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Modified Keratinocyte growth factor gene and its expression in yeast

A keratinocyte and growth factor technology, applied in the field of genetic engineering, can solve the problems of low expression, protein change, difficulty in renaturation, etc., and achieve the effects of short cycle, low cost and easy technology

Active Publication Date: 2011-11-16
QILU PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The KGF currently approved for marketing is expressed and produced by Escherichia coli, and the protein produced by Escherichia coli has difficulties in denaturation and renaturation; Heterologous expression of KGF, but the expression level is too low to be industrialized

Method used

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  • Modified Keratinocyte growth factor gene and its expression in yeast
  • Modified Keratinocyte growth factor gene and its expression in yeast
  • Modified Keratinocyte growth factor gene and its expression in yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] 1. Gene synthesis

[0064] The KGF gene sequence is from Genebank, accession number NM_002009, and its sequence is modified as follows: (1) delete 69 nucleotides at the 5' end of the gene; (2) follow the codon preference of Pichia pastoris without changing the amino acid sequence (3) Whole gene synthesis of keratinocyte growth factor sequence.

[0065] 2. Construction of recombinant plasmids

[0066] The pUC57-KGF plasmid was double cut with restriction endonucleases XhoI and EcoRI, the KGF target fragment was recovered by gel, and then ligated with the same cut pPIC9 plasmid. The ligation product was transformed into Escherichia coli JM109 competent cells, screened on LB plates (containing Amp), randomly selected strains to extract plasmids, and verified by XhoI / EcoRI double enzyme digestion (results are shown in the attached figure 1 ), after the verification is correct, further sequencing verification is carried out.

[0067] 3. Transformation and plate screening ...

Embodiment 2

[0076] 1. Gene synthesis

[0077] With embodiment 1.

[0078] 2. Construction of recombinant plasmids

[0079] The pUC57-KGF plasmid was double cut with restriction endonucleases XhoI and EcoRI, the KGF target fragment was recovered by gel, and then ligated with the pGAPZαA plasmid cut with the same enzyme to obtain the recombinant plasmid pGAPZαA-KGF. The recombinant plasmid pGAPZαA-KGF was transformed into Escherichia coli JM109 competent cells, and then plate screening, double enzyme digestion verification and sequencing verification were performed, and the next experiment was carried out after correctness.

[0080] 3. Transformation and plate screening of Pichia pastoris strain GS115

[0081] Recombinant plasmid pGAPZαA-KGF and blank plasmid pGAPZαA were digested with restriction endonuclease SacI, and 5 μg-20 μg of the plasmid was electroporated to transform Pichia pastoris GS115 competent cells. The specific method was the same as in Example 1.

[0082] 4. Shake flask...

Embodiment 3

[0085] 1. Gene synthesis

[0086] With embodiment 1.

[0087] 2. Construction of recombinant plasmids

[0088] The pUC57-KGF plasmid was double cut with restriction endonucleases XhoI and EcoRI, the KGF target fragment was recovered by gel, and then ligated with the pPICZαA plasmid cut with the same enzyme to obtain the recombinant plasmid pPICZαA-KGF. The recombinant plasmid pPICZαA-KGF was transformed into Escherichia coli JM109 competent cells, then screened on LB plates containing Zeocin antibiotics, double enzyme digestion verification and sequencing verification, and the next experiment was carried out after correctness.

[0089] 3. Transformation and plate screening of Pichia pastoris strain GS115

[0090] Recombinant plasmid pPICZαA-KGF and blank plasmid pPICZαA were digested with restriction endonuclease SacI, and 5 μg-20 μg of the plasmid was electroporated to transform Pichia pastoris GS115 competent cells. The specific method was the same as in Example 1, and the...

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Abstract

The invention discloses a Keratinocyte growth factor gene, vectors with the Keratinocyte growth factor gene and an application of the Keratinocyte growth factor gene, and belongs to the technical field of genetic engineering. The invention is characterized by comprising a gene segment which codes a Keratinocyte growth factor and has a nucleotide sequence shown in the formula SEQ ID NO.1, expression vectors with the gene segment and a system of expressing the Keratinocyte growth factor in pichia yeast through the expression vectors. Through the system of expressing a Keratinocyte growth factorin pichia yeast, the advantages of low cost, short cycle, easy realization of a technical scheme, and favorable expression level reaching to an industrialized production level are realized.

Description

technical field [0001] The invention relates to a keratinocyte growth factor expression gene and its expression and application, belonging to the technical field of genetic engineering. Background technique [0002] Keratinocyte growth factor (KGF) belongs to the fibroblast growth factor family (fibroblast growth factor, FGF), also known as FGF7. KGF was isolated and purified by Rubin from the conditioned medium of embryonic lung fibroblasts by ultrafiltration, affinity chromatography and reversed-phase high-pressure liquid phase (RP-HPLC) in 1989. The molecule has a strong Unlike other fibroblast growth factors that can affect the growth of almost all cells of mesoderm origin, the activity of KGF is mainly limited to epithelial cells, and has the effect of specifically promoting the mitosis of epithelial cells, especially keratinocytes , hence the name keratinocyte growth factor. [0003] The expression of KGF is limited to mesenchymal cells, while its receptors are expre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/63C12N1/19A61K38/18A61P35/00C12R1/84
Inventor 王晶翼李剑凤艾丽静王希菊刘克玲宫新江王庆民孙丽霞
Owner QILU PHARMA CO LTD
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