Human stem cell growth factor as well as production method and application of polyethylene glycol (PEG) modified human stem cell growth factor
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A growth factor and polyethylene glycol technology, applied in the field of medicine and biology, can solve the problems of reduced drug efficacy, achieve the effects of prolonging the half-life, increasing the activity of promoting red blood cell proliferation, and mild modification conditions
Inactive Publication Date: 2012-07-11
GUANGZHOU JINAN BIOMEDICINE RES & DEV CENT
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[0006] It can be seen that protein drugs are easily degraded by proteases in vivo, and PEG-modi
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Embodiment 1
[0042] Example 1: Recombinant expression of human stem cell growth factor
[0043] 1. Construction of SUMO-rhSCF-α fusion gene
[0044] 1. Primer design:
[0045] Primers were designed according to the SUMO gene sequence (GenBank no: NC_011677.1) and the rhSCF-α gene sequence (GenBank: AB009244.1) (see Table 1).
[0046] Table 1 is used to synthesize the primers of SUMO-rhSCF-full-length sequence
[0047]
[0048] Note: Bold is the SUMO gene sequence; underlined and bold is the His tag sequence; underlined is the 15-peptide linker gene sequence; italic is the added restriction site; underlined is the rhSCF-α gene sequence.
[0049] 2. PCR reaction:
[0050] (1) mRNA was extracted from human umbilical cord mesenchymal stem cells (hUCMSCs) by conventional methods, and cDNA of rhSCF-α was amplified by RT-PCR.
[0051] Use M-MLV reverse transcriptase to synthesize first-strand cDNA (20μl reaction system can be used for reverse transcription of 1ng~5μg total RNA or 1~500ngmR...
Embodiment 2
[0128] PEG Modification of Human Stem Cell Factor 1
[0129] 1) PEG modification: In pH=6.0, 50 mmol / L phosphate buffer, rhSCF-α protein concentration is 5 mg / mL, 20 kDa monomethoxypolyethylene glycol propionaldehyde ( mPEG-ALD-20kDa), reacted at 25°C for 12 hours, and added glycine to terminate the reaction.
[0130] 2) Separation and purification: use Superdex 200 molecular sieve column chromatography, use pH 7.0, 50mmol / L PB containing 150mmol / L NaCl as the mobile phase, collect the second wash peak, and obtain mPEG-ALD-20kDa- with a purity of 92.4%. rhSCF-a.
Embodiment 3
[0132] PEG Modification of Human Stem Cell Factor 2
[0133] 1) PEG modification: In pH=6.0, 50mmol / L phosphate buffer, rhSCF-α protein concentration is 5.5mg / mL, 20kDa monomethoxypolyethylene glycol propionaldehyde is added at a mass ratio of 2:1 (mPEG-ALD-20kDa), react at 4°C for 10 hours, and add glycine to terminate the reaction.
[0134] 2) Separation and purification: use Superdex 200 molecular sieve column chromatography, use pH 7.0, 50mmol / L PB containing 150mmol / L NaCl as the mobile phase, collect the second wash peak, and obtain mPEG-ALD-20kDa- with a purity of 93.5%. rhSCF-a.
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Abstract
The invention discloses a human stem cell growth factor as well as a production method and application of a polyethylene glycol (PEG) modified human stem cell growth factor. The production method comprises the following steps of: fusing an h-SCF (Stem Cell Factor)-alpha sequence and an SUMO (Small Ubiquitin-Related Modifier) sequence and constructing to obtain an SUMO-rhSCF-alpha fused gene expression vector; transferring the SUMO-rhSCF-alpha fused gene expression vector into a host bacteria to obtain engineering bacteria; culturing the engineering bacteria and inducing to express an SUMO-rhSCF-alpha fused protein; and cutting off an SUMO part to obtain an rhSCF-alpha protein. By using the production method, the high soluble expression and large-scale purification of the rhSCF-alpha protein in cell plasmas are realized, and the activity of the obtained rhSCF-alpha protein is high. The invention also discloses the production method of the PEG modified human stem cell growth factor; the half-life period of the PEG modified human stem cell growth factor obtained by using the method is remarkably prolonged, and the activity of the PEG modified human stem cell growth factor in promoting the proliferation of red blood cells is also remarkably improved; and the PEG modified human stem cell growth factor can be applied to the preparation of medicines for hypohemia therapy, reconstruction and recovery of a hematopoietic function after chemoradiotherapy and a bone marrow transplantation operation, stem cell ex-vivo expansion and gene therapy and cosmetics for promoting the metabolism of epidermal cells, repairing aged and damaged skin cells, delaying the aging of skin and the like.
Description
technical field [0001] The invention belongs to the technical field of medicine and biology, and specifically relates to a high-efficiency expression and purification method of human stem cell growth factor, a preparation method of polyethylene glycol-modified human stem cell growth factor and its application. Background technique [0002] Stem cell growth factor (SCF) is an acidic glycoprotein produced by stromal cells in the bone marrow microenvironment. It is an important hematopoietic cytokine that mainly acts on early hematopoietic stem cells and primitive hematopoietic progenitor cells, promoting their proliferation and Differentiation plays an important role in maintaining the survival of hematopoietic cells, promoting the proliferation and differentiation of hematopoietic cells, and regulating the growth and development of various hematopoietic cells. The human SCF gene is located on human chromosome 12q22-12q24, is 1.4kb long, and contains 7 introns and 8 exons. Na...
Claims
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