Use of human interleukin 32 in preparation of medicament for treating or preventing influenza A virus infection

A technology of influenza virus infection and human interleukin, applied in antiviral agents, medical preparations containing active ingredients, pharmaceutical formulas, etc., can solve the problems of undetectable IL-32 and undetectable IL-32 synthesis, and achieve The effect of avoiding toxic side effects

Inactive Publication Date: 2010-05-12
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] IL-32 can induce the production of IFN-γ in various human and other mammalian cell lines to stimulate human epithelial Wish cells, induce the production of IL-32 in a time-dependent manner in the cell lysate, and reduce it after 46h; IL-18 Stimulation of A549-Rβ (human lung cancer A549 cell line transfected with IL-18Rβ) with IL-1β also induced IL-32 production in a time-dependent manner in its lysate, but not in unstimulated A549-Rβ Synthesis of IL-32 was detected, and only IL-1β could induce IL-32 production in A549-WT (human lung cancer A549 cel

Method used

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  • Use of human interleukin 32 in preparation of medicament for treating or preventing influenza A virus infection
  • Use of human interleukin 32 in preparation of medicament for treating or preventing influenza A virus infection
  • Use of human interleukin 32 in preparation of medicament for treating or preventing influenza A virus infection

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0029] Example 1:

[0030] Construction and identification of plasmids

[0031] After extracting the mRNA of Jurkat cells, reverse transcription PCR to obtain cDNA, using it as a template to amplify the target fragments with the cloning primers of each indirect isoform. The vector Pcmv-Tag2A (Sigma) was digested with MluI and HindIII, purified and recovered, and ligated in a 16°C water bath overnight. Transform into Escherichia coli DH5α (China Type Culture Collection-Wuhan University Collection Center, Ordinary Escherichia coli) by electroporation, spread on ampicillin-resistant LB culture plate, cultivate at 37°C for 12-16h, select a single clone Perform PCR and restriction enzyme digestion identification, select 2 clones for sequencing, and identify whether the sequence and coding frame of the target fragment are correct.

[0032] A. DNA fragment recovery, glass-milk method

[0033] 1. Take the PCR product, add 1 / 10 volume of 10X loading buffer, and separate the digested fragment...

Example Embodiment

[0087] Example 2:

[0088] Mammalian cell culture and transfection

[0089] A. Mammalian cell culture

[0090] 1. Cell Recovery

[0091] 1) Take the cells out of the liquid nitrogen tank, and quickly thaw them in a 37°C water bath. During this time, shake the cryotube continuously to heat the cell liquid evenly;

[0092] 2) Transfer the thawed cells into a 25ml cell flask, and then add 3ml of the corresponding DMEM medium (manufacturer GIBCO, the same below);

[0093] 3) Place at 37℃, 5% (volume ratio) CO 2 Culture in an incubator;

[0094] 4) After the cells adhere to the wall, replace with new DMEM medium.

[0095] 2. Cell Passaging

[0096] 1) The flat space where the cells are overgrown, discard the DMEM medium;

[0097] 2) Add 8ml D-Hank’s buffer, shake the cells, and then remove the washing solution;

[0098] 3) Add 1ml of pancreatin and place at 37°C for 5 minutes;

[0099] 4) Add 4ml DMEM medium to neutralize pancreatin;

[0100] 5) Aspirate 2 / 3 of the cell suspension, then add 6ml DMEM...

Example Embodiment

[0119] Example 3:

[0120] A. EILSA method to detect IL-32 protein expression

[0121] 1. Serum, cell culture supernatant or cell lysate samples were diluted 1:50 with PBS (pH 7.4), 100 μl was added to the wells of the ELISA plate, and coated at 37°C for one hour or overnight at 4°C.

[0122] 2. Wash the ELISA plate 5 times with PBS (try to pat the liquid clean after each wash to ensure that it is cleaned as much as possible).

[0123] 3. Prepare 3% (volume ratio) PBST skimmed milk powder, dissolve it with PBST, add 100μl per well to the ELISA plate, and block at 37°C for one hour or at 4°C overnight.

[0124] 4. Wash with PBST more than 5 times.

[0125] 5. The IL-32 antibody prepared by the polypeptide immunized rabbits was diluted 1:500 with PBST, and 100 μl was added to each well at 37°C for one hour.

[0126] 6. Wash with PBST more than 5 times.

[0127] 7. Horseradish peroxidase-conjugated goat anti-rabbit IgG was diluted 1:5000 with PBST, 37°C for half an hour.

[0128] 8. Wash with ...

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Abstract

The invention discloses the use of human interleukin 32 in the preparation of medicaments for treating or preventing influenza A virus infection. Clones of splicing isoforms IL-32(alpha, beta, gamma, delta, epsilon and zeta) of IL-32 are cloned and established by using a molecular biological method; MDCK cells are transfected with the clones; and thus, the applicant has found that MDCK cells in which all subtypes of the human interleukin 32 are overexpressed are improved in resistance to influenza A viruses. Different IL-32 subtypes show different inhibition effects on the replication of the influenza A viruses. The IL-32 gamma has the most obvious anti-influenza A virus effect. And only the IL-32 gamma is of a secretor type. The human interleukin 32 is a critical antiviral gene in immunoreactions of a host. The applicant is the first one to put forward that the human interleukin 32 has an extremely effective anti-influenza A virus function and to point out that the human interleukin 32 has an application prospect in developing anti-influenza A virus medicaments for effectively preventing and treating influenza A virus infection.

Description

technical field [0001] The invention relates to the technical fields of molecular cloning, type A influenza virus titer and virus titer detection, and more specifically relates to an application of human interleukin-32 in a medicine for treating or preventing type A influenza virus infection. Background technique [0002] Typical influenza virus particles of type A are spherical, with a diameter of 80-120 nm, with an average of 100 nm. Influenza A viruses isolated from poultry, some strains are mainly spherical, while some strains are pleomorphic, some are filamentous, and some are odd-shaped particles. The pleomorphism of influenza A virus can be used as a genetic marker in the study of viral genetics, but the polymorphic virus particles initially isolated mainly become spherical after continuous passage in chicken embryos or cell cultures. The envelope of influenza A virus consists of fibrils, a lipid bilayer and matrix proteins. The fibrils on the surface of the capsule...

Claims

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Application Information

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IPC IPC(8): A61K38/20A61P31/16
Inventor 朱应黎玮李永奎孙伟吴建国
Owner WUHAN UNIV
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