Method for large-scale preparation of recombinant human acidic fibroblast growth factor (hFGF-1)

A technology for fibroblasts and growth factors, which can be applied in the fields of fibroblast growth factors, preparation methods of peptides, growth factors/inducing factors, etc. 1. Influence and other issues, to achieve the effect of production stability and applicability, good genetic stability, and stable expression level

Active Publication Date: 2019-08-02
WENZHOU MEDICAL UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] Although hFGF-1 has been successfully expressed in a variety of hosts, the Escherichia coli (E.coli) expression system is still an ideal expression system for the industrialization of genetically engineered bacteria, because it has the advantages of easy cultivation, short fermentation cycle, and low cost. However, whether hFGF-1 can be highly expressed in the E. coli

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  • Method for large-scale preparation of recombinant human acidic fibroblast growth factor (hFGF-1)
  • Method for large-scale preparation of recombinant human acidic fibroblast growth factor (hFGF-1)
  • Method for large-scale preparation of recombinant human acidic fibroblast growth factor (hFGF-1)

Examples

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Effect test

Embodiment 1

[0048] Example 1: Establishment and Identification of Engineering Bacteria Containing pET3c / rhFGF-1

[0049] 1.1 Construction and transformation of recombinant plasmid pET3c / rhFGF-1

[0050] In this example, codon optimization is performed on the gene sequence of hFGF-1 (GenBank, NM_001144892.2), which mainly includes excising 1-13 signal peptide amino acids at the N-terminus and 6 amino acids at the N-terminus of the mature protein (excluding the N-terminus). Initial amino acid methionine (M) total 19 amino acids to obtain hFGF-1 135 , the amino acid sequence of which is shown in SEQ ID NO:1. hFGF-1 135 Introduce NdeI and BamHI restriction enzyme sites (indicated in italics and underline) at both ends of the template, and design upstream and downstream primers: upstream primer PI: 3'; downstream primer PII: 3', for PCR amplification. The hFGF-1 gene and pET3c empty vector were treated with NdeI and BamHI enzymes respectively, digested at 37°C for 3 hours, and the corre...

Embodiment 2

[0057] Embodiment 2: engineering bacterium stability test

[0058] In order to study the stability of the engineered bacteria, this embodiment identified the stability of the plasmid and the expression stability of rhFGF-1 of the 10th, 20th, 30th, 40th, and 50th generation engineered bacteria during the propagation process.

[0059] Pick a single colony from the solid LB plate containing ampicillin sodium and inoculate it on the slant of the LB test tube containing ampicillin sodium, culture at 37°C for 12 hours as one generation, pick a little bacterial lawn and continue to streak and subculture until the 50th generation. During this process, appropriate dilution of the bacterial solution was taken every 10 generations and spread on the LB plate without ampicillin sodium. Incubate overnight at 37°C on solid plates and count.

[0060] The primary and 10th, 20th, 30th, 40th, and 50th generation engineering bacteria were inoculated into LB liquid medium containing 100 μg / ml amp...

Embodiment 3

[0065] Embodiment 3: Optimization of rhFGF-1 fermentation process

[0066] 3.1 Research and verification of rhFGF-1 small-scale fermentation factors

[0067] In this example, the factors and parameters in the rhFGF-1 shake flask fermentation process were first explored, including induction time, inducer concentration, induction timing, cultivation and induction temperature, pH value, dissolved oxygen, glucose concentration, chlorination The ammonium concentration was repeated three times to determine the optimal fermentation conditions.

[0068] Specifically: the engineering bacteria BL21 (DE3) plysS-pET3C / rhFGF-1, with 1:100 (V / V), inoculated in the 30ml liquid LB culture medium that contains ampicillin sodium (final concentration 100 μ g / ml), 37 Cultivate with shaking at 150rpm overnight; replant in a 250ml Erlenmeyer flask with 30ml of liquid LB medium at a ratio of 1:100, and culture with shaking at 200rpm for 12 hours at 37°C; put the bacterial solution in the logarithmi...

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Abstract

The invention aims to provide a method for large-scale preparation of a recombinant human acidic fibroblast growth factor (hFGF-1). The method optimizes relevant parameters of high-density cell fermentation of escherichia coli BL21(DE3)plysS-pET3c/rhFGF-1 under a pilot scale of 30 L, optimizes a purification process of rhFGF-1, and realizes stable large-scale production of the rhFGF-1. Specifically, according to the method, a large number of high-purity recombinant human acidic fibroblast growth factor proteins can be obtained through optimized escherichia coli BL21(DE3)plysS-pET3c/rhFGF-1 engineered strains, a fermentation process obtained by optimization of a response surface analysis method, and a column chromatography series purification process; the method is suitable for large-scaleproduction.

Description

technical field [0001] The invention belongs to the field of cell growth factors, in particular to a method for large-scale preparation of recombinant human acidic fibroblast growth factor. Background technique [0002] Human acidic fibroblast growth factor (hFGF-1) is a polypeptide substance that mediates cell-to-cell transmission, and it mainly has strong mitogenic and proliferation effects on cells derived from mesoderm and neuroectoderm role. Studies have shown that hFGF-1 has a wide range of biological functions. It not only plays an important role in promoting embryogenesis, development, angiogenesis, wound repair, bone growth, etc., but also plays a role in vasodilation, ischemic protection, brain injury and neural It plays a vital role in protection and other aspects, and can even improve glucose and lipid metabolism and treat chronic complications of diabetes. Therefore, hFGF-1 shows great clinical application value, so that people's demand for hFGF-1 is increasin...

Claims

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Application Information

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IPC IPC(8): C07K14/50C07K1/20C07K1/18C12N15/12C12N15/70
CPCC07K14/501C12N15/70C12N2800/22
Inventor 惠琦王晓杰李校堃黄臻余丙杰杨选鑫
Owner WENZHOU MEDICAL UNIV
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