mixture was transferred to a 6-well plate containing SFM4 medium, cultured in a 37°C incubator, and MTX (10nM) was added after 48h for pressurized screening , when the cell viability recovered to more than 90%, MTX was increased to 100nM; the cells were added with MTX to 500nM, and after the viability recovered to more than 90%, the cells were transferred to shake flasks for culture. On the third and fifth days of culture, respectively, Glucose (4g / L) was added, and the cultivation was continued until the 7th day. The culture solution was taken and centrifuged at 5000 rpm for 15 min at 4°C, and the supernatant was collected and filtered through a 0.45 μm filter membrane for purification.

Embodiment 3

[0041] Purification of rhIDUA

[0042] The supernatant of the above expression cells was purified by ion exchange chromatography, and the specific operations were as follows:

[0043] Anion exchange chromatography: 1mL HiTrap TM The Q HP prepacked column is connected to the AKTA purifier chromatography system. Mobile phase A: 20mM Tris-HCl, pH=8.0; mobile phase B: 20mM Tris-HCl, containing 1M sodium chloride, pH=8.0; flow rate 1.5mL / min, detection wavelength 254nm and 280nm. First equilibrate the chromatography column with mobile phase A for 5-10CV, and load the collected and treated cell supernatant. The target protein is bound to the medium, and some impurities penetrate. After loading the sample, use mobile phase A to wash the chromatography column for 3-5CV, and finally use mobile phase B to carry out gradient elution (0-50% B, 20min) for the target protein, collect the eluted samples and replace the buffer to 20mM citric acid (pH=6.0). The column was regenerated wi...

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Abstract

The invention relates to a human alpha-L-iduronidase expression vector which is a pCMV-IDUA expression plasmid and a preparation method of recombinant human alpha-L-iduronidase. The method includes that by cloning, expressing and coding genes of human alpha-L-iduronidase, a Chinese hamster ovary cell line DG44 is transfected, and stable expression of alpha-L-iduronidase in cells is realized, so that alpha-L-iluronidase which is obtained finally has bioactivity of catalyzing substrate alpha-Liduronide enzymatic reaction and is suitable for being used to treat mucopolysaccharidosis I typr (MPS-I) caused by glycosaminoglycan catabolism disturbance due to alpha-L-iduronidase deficiency.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a human α-L-iduronidase gene recombination vector, a method and an application for recombinantly expressing human α-L-iduronidase in a host transformed with the vector. Background technique [0002] Mucopolysaccharide storage disease (Mucopolysaccharide, MPS) is caused by the deficiency of acidic mucopolysaccharide degrading enzymes, resulting in glycosaminoglycan degradation metabolism disorders. Ⅰ is the type with a relatively high incidence in my country. Lysosomal α-L-iduronidase (α-L-iduronidase, IDUA) deficiency causes glycosaminoglycans (glycosaminoglycans, GAGs) degradation metabolism disorder, which leads to MPS-Ⅰ, which mostly occurs in infancy or childhood. A disabling and fatal inherited metabolic disorder. Recombinant α-L-iduronidase (rhIDUA) as an enzyme replacement therapy (ERT) drug in clinical treatment shows that all patients can safely pass through c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N9/24A61K38/47A61P3/00
Inventor 刘小章高小平
Owner CHENGDU UNIV OF TRADITIONAL CHINESE MEDICINE
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