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Recombinant human keratinized cell growth factor production method

A technology for keratinocytes and growth factors, applied in the field of bioactive polypeptides of human keratinocyte growth factors prepared by gene recombination technology, can solve the problems of toxicity, complicated process steps, low KGF expression efficiency, etc., to simplify the production process and improve production efficiency effect

Inactive Publication Date: 2009-09-30
信立泰(苏州)药业有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The process steps of the prior art are extremely complicated. At the same time, KGF is extremely toxic to E. coli hosts, and the expression efficiency of KGF in ordinary E. coli is very low. In order to overcome the above defects, this patent provides a method for the preparation of Preparation method of recombinant KGF

Method used

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  • Recombinant human keratinized cell growth factor production method
  • Recombinant human keratinized cell growth factor production method
  • Recombinant human keratinized cell growth factor production method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. Selection of expression vector

[0043] Choose Novagen’s pGEX series expression vectors such as pGEX-4T-3 as the expression vector. pGEX-4T-3 is an E. coli expression vector system for efficient fusion expression of proteins and polypeptides. The target protein or polypeptide is 225 amino acid residues Glutathione S-transferase (GST) was used as the fusion partner. (see attached figure 1 ).

[0044] 2. Gene design and synthesis:

[0045]According to the KGF1 DNA coding sequence reported in the literature (the sequence can be changed according to the principle of universality and degeneracy of the genetic code, but the amino acid composition and arrangement are not changed), the code is designed to mature into the KGF1 sequence, and BamHI is added to the 5-end of the sequence Recognition sequence GGATCC and enterokinase (Enterokinase) cleavage site coding sequence or thrombin (thrombin) cleavage site coding sequence; add the recognition sequence CTCGAG of Xho I at ...

Embodiment 2

[0051] According to the physicochemical property determination of the rhKGF of embodiment 1 gained

[0052] 1. Determination of the N-terminal 15 amino acid sequences

[0053] The recombinant human keratinocyte growth factor obtained by the above two methods was subjected to N-terminal assay, and the assay results were as follows:

[0054] N-Ser-Tyr-Asp-Tyr-Met-Glu-Gly-Gly-Asp-Ile-Arg-Val-Arg-Arg-Leu-C

[0055] exactly as expected.

[0056] 2. Quantitative mass spectrometry

[0057] The molecular weight of the recombinant human keratinocyte growth factor obtained by the two methods was determined by FAB method, which was 16278.5, which was consistent with the theoretical prediction.

[0058] 3. Determination of biological activity:

[0059] The biological activity of KGF1 was determined by using the characteristics of KGF1 to promote the proliferation of 4MBr-5 cells.

[0060] The measured results are:

[0061]

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Abstract

The present invention relates to a production method of recombinant human keratinocyte growth factor, in particular to a method for preparing a biologically active polypeptide of human keratinocyte growth factor using gene recombination technology, the method comprising the following steps: a. Artificially synthesizing human keratinocyte growth factor The gene corresponding to the factor amino acid coding region; b. the gene obtained in step a is fused with the 3' end of the glutathione S-transferase protein gene on a suitable expression vector, and the middle part of the gene contains thrombin or enterokinase recognition site point; c. transforming a suitable expression vector containing the gene encoding the fusion protein into a suitable Escherichia coli to obtain a genetically engineered bacterium; d. after the genetically engineered bacterium is fermented, the GST-KGF fusion protein is prepared through extraction and chromatography purification steps; e. After cleavage with thrombin or enterokinase, separate and purify to obtain recombinant keratinocyte growth factor.

Description

Technical field: [0001] The invention relates to a preparation method of a biologically active polypeptide, in particular to a method for preparing the biologically active polypeptide of human keratinocyte growth factor by using gene recombination technology. Background technique: [0002] Human keratinocyte growth factor (English name: Human Keratinocyte Growth factor, abbreviated: KGF or KGF1) is currently known as a cytokine that can promote the proliferation of non-fibroblast epithelial cells (especially keratinocytes). The histological distribution of KGFmRNA has its particularity: KGFmRNA is expressed in several stromal fibroblast cell lines from embryonic, neonatal and adult epithelial tissues, and it is also expressed in human kidney and gastrointestinal tract, while it is expressed in glial cells, lung KGF mRNA was not detected in , brain and various epithelial cells (including squamous cell carcinoma, bronchial epithelial cells, etc.). This histological distributi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/12C12N15/70C07K1/14C07K14/435C12P21/02A61K38/18
Inventor 赵波
Owner 信立泰(苏州)药业有限公司
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