Construction and screening method for recombinant human epidermal growth factor engineering bacteria

An epidermal growth factor and screening method technology, which is applied in the field of construction and screening of high-efficiency intracellular soluble expression recombinant human epidermal growth factor engineering bacteria, can solve the problems of low expression soluble rate and plasmid loss, and achieves high soluble rate , Improve the stability, simplify the effect of purification operation

Inactive Publication Date: 2013-03-20
天津强微特生物科技有限公司
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Problems solved by technology

[0007] The purpose of the present invention is to solve the problems of low soluble rate of intracellular expression of recombinant epidermal growth factor and loss of plasmid in fermentation culture, and provide a method for constructing and screening highly efficient, stable and highly soluble hEGF engineering bacteria

Method used

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  • Construction and screening method for recombinant human epidermal growth factor engineering bacteria
  • Construction and screening method for recombinant human epidermal growth factor engineering bacteria
  • Construction and screening method for recombinant human epidermal growth factor engineering bacteria

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Embodiment Construction

[0020] The specific process of recombinant hEGF engineering bacterium construction of the present invention is as follows:

[0021] 1. Containing the construction of kanamycin resistance gene and cspA gene functional element

[0022] 1.1 Acquisition of Kanamycin Resistance Gene Fragment

[0023] 1.1.1 Design primers, the sequence is as follows:

[0024] Primer Kan-1: 5′-ggcaaatt agatct cttttctacggggtctgacg-3' (SEQ ID NO: 1)

[0025] Primer Kan-2: 5′-aatttgcc catatg cgatttcggcctattggtta-3' (SEQ ID NO: 2)

[0026] 1.1.2 PCR reaction to obtain kanamycin resistance gene fragment

[0027] The DNA polymerase Vent used in the PCR reaction was purchased from NEB Company, and the pET42a plasmid was purchased from Merck Millipore. The PCR reaction system was as follows:

[0028]

[0029] The conditions of the PCR reaction are: denaturation: 94°C, 30 seconds; annealing: 56°C, 30 seconds; extension: 72°C, 1 minute 20 seconds; number of cycles: 30

[0030] 1.2 Acquisition of fu...

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Abstract

The present invention provides a construction and screening method for recombinant human epidermal growth factor (hEGF) engineering bacteria. The construction and screening method comprises the following main steps: 1, adopting a PCR technology to obtain kanamycin resistance gene, and adopting a chemical synthesis method to obtain an escherichia coli cspA gene function element; 2, respectively optimizing gene sequences encoding SUMO protein and hEGF, and then adopting chemical synthesis to obtain a large amount of expressed fusion protein gene sequences OPT-SUMO-hEGF; 3, linking the fragments obtained from the previous two steps into pET21 plasmid to obtain pET-Kan-cspA-SE plasmid; 4, adopting the pET-Kan-cspA-SE plasmid as a template to construct a transposition body; 5, transforming the transposition body into escherichia coli Origami 2 (DE3); and 6, screening a strain having high fusion protein SUMO-hEGF expression. According to the present invention, genome of the obtained strain is stable, a soluble rate of the expressed hEGF is high, and the method is suitable for large-scale industrial production.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a construction and screening method of highly efficient intracellular soluble expression of recombinant human epidermal growth factor (hereinafter referred to as hEGF) engineering bacteria. Background technique [0002] Human epidermal growth factor (Human epidermal growth factor, hEGF) is a single-chain polypeptide containing 53 amino acid residues, with a molecular weight of 6216 Daltons; hEGF sequence contains 6 cysteine ​​residues, which can form three pairs of intramolecular two Sulfur bonds, these disulfide bonds stabilize the conformation of hEGF and play an important role in its biological functions. [0003] In vivo, hEGF induces epithelial development, promotes angiogenesis, and inhibits gastric acid secretion. In vitro, hEGF is a mitogen for fibroblasts, epithelial cells, and endothelial cells, and can promote colony formation of epidermal cells. Currently, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12R1/19
Inventor 郑春阳王磊王巍徐彬
Owner 天津强微特生物科技有限公司
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