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Method for preparing recombinant human keratinocyte growth factor-2 by secretion expression

A technology for keratinocytes and growth factors, which is applied in the field of preparation of optimized secretion and expression of recombinant human keratinocyte growth factor, can solve the problems of protein destruction, increase the difficulty and cost of purification, etc.

Inactive Publication Date: 2009-09-23
WENZHOU MEDICAL UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the production process of the rhKGF-2 E. coli expression system in the world, whether the expression product exists in the form of soluble protein or not, is expressed in the cytoplasm, and the protein must be broken by ultrasonic waves to release the protein, which not only affects the protein itself Has a destructive effect, but also increases the difficulty and cost of purification

Method used

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  • Method for preparing recombinant human keratinocyte growth factor-2 by secretion expression
  • Method for preparing recombinant human keratinocyte growth factor-2 by secretion expression
  • Method for preparing recombinant human keratinocyte growth factor-2 by secretion expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Construction of engineering bacteria expressing rhKGF-2

[0052] 1. Synthesis of the target gene

[0053] According to the known natural amino acid sequence of rhKGF-2, the coding sequence of rhKGF-2 was designed without changing the amino acid sequence according to the codon preference of E. primers. The full-length sequence of rhKGF-2 was obtained by bridging PCR.

[0054] When artificially synthesizing the complete sequence of the recombinant human keratinocyte growth factor-2 (rhKGF-2) gene, a .NcoI restriction site is introduced at the 5' end of the gene and a HindIII restriction site is introduced at the 3' end.

[0055] 2. Construction of expression plasmid-pET22b(+)-rhKGF-2 and transformation of host bacteria

[0056] The expression plasmid is pET22b(+) expression vector purchased from InVitrogen (the expression vector contains an IPTG-induced promoter and peIB secretion signal state), and the host bacteria is Escherichia coli BL21(DE3).

[0057] The gene rh...

Embodiment 2

[0062] Selection of Appropriate Expression Vectors

[0063] According to the method similar to Example 1, the KGF-2 sequence was inserted into several different expression vectors, such as pET28a(+) (purchased from Invitorgen), pET29a(+) (purchased from Invitrogen), pET-30Ek / LIC purchased from Invitorgen) with the same restriction site (NcoI / HindIII) to obtain plasmids pET28a(+)-rhKGF-2, pET29a(+)-rhKGF-2, and pET-30Ek / LIC-rhKGF-2.

[0064] The plasmids pET28a(+)-rhKGF-2, pET29a(+)-rhKGF-2, and pET-30Ek / LIC-rhKGF-2 were respectively transformed into corresponding host bacteria, and resistant strains were selected. Shake flask test was carried out together with engineering bacteria containing plasmid pET22b(+) / rhKGF-2. After 2 hours of IPTG (or arabinose) induction, the expression of the target protein expressed by engineering bacteria containing plasmid pET22b(+)-rhKGF-2 The amount accounts for about 20% of the total protein, while the expression amount of the target protein ...

Embodiment 3

[0067] Choose the best medium

[0068] Pick the Escherichia coli BL21 (DE3) monoclonal that transfers to pET22b (+)-rhKGF-2 prepared in Example 1, inoculate in the 250ml Erlenmeyer flask that contains 50ml LB seed liquid, cultivate 3~4hr, wait for OD 600 reach 0.8-1.0, inoculate in a 1000ml Erlenmeyer flask containing 250ml improved seed solution at a ratio of 1:10, cultivate for about 8-10h, and wait for OD 600 When it reaches 6-8, put it into the tank for fermentation according to 1:10, add carbon source (glycerol), magnesium salt, and ammonia water to adjust pH 6.8-7.0, temperature 37°C, DO>30%, wait for OD 600 When it reaches 15~18, add 0.5~0.8mMIPTG to start induction, add carbon source (glycerol), nitrogen source, and phosphate buffer to adjust pH to 7.2~7.4, DO>50%, temperature 30~32℃, induction 4~ 5hrs are over. The samples were detected by SDS-PAGE (and scanned) to determine the protein content.

[0069]

[0070]

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Abstract

The invention provides a method for preparing recombinant human keratinocyte growth factor-2 by secretion expression. The method provides an expression vector of the recombinant human keratinocyte growth factor-2, which contains an secretion signal peptide coding sequence and can secrete KGF-2 protein in periplasm for expression; and when a host cell containing the recombinant plasmid is subjected to repeated freeze thawing at a low temperature, the recombined protein can be released in a buffer solution under the condition of not damaging inner membranes of bacilli and the activity of the protein.

Description

technical field [0001] The invention relates to a preparation method for optimally secreting and expressing recombinant human keratinocyte growth factor (rhKGF-2), as well as an expression vector and a host cell used in the method. Background technique [0002] Keratinocyte growth factor (keratinocyte growth factor, KGF) was isolated and purified from the culture supernatant of human fetal lung fibroblasts by Rubin et al. in 1989, and was found to have the effect of promoting mouse keratinocyte mitosis, so it was named keratinocyte growth factor. In 1996, Yamasaki et al. amplified a new growth factor from mouse embryos, another new cytokine closely related to KGF, called KGF-2, also known as fibroblast growth factor-10 (FGF -10). Afterwards, Emoto et al. cloned the cDNA of human KGF-2 from human lungs. The gene is located at 5p12-5p13 of human chromosome 5. Human FGF-10 consists of 624 nucleotides, encodes 208 amino acids, and has a molecular weight of 19.3KD. It has 57% ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C12N1/21C07K14/475C12R1/19
Inventor 李校堃王晓杰张云龙田海山王会岩黄志锋黄亚东王艳芳姚娜杨萍肖业臣刘孝菊肖健
Owner WENZHOU MEDICAL UNIV
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