Recombinant expression vector, engineering bacteria, preparation method and application thereof

A recombinant vector and engineering bacteria technology, applied in the field of genetic engineering, can solve the problems of low purity, complex IL-18 purification process, low recovery, etc., and achieve the effects of high purity, high recovery rate and simple process characteristics

Active Publication Date: 2020-04-28
HE EYE HOSPITAL SHENYANG
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  • Abstract
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Problems solved by technology

The present invention utilizes gene recombination technology to construct a genetically engineered bacterium that highly expresses human IL-18, establishes a simple and effective technology for purifying IL-18, solves the problem of preparing a large amount of IL-18, and can meet future clinical needs
[0007] The present invention mainly solves the problems of complex IL-18 purification process, low recovery and low purity

Method used

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  • Recombinant expression vector, engineering bacteria, preparation method and application thereof
  • Recombinant expression vector, engineering bacteria, preparation method and application thereof
  • Recombinant expression vector, engineering bacteria, preparation method and application thereof

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preparation example Construction

[0090] The invention provides a recombinant expression vector, engineering bacteria, preparation method and application thereof. The present invention utilizes gene recombination technology to construct a genetically engineered bacterium that highly expresses human IL-18, establishes a simple and effective technology for purifying IL-18, solves the problem of preparing a large amount of IL-18, and can meet future clinical needs. A stable and simple process route for inclusion body renaturation and IL-18 purification has been established, and the protein purity can reach more than 97%; a method for identifying IL-18 activity has been established.

[0091] The invention mainly solves the problems of complicated IL-18 purification process, low recovery and low purity. The process features simplicity, rapidity, high recovery rate and high purity (97%).

[0092] The invention also provides a method for detecting IL-18 activity. The identification of interleukin-18 activity usually ...

Embodiment 1

[0095] Example 1 Construction of the prokaryotic expression vector pBV220-IL-18 of IL-18

[0096] The gene containing IL-18 obtained by RT-PCR was digested with EcoRI and BamHI, and then ligated with the vector pBV220 which was digested with EcoRI and BamHI; the prokaryotic expression vector pBV220-IL-18 containing the IL-18 gene was constructed, Transform Escherichia coli DH5α to construct engineering strain DH5α-pBV220 IL-18; (see figure 2 , 3 ).

Embodiment 2

[0097] Fermentation and high-efficiency expression of embodiment 2hrIL-18

[0098] Activation, fermentation, induction and recovery of inclusion bodies of engineered bacteria: inoculate engineered strains stored at -70°C on nutrient agar plate medium containing Amp, and culture at 37°C for more than 12 hours; pick a single colony and inoculate into LB containing Amp In the liquid medium, culture on a shaking table at 37°C for more than 12 hours; inoculate the bacterial liquid into the fermentation medium containing Amp at a ratio of 1-10%, and cultivate it on a shaking table at 28-32°C for more than 12 hours; inoculate the bacterial liquid at a ratio of 5-10% Proportionally inoculated into the fermenter and cultured at 28-32°C; wait until the concentration of the bacterial solution reaches OD 600 After about 2.0-3.0, the temperature was raised to 42°C to induce culture for 3-6 hours, and at the same time, a small amount of filler solution was continuously added, and the ammoni...

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Abstract

The invention relates to the field of gene engineering, and in particular relates to a recombinant expression vector, engineering bacteria, a preparation method and application thereof. Human IL-18 gene engineering bacteria can be effectively expressed by a gene recombination technology, a simple and effective IL-18 purification technology is established, the problem of preparation of a large amount of IL-18 can be solved, and the clinical needs in the future can be met. A stable and simple inclusion body renaturation and IL-18 purification technology is established, and protein purity can reach more than 97%; and an IL-18 activity identification method can be established. The problems of complex IL-18 purification process, low recovery rate and low purity and the like can be mainly solved, and the process has the advantages of being simple, fast, high in recovery rate and high in purity (97%).

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to recombinant expression vectors, engineering bacteria, preparation methods and applications thereof. Background technique [0002] Wet age-related macular degeneration (AMD) and diabetic retinopathy (DR) lack ideal treatment measures. Clinically used anti-VEGF therapy involves regular intraocular injection of anti-VEGF monoclonal antibody (such as Lucentis), which not only has certain damage In addition, although the incidence is low, each injection carries a serious risk of intraocular infection, retinal hemorrhage and detachment, and this treatment has no end point. In 2013, there were 93 million diabetic retinopathy (DR) patients worldwide, including 17 million proliferative DR (PDR) patients, 21 million diabetic macular edema patients, and 28 million vision-threatening DR patients. The incidence of diabetic retinopathy is 25% after 5 years of diabetes onset, increases to 60...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12P21/02C07K14/54C07K1/16A61K38/20A61P9/10A61P27/02C12R1/19
Inventor 赵洪礼王卓实何伟
Owner HE EYE HOSPITAL SHENYANG
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