Usage of differentiation protein of medullary cell MyD88 in preparing medicinal preparation

A technology of cell differentiation and pharmaceutical preparation, applied in the biological field

Inactive Publication Date: 2005-01-12
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report about the antiviral effect of MyD88

Method used

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  • Usage of differentiation protein of medullary cell MyD88 in preparing medicinal preparation
  • Usage of differentiation protein of medullary cell MyD88 in preparing medicinal preparation
  • Usage of differentiation protein of medullary cell MyD88 in preparing medicinal preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Establishment of MyD88 Stable Expression Cell Line

[0043] Human liver cancer HepG2 cells were cultured in 10% fetal bovine serum, 10 5 u / L penicillin, 0.1g / L streptomycin in DMEM medium (Gibco). The empty vector pcDNA3.1 / myc-His or pcDNA-MyD88 (8 μg each) was transfected into HepG2 cells (1×10 6 After 36 hours, the cells were inoculated into selective medium containing 200 μg / ml G418 (Gibco Company) at a ratio of 1:4. After 3 weeks of selection by G418, single-cell clones resistant to neozyme were selected, and a neozyme-resistant control cell line (Neo) was selected from cells transfected with the empty vector pcDNA3.1 / myc-His , The cells transfected with pcDNA-MyD88 were selected to select two cell lines with stable expression of MyD88, namely MyD88-6 and MyD88-8. The MyD88 monoclonal antibody was used to detect the expression level of MyD88 protein in the above two MyD88 stable expression cell lines and Neo cell lines treated or untreated by IFN-α by W...

Embodiment 2

[0044] The influence of embodiment 2MyD88 on HBV protein synthesis

[0045] Inoculate HepG2 cells, MyD88 stable expression cell lines and Neo cell lines in 6-well plates, 3×10 per well 5 After culturing overnight, pHBV3.8 (3 μg) and pcDNA-CAT (0.5 μg) were transiently transfected into the above cells by calcium phosphate precipitation method. After 16 hours, fresh medium was replaced, and 1000 IU / ml recombinant human IFN-α (R&D Company) was added to the medium of cells in the IFN treatment group. After continuing to culture for 1 day, 2 days, and 3 days, the cell culture supernatants were collected respectively, and the cells were lysed after three days of culture. The levels of HBsAg and HBeAg were detected by CAT ELISA kit (Roche Company) to detect the CAT activity in the transfected cells to balance the transfection efficiency. The experiment was repeated three times, with multiple wells each time.

[0046] The results confirmed that compared with the Neo cell line trans...

Embodiment 3

[0047] Example 3 Effect of MyD88 protein on HBV replication

[0048] HepG2 cells, MyD88 stable expression cell lines and Neo cells were divided into 1×10 6 cells / dish, inoculated on 60mm plate, transiently transfected the above cells with pHBV3.8 (10μg) and pcDNA-CAT (1.5μg) by calcium phosphate precipitation method, and added 1000IU / ml recombinant Human IFN-alpha. After 48 hours of transfection, the cells were washed once with PBS, and 650 μl of freshly prepared cell lysate (10 mmol / L Tris-HCl pH7.9, 1 mmol / L EDTA, 50 mmol / L NaCl, 1% NP40, 8% sucrose ), let stand at 37°C for 10min, repeatedly blow and blow to disperse the cell lysate, and centrifuge at 12000r / min for 2min at 4°C. Take 50 μl of the supernatant and use CAT ELISA to detect the CAT activity in the cells to balance the transfection efficiency, and extract the transfected cells according to the method of Lin Xu et al. HBV core particle DNA in the cytoplasm. According to the transfection efficiency, the loading ...

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Abstract

An application of the myeloid cell differentiation protein, MyD88 in preparing the medicines for treating the infection of HVB, such as hepatitis B, is disclosed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the application of a myeloid cell differentiation protein in the preparation of pharmaceutical preparations, in particular to the application of the myeloid cell differentiation protein MyD88 protein in the preparation of anti-hepatitis B virus pharmaceutical preparations. Background technique [0002] Hepatitis B virus is a pathogen that seriously harms the human body. It can cause chronic active hepatitis and increase the probability of liver cell cancer [Kao, J.H.et al.Lancet Infect.Dis.2002, 2: 395-403] . Although recombinant HBV vaccines are now widely used, HBV infection remains a major challenge to countries around the world. Currently, only interferon-α (Interferonalpha, IFN-α) and some nucleoside analogs such as lamivudine are the only drugs that can inhibit HBV replication. However, the cure rate of interferon therapy is limited, and relapses usually occur after discontinua...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P1/16A61P31/12
Inventor 袁正宏熊炜
Owner FUDAN UNIV
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