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Pharmaceutical preparation containing p40 molecule or its gene

A molecular and preparation technology, applied in the field of pharmaceutical preparations, can solve the problems of prone to infection death, weakened anti-bacterial infection, weak anti-infection ability, etc.

Inactive Publication Date: 2006-03-22
FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For p35 gene-deficient mice that cannot produce IL-12 but can produce IL-23, or p40 gene-deficient mice that cannot produce IL-12 or IL-23, the ability of both to resist bacterial infect

Method used

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  • Pharmaceutical preparation containing p40 molecule or its gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Production of recombinant IL-23 and p40 molecules

[0043] Total RNA was extracted from the cells of BALB / c mice subcutaneously implanted tumors, and the full-length cDNAs of p19 and p40 were obtained by RT-PCR, and inserted into the cloning site of the animal cell expression vector pcDNA3 (Invitrogen Company) ( Eco RI / Bam HI), and the two cDNAs are connected by an internal ribosomal entry site (IRES) (Duke, GM et al., J. Virol., 66, 1602-1609, 1992). , The IL-23 expression vector is inserted in the downstream of the CMV promoter with the gene arranged in the order of p19 / IRES / p40, and the transcription of p19 and p40 cDNA is controlled by the CMV promoter. In the vector used to express soluble p40 molecules, only p40 cDNA is inserted, and its transcription is controlled by the CMV promoter. The above-mentioned vector was introduced into monkey kidney cells COS-7 (American Type Culture Collection, ATCC) with Lipofectin reagent (Invitrogen Co.), cultured for 48 hours, ...

Embodiment 2

[0046] p40 inhibits IL-23-induced splenocytes to produce IFN-γ

[0047] Spleen cells of C57BL / 6 mice were stimulated with Concanavalin A (5 μg / ml, Sigma Company). After 48 hours, living cells were recovered, rinsed twice with phosphate buffer, and inoculated on 24-well plates (2.5×10 6 per well), adding the culture supernatant of COS-7 cells containing IL-23 for culturing, and adding the culture supernatant of COS-7 cells introduced with p40 gene or the culture supernatant of COS-7 cells without introducing any gene. After culturing for 48 hours, the culture supernatant was recovered, and the content of IFN-γ was determined by ELISA (eBioscience).

[0048] The result is as figure 1shown. When the culture supernatant containing p40 molecule was added, the secretion of IFN-γ was significantly decreased compared with the group added with the culture supernatant not containing p40 molecule. It indicated that p40 molecule significantly inhibited the effect of IL-23 on IFN-γ p...

Embodiment 3

[0050] Will IL-23 or p40 gene transfer into cells

[0051] Mouse p19 and p40 cDNAs were inserted into the cloning site (Eco RI / Bam HI) of the retroviral vector LXSN using IRES. The IL-23 expression vector is a gene fragment arranged in the sequence p19 / IRES / p40 inserted downstream of the 5'LTR, and only the cDNA of p40 is inserted into the expression vector of the soluble p40 molecule. Both IL-23 and p40 transcription are controlled by the 5'LTR. After the above-mentioned expression vector was introduced into the packaging cell Psi-2 (ATCC) with Lipofectin reagent (Invitrogen Company), selection culture was carried out with a culture medium supplemented with G418 (400 μg / ml, Invitrogen Company), and then the culture supernatant and multiple Polyquaternary amine (polybrene, 8 μg / ml, Aldrich Company) was further infected with PA317 cells (ATCC), and G418 (400 μg / ml, Invitrogen Company) was used for selective culture, and the PA317 cell culture supernatant contained retroviru...

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Abstract

The invention belongs to the pharmaceutical preparation field and relates to a pharmaceutical preparation which takes molecule p40 or a gene encoding molecule p40 as the active ingredient and is used for antagonism against immunoreaction caused by interleukin-23 (IL-23). In particular the invention takes soluble molecule p40 or a gene which can express and secrete molecule p40 as the active ingredient, antagonizes the immunoreaction which is mediated by IL-23 and unfavorable to organisms, can be widely applied to treat immunological diseases such as collagenosis, multiple sclerosis, rheumatoid arthritis, ulcerous colitis, I type diabetes mellitus, chronic thyroiditis and so on caused by such as immunoreaction abnormal hyperfunction, and also can be used to inhibit rejection reactions of the organisms to implants.

Description

technical field [0001] The invention relates to a pharmaceutical preparation for resisting immune response caused by interleukin-23 (IL-23), which takes p40 molecule or gene encoding p40 molecule as active ingredient. Specifically, the body's soluble p40 molecule or the gene that can express and secrete the p40 molecule is used as the active ingredient to resist the immune response mediated by IL-23 that is unfavorable to the body, and can be widely used, for example, in the treatment of autoimmune diseases and ease of graft rejection. Different reactions, etc. The invention belongs to the field of pharmaceutical preparations. Background technique [0002] In the past, the use of immunosuppressants was the only way to fight against harmful immune responses to the body (such as autoimmune diseases that target their own organs or rejection after organ transplantation, etc.). Such immunosuppressants include anti-T lymphocyte antibodies and steroid hormones, which can inhibit ...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00A61P37/06C12N15/09A61K35/12A61K35/76A61K38/00A61P1/04A61P3/10A61P19/02A61P29/00C07K14/54
Inventor 田川雅敏王彦青
Owner FUDAN UNIV
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