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Method for preparing recombinant human nerve growth factor by using Escherichia coli expression system

A technology of growth factor and Escherichia coli, which is applied in the field of preparation of recombinant human nerve growth factor, can solve problems such as inconsistency, small prokaryotic cells, and difficulty in achieving renaturation effects, and achieve simple and easy preparation methods, high purification efficiency, and easy implementation Effect

Active Publication Date: 2013-10-16
WUHAN HITECK BIOLOGICAL PHARMA
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Problems solved by technology

For example: HE Ling-bing et al. (HE Ling-bing, WANG Yanz, WANG Ge, CHEN Chao, and CAO Shu-gui, Expression and Purification of Active Recombinant Human Nerve Growth Factor from Escherichiu coli. CHEM. RES. CHINESEU, 2007 , Vol.23, No.2) fused thioredoxin and histidine-tagged protein with hNGF β subunit, separated and purified β-NGF by affinity chromatography that specifically adsorbed the tag protein, and used thioredoxin protein to help β-NGF to be expressed in a soluble form, but prokaryotic cells are small, and only when the expression amount is small, the protein can exist in a soluble form. When a large amount of foreign protein is expressed, most of the protein will still aggregate Denaturation; Sun Weiguo et al. (Sun Weiguo, Liu Nongle, Ding Hongmei, Chen Liucun, Zhao Qiang, Xu Hua, Shen Beifen, Shao Ningsheng, Research on prokaryotic fusion expression and activity of recombinant human nerve growth factor β subunit. Biotechnology Communications, 2009, Vol .20, No.4) DsbA, DsbC and thioredoxin (Trx) in the disulfide bond-forming protein family (Dsb) were used as fusion molecules, and hNGF β subunits were fused and expressed in the prokaryotic expression system, although the two Sulfur-bond-forming proteins can assist β-NGF in vitro renaturation, but cannot recognize the exact position of the 3 pairs of disulfide bonds in β-NGF, so it is difficult to achieve the ideal renaturation effect, and the final product of this study carries a tagged protein, Inconsistent with natural β-NGF, cannot be used for drug production

Method used

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  • Method for preparing recombinant human nerve growth factor by using Escherichia coli expression system
  • Method for preparing recombinant human nerve growth factor by using Escherichia coli expression system
  • Method for preparing recombinant human nerve growth factor by using Escherichia coli expression system

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1: Obtaining genetically engineered bacteria expressing the modified human nerve growth factor β subunit

[0051] 1. Referring to the preferred codon table of prokaryotes, without changing the amino acid sequence, the base sequence of the leader peptide and mature peptide of the natural human nerve growth factor β subunit was modified, and enterokinase was designed between the two sequences Cutting site, the sequence was synthesized by chemical synthesis (the base sequence is shown in SEQ ID NO: 3, and the expressed amino acid sequence is shown in SEQ ID NO: 4)

[0052] The codons formed by the bases in the sequence are all high-frequency codons of the E. coli expression system, which can maximize the expression of the target gene without changing the amino acid sequence. The 5' end GGATCC and the 3' end CTCGAG in the sequence are BamHI and XhoI restriction endonuclease sites respectively, which can facilitate the insertion of the target gene into the pET28a(+) ...

Embodiment 2

[0059] Example 2: Screening of Induction Conditions for rhβNGF Genetically Engineered Bacteria

[0060] Inoculate 0.2% recombinant engineered bacteria frozen in glycerol tubes into fresh LB-resistant medium (containing Kana 50 μg / ml), culture overnight at 37°C with shaking at 220 rpm, and activate the engineered bacteria.

[0061] 1. Effect of IPTG concentration on protein expression

[0062] Inoculate the activated engineered bacteria into 2ml of LB-resistant medium (containing Kana 50μg / ml), shake culture at 37°C and 220rpm until A550=0.6, then add IPTG to make the final concentrations 0.1 mmol / L, 0.5 mmol / L, respectively / L, 1.0 mmol / L, 1.5 mmol / L, 2.0 mmol / L, 3.0 mmol / L, 4.0 mmol / L, 5.0 mmol / L, after 3 hours of induction culture, centrifuge to collect the precipitate, and use 0.5ml, 20mmol / L Tris -HCl (pH8.0) resuspended cells, repeated freezing and thawing at -80°C for three times, centrifuged to take the precipitate, resuspended with 0.1ml protein electrophoresis sample...

Embodiment 3

[0070] Example 3: Induced expression of rhβNGF genetically engineered bacteria and isolation and purification of inclusion bodies

[0071] 1. Pick a monoclonal genetically engineered colony and inoculate it in 50mL LB liquid medium, shake at 220rpm at 37°C for 14h;

[0072] 2. Cultivate the above bacteria in 3L liquid LB medium at 37°C and 220rpm shaking until A550=0.6 (about 3h), according to the results of the induction conditions (such as figure 1 , figure 2 , image 3 ), adding IPTG with a final concentration of 1mM, maintaining the pH value of the medium at 7.0, and inducing at 37°C for 3.5 hours;

[0073] 3. Centrifuge (4°C, 8000rpm, 10min), collect the cells, add 20mM Tris-HCl (pH=8.0) lysis buffer to resuspend at a ratio of 20ml per gram of cell wet weight, and freeze at -20°C;

[0074] 4. Dissolve the bacterial suspension at 37°C, and perform ultrasonic lysis, with a power of 400W, ultrasonic for 15 seconds, with an interval of 15 seconds, for 30 minutes.

[0075...

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Abstract

The invention discloses a method preparing a recombinant human nerve growth factor by using an Escherichia coli expression system. Beta-subunit genes of the recombinant human nerve growth factor comprise 6 histidine purification sites, an enterokinase cleavage site and a beta-subunit gene encoding mature peptide of modified human nerve growth factor and a molecular chaperone gene. The beta-subunit genes of the recombinant human nerve growth factor have high expression quantity in the Escherichia coli; generated inclusion body protein has high-efficiency renaturation under the assist of the molecular chaperone; and the recombinant human nerve growth factor obtained by nickel column affinity chromatography after the molecular chaperone is cleavaged accurately by the enterokinase, has a purity greater than 99% and biological activity greater than 500,000 AU / mg.

Description

technical field [0001] The invention relates to a method for preparing recombinant human nerve growth factor by using an Escherichia coli expression system, belonging to the technical field of biomedicine. Background technique [0002] Nerve Growth Factor (NGF) is the earliest discovered and most typical neurotrophic factor, and it is also one of the most important bioactive molecules in the nervous system. It is composed of three subunits, α, β, and γ, of which the β subunit (β-NGF) is the only subunit with biological activity. expression has an important regulatory role. At present, β-NGF has been developed into a drug for the treatment of peripheral nerve injury and is widely used in the fields of neurological diseases such as nerve injury, hemiplegia, stroke, craniocerebral injury, and cerebral palsy in children. But commercial NGF is mouse-derived nerve growth factor (mNGF). Such animal-derived protein products often have high immunogenicity, which can easily cause a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/70
Inventor 李佳楠汤华东陈亚李汝霖陈煌
Owner WUHAN HITECK BIOLOGICAL PHARMA
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