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Human interleukin-2 (IL-2)/Fc fusion protein

A fusion protein and human interleukin technology, applied in the field of DNA recombination, can solve the problems of limited antiviral treatment effect, deterioration of virus disease, difficult treatment, etc., and achieve the effect of inducing immune tolerance and prolonging half-life of islet transplantation.

Active Publication Date: 2011-09-07
上海百英生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 3. Chronic hepatitis B develops to the third stage, that is, the inactive virus carrier state. During this period, due to the low immunity of the body, the virus is low in replication, and it is difficult to clear it. After several years, the virus may become active again and cause the condition to worsen. treat;
[0006] 4. The effect of existing antiviral treatment is limited, and the clearance rate of interferon on hepatitis B is only 30%
At present, there is no effective and widely used method that can break "immune tolerance", activate the body's protective immune response against hepatitis B virus, and protect liver cells and functions at the same time, so it cannot achieve the purpose of clearing the virus and making the patient recover

Method used

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  • Human interleukin-2 (IL-2)/Fc fusion protein
  • Human interleukin-2 (IL-2)/Fc fusion protein
  • Human interleukin-2 (IL-2)/Fc fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of Human Interleukin 2 and Fc Fusion Protein Expression Plasmid

[0046] 1. Cloning of human interleukin-2 functional gene

[0047] Adherent mononuclear cells were isolated from peripheral blood of normal people, stimulated by adding LPS for 4 hours, total RNA was extracted by one-step method of guanidine isothiocyanate, the first strand of cDNA was synthesized by MMLV reverse transcriptase, and then used as a template The entire sequence of the extracellular region of human interleukin 2 (GeneBank: NM_000586.3) was amplified, including the secretion signal peptide sequence, and the 145th amino acid Cys was changed to Ser by using the downstream primers, and the upstream and downstream primers were respectively introduced into the NotI and BamHI sites. The primer sequences used are as follows:

[0048] Upstream primers:

[0049] P1:5'ATATGGCGGCCGCTAACCTCAACTCCTGCCACA3'

[0050] Downstream primers:

[0051] P2: 5'CTCTGGGATCCGTCAGTGTTGAGATGATGCT...

Embodiment 2

[0078] Example 2. Production of Human Interleukin 2 and Fc Fusion Protein

[0079] 1. Screening of stable high-expression cell lines

[0080] Dilute CHO cells in logarithmic phase to 8 x 10 with cold 1 x PBS 6 After / ml,

[0081] Take 0.4ml of the suspension, add it to the electric shock cup, then add 15ug of linearized expression plasmid, mix and let stand on ice for 10-15min. Set the voltage of the electroporator to 800V, and the capacitance to 25uF. After spotting, the electric shock cup was placed on ice for 10 minutes, and the cells were transferred to a 10cm culture dish containing 5% 1×FBS culture solution with a pipette for culture. Two days later, when the growth state of the cells was restored, negative cells were screened with 400 ug / ml of G418. Observe the state of the cells, and change the medium every 4 days. After two weeks of culture, use the limiting dilution method to screen high-expression cell lines with 96-well culture plates. Finally, a stable high-ex...

Embodiment 3

[0088] Example 3. Detection of biological function of human interleukin 2 and Fc fusion protein

[0089] 1. Detection of biological activity of IL-2 in human interleukin 2 and Fc fusion protein

[0090] The detection method is as follows (reference, Nature, 1977: 268, 154-156): IL-2 receptor-positive CTLL-2 cells are the target cells of the receptor combination, and the CTLL-2 cells are treated with PH 3 RPMI for 20 seconds to remove the binding IL-2 on the receptor. Cells were washed twice with RPMI (PH 7) and then mixed with 125 I (Dupont, Boston, MA) was co-cultured for 60 minutes in a 37°C incubator, and the radiolabeled CTLL-2 cells were washed twice, and incubated with different concentrations of rIL-2 or IL-2 / Fc for 60 minutes on ice , and the supernatant was transferred to a glass test tube for radioactivity determination.

[0091] Test results:

[0092] 1) if Figure 4 As shown, in the classic assay for detecting IL-2 activity, IL-2 / Fc and recombinant IL-2 (recom...

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Abstract

The invention provides human interleukin interleukin-2 (IL-2) / Fc fusion protein. The human IL-2 of the fusion protein comprises all sequences of a human IL-2 extracellular region; the Fc fragments comprise a hinge region, a CH2 region and a CH3 region; the human IL-2 / Fc sequences are fused directly or through a connection sequence; and the Fc fragments are human or animal IgG, IgM, IgD and IgA orsubtypes thereof. The ADCC and CDC effective factor action can be eliminated, and in addition, the human IL-2 / Fc fusion protein has the compatibility with a recombinant IL-2 receptor so that the half-life period is obviously prolonged and also has all the biological activity of the IL-2 receptor. The IL-2 / Fc obviously improves the humoral immune response stimulated by the hepatitis B vaccine and the immunity of the CD8+T cells targeted to the hepatitis B vaccine. Moreover, the balance immune (suppression) of the effective T cells and the regulatory t cells can be adjusted under the action of the cyclosporine A so that the pancreatic islet transplantation immune tolerance is induced.

Description

technical field [0001] The invention relates to a fusion protein, in particular to a human interleukin 2-Fc fusion protein and belongs to the technical field of DNA recombination. Background technique [0002] Hepatitis B is a global infectious disease, especially in my country. According to the World Health Organization, about 2 billion people in the world have been infected with HBV, of which 350 million are chronic HBV infections, and 1 million people die every year. In HBV infection. The hepatitis B infection rate in our population is 57%, the HBsAg positive rate is 9.09%, and about 300,000 people die from HBV-related diseases every year. Therefore, hepatitis B is a disease that is very harmful to people's health. However, the treatment of chronic hepatitis B has so far been difficult, especially in the following situations: [0003] 1. For the control of hepatitis B, neonatal hepatitis B vaccine vaccination is undoubtedly very important, but the current protection rat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/79C12N5/10A61K38/20A61K39/39A61P1/16A61P31/20A61P37/04A61P37/06
Inventor 郑心校田燕卞春东查长春刘智
Owner 上海百英生物科技股份有限公司
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