Production process of human horny cell growth factor-2

A technology for keratinocytes and growth factors, which can be used in biochemical equipment and methods, botanical equipment and methods, genetic engineering, etc., and can solve problems such as low yield, large workload, and complicated purification procedures.

Inactive Publication Date: 2003-08-20
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 b...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Construction of KGF-2 Secretion and Expression System

[0074] 1. Obtaining the target gene

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

[0076] According to the KGF-2 cDNA sequence reported in the literature, 20 complementary oligonucleotides were synthesized, and specific enzyme cutting 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 Treat with T4 phage polynucleotide kinase at 37°C for 30min. The phosphorylated oligonucleotide fragments were mixed in equal molar numbers, denatured at 94°C for 5 minutes, and immediately annealed at 65°C for 10 minutes, then added T4 ligase, and reacted overnight at 16°C.

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

[0078] The pSE380 plasmid was digested with EcoR I and BamH I, and the large fragment was reclaimed, connec...

Embodiment 2

[0082] Medium selection

[0083] Element

[0084] The results showed that low concentration of glucose (0.2-0.5%) was helpful to increase the expression level, but too high concentration of glucose was not conducive to the expression. Under the same conditions, No. 3 medium had the best effect.

Embodiment 3

[0086] Express on the tank (5L)

[0087] 1. Bacteria:

[0088] Escherichia coli BL21(DE3) / pSE380-KGF-2 was cultured in LBA for 20hr, OD 600 up to 2.7.

[0089] Two, basal medium: medium 2 in embodiment 2

[0090] 3. Fermentation and cultivation stage:

[0091] Temperature: 35°C

[0092] pH: 6.8

[0093] OD 600 : 2.0

[0094] Co-culture for 5.5 hours

[0095] 4. Fermentation induction stage:

[0096] Temperature: 35°C

[0097] pH: 6.8

[0098] IPTG: Lactose added amount: 3:1 (quantity ratio, where IPTG is 0.5mM)

[0099] end of induction OD 600 : 5.8

[0100] Total induction 2 hours

[0101] 5. Experimental results:

[0102] After 2 hours of induction, the expression level of KGF-2 accounts for about 18% of the total protein in the fermentation supernatant, and the expression level reaches 100-200ug / ml fermentation broth (measured by the improved 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 invention relates to a production method of human keratinocyte growth factor-2 (Keratinocyte Growth Factor-2, KGF-2), an expression vector and a host cell used in the method. Background technique [0002] Treatment of chronic trauma varies with the severity of the injury. Partial and full-thickness lesions are generally treated with dressings and medical or surgical removal of necrotic tissue known as debridement. Antibiotics are available to prevent infection, and partial-thickness to full-thickness lesions represent the largest group of patients with chronic injuries, most in need of treatment with cytokines such as KGF-2. Patients with full-thickness injuries that extend to muscle, tendon, or bone are at great risk of sepsis and are usually treated surgically. The treatment of chronic trauma has always been a clinically difficult problem, and there is no effective means. There are nearly 3 million patients in the United States every year, and...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N15/79C12P21/02
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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