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Human keratinized cell growth factor-1 analogue preparation method and application thereof

A keratinocyte, growth factor technology, applied in the field of genetic engineering, can solve the problems of unsatisfactory KGF yield, high cost, time-consuming, etc., and achieves the effects of increasing activity, improving production efficiency, and reducing costs

Active Publication Date: 2011-02-23
吉林农大生物反应器工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the solubility and yield of the protein are improved, time-consuming, expensive protease costs and the risk of protease hydrolysis of the target protein often fail to obtain a satisfactory KGF yield

Method used

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  • Human keratinized cell growth factor-1 analogue preparation method and application thereof
  • Human keratinized cell growth factor-1 analogue preparation method and application thereof
  • Human keratinized cell growth factor-1 analogue preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] According to the nucleotide sequence (SEQ ID No.5) of human KGF1 on the NCBI network, its amino acid sequence is composed of 163 amino acids, as shown in (SEQ ID No.6), use DNAStar software to design and synthesize 13 primers, and apply the recursive PCR method Artificially synthesized the whole gene sequence of KGF, and designed a pair of primers at both ends of KGF to synthesize Δ23KGF, then designed a pair of primers at position 40, and mutated cysteine ​​to serine to obtain Δ23KGF (40S). And remove the NdeI and BamH I sites contained inside it. Escherichia coli preferred codons were selected for artificial synthesis in order to increase the level of recombinant expression.

[0027] F1 (SEQ ID No. 9): GACATACCCGTAGCTATGATTATATGGAAGGTGGTGATATTCGTGTGCGTCGTCTGTTT (79-137)

F2 (SEQ ID No. 10): AACAGATGGCGACGAATGTGAATTGCAGCAGCCCAGAAAGACATACCCGTAGCTATGAT (40-98)

F3 (SEQ ID No. 11): ACAGAGAACAGATTGGTGGTTGTAATGATATGACCCCGGAACAGATGGCGACGAATGTG (1-59)

...

Embodiment 2

[0083] SEQ ID NO: 27: Ulp-F2 (5'-3', 59 bases in total)

[0084] SEQ ID NO: 28: Ulp-F3 (5'-3', 59 bases in total)

[0085] SEQ ID NO: 29: Ulp-F4 (5'-3', 59 bases in total)

[0086] SEQ ID NO: 30: Ulp-F5 (5'-3', 46 bases in total)

[0087] SEQ ID NO: 31: Ulp-F6 (5'-3', 33 bases in total)

[0088] SEQ ID NO: 32: Ulp-F7 (5'-3', 59 bases in total)

[0089] SEQ ID NO: 33: Ulp-F8 (5'-3', 59 bases in total)

[0090] SEQ ID NO: 34: Ulp-F9 (5'-3', 59 bases in total)

[0091] SEQ ID NO: 35: Ulp-R1 (5'-3', 59 bases in total)

[0092] SEQ ID NO: 36: Ulp-R2 (5'-3', 59 bases in total)

[0093] SEQ ID NO: 37: Ulp-R3 (5'-3', 59 bases in total)

[0094] SEQ ID NO: 38: Ulp-R4 (5'-3', 59 bases in total)

[0095] SEQ ID NO: 39: Ulp-R5 (5'-3', 59 bases in total)

[0096] SEQ ID NO: 40: Ulp-R6 (5'-3', 59 bases in total)

[0097] SEQ ID NO: 41: Ulp-R7 (5'-3', 59 bases in total)

[0098] SEQ ID NO: 42: Ulp-R8 (5'-3', 59 bases in total)

[0099] SEQ ID NO: 43: Ulp-R9 (5'-3', 59 bases in to...

Embodiment 3

[0108] As far as the tandem connection between the two transcription units is concerned, the recombinant expression vector of the present invention first includes a mature peptide encoding a small molecule ubiquitin-related modifier operatively connected to the first promoter, which is similar in structure to human KGF-1 The nucleotide sequence of the fusion protein composed of the substance, and an insertion site for an operable second transcription unit. With this insertion site, the second transcription unit consisting of the nucleotide sequence encoding the ubiquitin-associated modifier protease and the second promoter upstream thereof can be easily inserted into the expression vector.

[0109] In order to construct the cDNA sequence of the fusion protein containing the mature peptide encoding small molecule ubiquitin-related modification factors under the control of the T7 promoter and the human KGF-1 structural analogue connected in tandem under the control of the T7 prom...

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Abstract

The invention provides a human keratinocyte growth factor -1 structure analogues KGF-1delta 23KGF(40S), an N end of an amino acid sequence of which lacks 23 amino acids, while the 40-bit cysteine point of which is mutated into a nonpolar amino acid. The invention also relates to a production method of the structure analogues, which carries out the fusion expression with a small ubiquitin related modifier gene mature peptide, while a fusion protein and the ubiquitin related modifier gene protease 1 co-express in the prokaryotes. In the process of fermentation expression, the ubiquitin related modifier gene protease 1 can hydrolyze the fusion protein to produce a soluble KGF-1delta 23KGF (40S). The human keratinocyte growth factor structure analogues can facilitate the proliferation of the keratinocyte cells, the growth of the hair follicle cells and inhibit the growth of the fibroblast cells, and has the functions of anti-scar, anti-fibrosis, epidermis healing facilitation and corneal wound reparation, etc.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically, the invention relates to a human keratinocyte growth factor-1 (Keratinocyte Growth Factor, KGF) structural analog protein, a gene encoding the protein, and a recombinant expression vector containing the gene. The present invention also relates to a method for expressing and producing human keratinocyte growth factor-1 structural analogue protein in a soluble form. Background technique [0002] Keratinocyte growth factor (KGF) is a kind of peptide growth factor with a wide range of biological activities. It belongs to the FGF family member (FGF-7). Repair and healing of chronic tissue damage (persistent damage in diabetic patients) and venous ulcers (including chronic gastrointestinal ulcers). Studies have shown that systemic administration of KGF is safe, and the normal human body has a good tolerance to it. Such as the intravenous injection Kepivance of Ameri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/475C12N15/12A61K38/17A61P27/02A61P17/02A61P17/14
Inventor 李校堃刘孝菊苏志坚
Owner 吉林农大生物反应器工程有限公司
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