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Production process of human horny cell growth factor-2

A technology of keratinocytes and growth factors, applied in biochemical equipment and methods, botany equipment and methods, genetic engineering, etc., can solve problems such as low yield, complicated purification process, and high cost

Inactive Publication Date: 2006-03-29
武汉光谷亚太药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] At present, in this field, Escherichia coli expresses KGF-2 mostly in the form of inclusion bodies, and the expression amount accounts for 10-15% of the total protein of the bacteria, which requires denaturation and renaturation means, heavy workload, low yield and high cost
For example, HGS uses the pREP4 plasmid as a vector, and bacterial OD 600 When it grows to 0.4-0.6, it is induced with IPTG. After 3-4 hours of induction, the bacteria are obtained, dissolved in 6M guanidine hydrochloride, and then purified. This process can not be high in KGF-2 expression, and the purification process is complicated (Ruben; Steven M., et al. al., Keratinocyte growth factor-2, US6077692)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Construction of KGF-2 secretion expression system

[0074] 1. Acquisition of target gene

[0075] The expression plasmid is pSE380 (Invitrogen), and the host strain is E. coli BL21 (DE3).

[0076] According to the KGF-2 cDNA sequence reported in the literature, 20 complementary oligonucleotides were synthesized, and specific restriction sites EcoR I and BamH I were introduced at the 5'end and 3'end respectively. According to the conventional method of molecular cloning, first Treated with T4 phage polynucleotide kinase at 37°C for 30 min. The phosphorylated oligonucleotide fragments were mixed in equal mols, denatured at 94°C for 5 minutes, and immediately changed to 65°C for annealing for 10 minutes, and then T4 ligase was added and reacted at 16°C overnight.

[0077] 2. Construction of expression plasmid pSE380-KGF-2 and transformation of host bacteria

[0078] After pSE380 plasmid was digested with EcoR I and BamH I, the large fragment was recovered, and the synthesized ...

Embodiment 2

[0082] Medium selection

[0083] Ingredients

[0084] The results show that low concentration of glucose (0.2-0.5%) helps to increase expression, but too high glucose concentration is not conducive to expression. Under the same conditions, medium 3 has the best effect.

Embodiment 3

[0086] Can expression (5L)

[0087] 1. Bacteria:

[0088] Culture E. coli BL21(DE3) / pSE380-KGF-2 in LBA for 20hr, OD 600 Up to 2.7.

[0089] 2. Basic medium: Medium 2 in Example 2

[0090] 3. Fermentation culture stage:

[0091] Temperature: 35℃

[0092] pH: 6.8

[0093] OD at induction 600 : 2.0

[0094] Co-cultivation for 5.5 hours

[0095] 4. Fermentation induction stage:

[0096] Temperature: 35℃

[0097] PH: 6.8

[0098] IPTG: Addition of lactose: 3:1 (amount ratio, where IPTG is 0.5mM)

[0099] End of induction OD 600 : 5.8

[0100] 2 hours total induction

[0101] 5. Experimental results:

[0102] After 2 hours of induction, the expression of KGF-2 accounted for about 18% of the total protein of the fermentation supernatant, and the expression reached 100-200ug / ml fermentation broth (measured by the modified Lowry method).

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Abstract

The production process of human horny cell growth factor-2 includes the steps of: culturing engineering colibacillus carrying expression vector selected from pSE280, PSE380 and pSE420 under the proper expression condition and inserting the encoding sequence of human horny cell growth factor in the polyclonal site of the said expression vector so as to express human horny cell growth factor; and separating and purifying the expressed human horny cell growth factor-2. The production process of the present invention has high expression amount and simple separation and purification steps. The present invention provides also corresponding expression vector and engineering bacterial.

Description

Technical field [0001] The present invention relates to a production method of human keratinocyte growth factor-2 (Keratinocyte Growth Factor-2, KGF-2), and an expression vector and host cell used in the method. Background technique [0002] The treatment of chronic trauma varies with the severity of the injury. Partial and full-thickness injuries are generally debrided by dressing and removing necrotic tissue with drugs or surgery. Antibiotics can be used to prevent infection. Partial thickness damage to full thickness damage represents the largest group of chronically injured patients, and the most need for treatment with cytokines such as KGF-2. In patients with full-thickness injuries, the injury extends to muscles, tendons, or bones. There is a great risk of sepsis, and surgical treatment is usually used. The treatment of chronic trauma has always been a clinically difficult problem, and there is no effective means. There are almost 3 million patients in the United States ea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C12N15/12C12N15/63C12N15/79
Inventor 黄阳滨朱建欣邱林峰
Owner 武汉光谷亚太药业有限公司
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