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Recombinant interleukin 18 as well as preparation method and application thereof

A technology of interleukin and host cells, applied in the field of recombinant interleukin 18 and its preparation, can solve the problems of low renaturation rate, high price, low yield, etc., and achieve the effect of promoting activity and promoting the formation of IFN-γ

Active Publication Date: 2018-06-29
HE EYE HOSPITAL SHENYANG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the preparation of recombinant human IL-18 mainly adopts the method of bioengineering, but due to the special structure of IL-18 sequence, it is not easy to purify during the biosynthesis process, and the activity after synthesis is very low
Therefore, some problems such as low purity, low renaturation rate and low yield have led to an increase in the preparation cost of IL-18, which in turn leads to expensive

Method used

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  • Recombinant interleukin 18 as well as preparation method and application thereof
  • Recombinant interleukin 18 as well as preparation method and application thereof
  • Recombinant interleukin 18 as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0098] Example 1 The prokaryotic expression vector pBV220-IL-18 of IL-18 was constructed.

[0099] The IL-18-containing gene (SEQ ID NO:2) obtained by RT-PCR was double-digested with EcoRI and BamHI, and then ligated with the vector pBV220 cut with EcoRI and BamHI; the prokaryotic expression of the IL-18-containing gene was constructed The vector pBV220-IL-18 was transformed into Escherichia coli DH5α, and the engineering strain DH5α-pBV220IL-18 was constructed; ( Figure 1~2 ).

Embodiment 2

[0100] Fermentation and high-efficiency expression of embodiment 2hrIL-18

[0101] 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 1:100, and culture on a shaking table at 32°C for more than 12 hours; inoculate the bacterial liquid into the fermentation medium at a ratio of 1:10 Cultivate in the tank at 32°C; wait until the concentration of the bacterial solution reaches OD 600 After about 2.0, the temperature was raised to 42°C to induce culture for 4 hours, and at the same time, a small amount of filler solution was continuously added, and the ammonia water automatically adj...

Embodiment 3

[0102] Refolding and purification of embodiment 3hrIL-18

[0103] 3.1 Washing, dissolution and renaturation of inclusion bodies: Weigh an appropriate amount of inclusion bodies, add inclusion body washing solution to wash twice, then add inclusion body dissolving solution at a ratio of 2%, and dissolve with magnetic stirring for more than 4 hours at 4°C; 4 ℃, 9000rpm, centrifuge for 10min, collect the supernatant; slowly add the refolding solution at a ratio of 1:8 to reduce the protein concentration to 0.2%, dissolve with magnetic stirring at room temperature for 4h; refold at 4°C for more than 20h; purify immediately.

[0104] 3.2 Purification of hrIL-18: equilibrate the hydroxyapatite chromatography column with the balance solution, load all the above-mentioned diluted refolding solution at a flow rate of 10ml / min; Elute with equilibrium solutions containing 0.1, 0.2, 0.5 and 1.0M NaCl, collect the elution peaks, and identify by SDS protein electrophoresis, the target prote...

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Abstract

The invention relates to the field of biotechnology and particularly relates to a recombinant interleukin 18 as well as a preparation method and application thereof. The rhIL-18 provided in the invention is mutated from a serine to an amino acid residue capable of being hydroxylated at a 10 site of N-terminus; hrIL-18 shown in SEQ ID NO: 1 is used for experiments and solves problems of low solubility, poor stability, low bioactivity and the like of wild IL-18; the hrIL-18 shown in SEQ ID NO: 1 is easier to purify and is suitable for industrial production. The rhIL-18 provided in the invention can inhibit generation of microtubules in human endothelial cells, can promote activity of NK cells and promote formation of IFN-gamma, but does not affect the activity of human retinal pigment epithelial cells, so as to indicate that the rhIL-18 can be used as a medicine for treating neovascularization diseases and / or malignant tumors.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant interleukin-18 and its preparation method and application. Background technique [0002] Abnormal neovascularization behind the retina in the macular area is called wet age-related macular degeneration (AMD); specific fundus lesions in diabetic microangiopathy, such as retinal neovascularization, are called diabetic retinopathy (DR). 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% after 10 years, and can be as high as 75% to 80% after 15 years. It can be seen that accelerating the development of new preparations for the treatment of fundus vascular lesions will not only have social benefits, but will also...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/54C12N15/24C12N15/11C12N15/70C12N1/21A61K38/20A61P27/02A61P35/00
CPCA61K38/00C07K14/54
Inventor 赵洪礼王卓实周艳楠刘畅张鹏何伟
Owner HE EYE HOSPITAL SHENYANG
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