IL-18 mutant polypeptide as well as preparation and usage

A mutant and expression vector technology, applied in the field of genetic engineering, can solve the problem of not being able to be converted into active

Inactive Publication Date: 2008-05-14
郑骏年
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Experiments have shown that ICE-deficient mice can produce IL-18 precursors, but

Method used

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  • IL-18 mutant polypeptide as well as preparation and usage
  • IL-18 mutant polypeptide as well as preparation and usage
  • IL-18 mutant polypeptide as well as preparation and usage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Preparation of IL-18 Mutant Polypeptide IL-18E121R

[0043] (1) Construction of IL-18 mutant expression vector

[0044] IL-18 is structurally similar to the IL-1 family, but not functionally the same. A detailed comparison of the amino acid sequences of IL-18 and IL-1 in 7 strains (human, rat, mouse, horse, cow, pig, dog) shows that IL-18 and IL-1 have high homology The sequence of the two suggests that the two have the same characteristics, while some sequences with low homology may be related to the unique function of IL-18, especially the highly conserved sites in various IL-18 lines that are different from IL-1 . Therefore, Glu121 was selected for mutation in the loop7 region of IL-18. Except for dog IL-18, this site is highly conserved among the six IL-18 strains, and this site has a negative charge, which may bind to the IL-18 receptor through an ionic bond. We mutated Glu121 to Arg, changed the charge properties of this site, and observed the effect...

Embodiment 2

[0066] Example 2 IL-18 Mutant Polypeptide E121R Treats Arthritis in Mice

[0067] (1) Establishment of collagen-induced arthritis (CIA) mouse model

[0068] DBA / 1 mice were immunized with type II natural bovine collagen to induce CIA. From 30 days after immunization, the mice were examined for the incidence of CIA every day. All mice were sacrificed 10 days after the onset of disease, and the degree of swelling of the first paw showing disease symptoms was measured with a precision caliper and the level of IL-6 was detected.

[0069] (2) Treatment of arthritis with IL-18 mutants

[0070] Treatment of DBA / 1 mice immunized with type II native bovine collagen was initiated at the first clinical sign of disease. IL-18E121R was intraperitoneally injected daily with two different concentrations of 1 and 0.1 mg / kg for 10 days. The mice in the control group were divided into two groups, one group was injected with IL-18 binding protein (IL-18BP) as a treatment control, and the othe...

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Abstract

The invention discloses an IL-18 mutant peptide and the preparation method and the usage, which,belongs to the biological technical field. The invention is characterized in that the PCR site-directed mutagenesis technology is adopted to construct the human IL 18 mutant (hIL-18), which is cloned into the temperature-controlling carrier with the PR-PL promoter, and expressed with high efficiency among the Escherichia coli. The expressed hIL-18 mutant D134R is used to cure the impairment caused by IL-18.

Description

technical field [0001] The invention relates to the field of genetic engineering. Specifically, it is an IL-18 mutant polypeptide, its preparation and use. The present invention relates to the use of PCR site-directed mutation technology to construct human interleukin 18 (hIL-18) mutants, which are cloned into R -P L The temperature-controlled vector of the promoter, and high-efficiency expression in Escherichia coli. Utilization of expressed hIL-18 mutants to treat diseases caused by IL-18 damage. Background technique [0002] (1) Discovery and structure of IL-18 [0003] In 1989, Nakamura et al. found that splenocytes of mice with endotoxic shock caused by Propionibacterium acnes (P.acnes) and lipopolysaccharide (LPS) could induce interferon-gamma (IFN-γ )produce. Neutralization of the protein after purification of splenocyte culture supernatant with IL-1, IL-4, IL-5, IL-6 or tumor necrosis factor (tumor necrosis factor, TNF) antibody could not be eliminated, suggest...

Claims

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Application Information

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IPC IPC(8): C07K14/54A61K38/20C12N15/09
Inventor 郑骏年裴冬生孙亚峰付奕赵惠仁陆梁胡书群周虎
Owner 郑骏年
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