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Production process of neurotrophic factor

A technology of neurotrophic factors and production methods, which is applied in the field of genetic engineering, can solve problems such as not being suitable for large-scale preparation, and achieve the effects of shortening renaturation time and reducing polarization phenomena

Inactive Publication Date: 2004-05-26
HANGZHOU JIUYUAN GENE ENG
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Problems solved by technology

CNTF has been cloned and expressed by bacterial systems (Masiakowski, et al.1991, J.Neurosci.57:1003-1012; Annalise Di Marco et al.1996, Proceedings of the National Academy of Sciences of the United States of America), but these methods are only suitable for preparing a small number of samples For scientific research, not suitable for large-scale preparation

Method used

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  • Production process of neurotrophic factor
  • Production process of neurotrophic factor
  • Production process of neurotrophic factor

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Experimental program
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Embodiment

[0020] Construction of CNTF engineering bacteria

[0021] Synthesized and cloned on the T-vector by Gene Synthesis Company and verified by sequencing, purified and recovered, digested with NdeI, BamHI and then purified and recovered; pGENS plasmid was also digested with NdeI, BamHI and XhoI, and the large fragment was recovered; The recovered CNTF gene fragment and the large vector fragment were ligated in vitro to form the recombinant plasmid pGENS CNTF, as shown in FIG. 2 . The recombinant plasmid was transformed into the Escherichia coli recipient strain BL21(DE3)pLYSS, and a transformant with a completely correct structure was screened through the verification process of enzyme digestion, PCR, and DNA sequencing. After shake flask expression screening, it was determined that pGENS CNTF2 was the original engineering bacterium for production.

[0022]The pGENS CNTF plasmid is an expression plasmid constructed by our company, including the following components: ColEl replico...

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Abstract

The present invention discloses the production process of neurotrophic factor. The production process includes computer-aided design, modifying and altering the gene of ciliary nerve trophic factor to obtain modified mutation human ciliary nerve trophic factor cDNA, artificially synthesizing the cDNA, constituting recombinant plasmid, converting to bacillus coli, high density expression in automatic fermentation tank; renaturation in tangent flow superfiltering mode; ion exchange chromatography, hydrophobic chromatography and molecular sieve chromatography to purify and thus to obtain high-purity ciliary nerve trophic factor. The present invention has the advantages of stable and efficient expression of mutant in bacillus coli, less polarization insuperfiltering course and high purity of renaturated ciliary nerve trophic factor.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a production method of neurotrophic factors. Background technique [0002] CNTF was first discovered in vitro to promote the survival of ciliary ganglion in chick embryos (Manthorpe et al., 1980, J.Neurochem.34:69-75). In the following decades, many more Related biological functions were discovered. Hughes et al. found that it can promote the differentiation of glial progenitor cells in the visual and cranial nerves of perinatal mice (Hughes et al., 1988, Nature 335:70-73), and also found that it can promote the differentiation of dorsal root ganglia in chick embryos. Growth (Skaper and Varon, 1986, Brain Res. 389:39-46). In addition, he can also promote the growth and differentiation of motor neurons, hippocampal neurons and parasympathetic spinal cord neurons (Ip, et al.1991, J.Neurosci.11: 3124-3134; Blottner, et al.1989, Neurosci.Lett .105:316-3...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K14/48C12N15/12C12P21/02
Inventor 黄岩山王昌梅裘霁宛李辉孙瑛邹晔孙汉栋丁海
Owner HANGZHOU JIUYUAN GENE ENG
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