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Fusion protein comprised of human serum and interferon and its coding gene and application

A technology of human serum albumin and fusion protein, which is applied in the field of fusion protein and its coding gene and application, can solve the problems of unbearable for patients, short half-life of natural alpha interferon, long treatment cycle, etc., and achieve easy purification, long half-life, good curative effect

Inactive Publication Date: 2007-10-31
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the half-life of natural interferon-alpha is relatively short, and only frequent administration (in clinical treatment, three injections per week are often used) can obtain satisfactory curative effect.
Such frequent medication combined with a longer treatment cycle (6-12 months) makes it more difficult for patients to bear

Method used

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  • Fusion protein comprised of human serum and interferon and its coding gene and application
  • Fusion protein comprised of human serum and interferon and its coding gene and application
  • Fusion protein comprised of human serum and interferon and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, the preparation of human serum albumin and interferon fusion protein

[0064] 1. Acquisition of human serum albumin and interferon fusion protein genes

[0065] (1) Interferon-α2b gene

[0066] The interferon-α2b gene is an artificial gene synthesized according to the preferred codons of Pichia pastoris. The synthesis of the interferon-α2b gene was described in the literature (Martinez et al: PCR-based gene synthesis as efficient approach for expression of the A+T-rich malaria genome. Protein Engineering. 1999, 12(12): 1113-1120) method to proceed.

[0067] (2) HSA gene

[0068] The HSA gene is an artificial gene synthesized by Liu Zhimin et al. based on the preferred codons of Pichia pastoris. The synthesis of the HSA gene was carried out according to the method described in the literature (Liu Zhimin et al.: human serum albumin modified gene, expression vector and its host. Chinese invention patent number CN 99102794.9).

[0069] (3) Acquisition of ...

Embodiment 2

[0101] Embodiment 2, comparison of fusion protein stability

[0102] After adjusting the protein concentration of the fusion protein to 1 mg / ml, the samples were placed at 4°C and 37°C for different times, and 10 μl samples were taken to detect the content of covalent polymers by non-reducing SDS-PAGE. The results are shown in Figure 3. In this figure, lane 1 is the HSA-IFN-α2b sample that was electrophoresed immediately after the last step of purification, lane 2 is the HSA-IFN-α2b sample that was stored at 4°C for 60 days, and lanes 3 and 4 are the samples that were stored at 37°C, respectively. HSA-IFN-α2b samples after 1 day and 10 days. Lane 5 is the IFN-α2b-HSA sample that was electrophoresed immediately after the last step of purification, lane 6 is the IFN-α2b-HSA sample that was stored at 4°C for 60 days, and lanes 7 and 8 are the samples that were stored at 37°C for 1 day and IFN-α2b-HSA samples after 10 days. The results of electrophoresis showed that HSA-IFN-α2b ...

Embodiment 3

[0103] Embodiment 3, the determination of antiviral activity of fusion protein

[0104] The activities of IFN-α2b (Anfulon from Tianjin Hualida Bioengineering Co., Ltd.) and the fusion proteins HSA-IFN-α2b and IFN-α2b-HSA purified in the last step were determined by cytopathic inhibition method.

[0105] Cell culture: Wish cells (National Institute for the Control of Pharmaceutical and Biological Products) are adherent cells, which are digested with 0.25% (mass percentage) trypsin, and RPMI 1640 medium containing 10% (volume percentage) calf serum is added to double Resistance (penicillin and streptomycin each 100 units), cultured at 37°C, 5% carbon dioxide and passaged every other day.

[0106] Gray pigeon variant VSV virus preparation: get 9-10 day-old chicken embryos, remove the head, wings, and viscera to make a cell suspension, inoculate in a cell culture bottle and cultivate for 24 hours to form a dense single-cell layer, and inoculate VSV virus (China Institute for the...

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Abstract

The invention discloses a fusing protein, encoding gene and appliance, which is characterized by the following: comprising with albuminar and interferon; comprising (a) and (b) protein; choosing amino acid residue sequence from sequences 3 in sequence table as the protein (a); replacing or deleting or adding amino acid residue sequence from sequences 1 trough one or several amino acid residue sequence; possessing interferon alpha 2b active; deriving from the protein (a) as the protein (b). The interferon part of the fusing protein is placed N on end of the fusing protein, but a part of albuminar is placed on the end of C. This fusing protein possesses better homogeneity, higher stability and higher receiving rate, which can be used to cure multiple tumors and virus disease.

Description

technical field [0001] The invention relates to a fusion protein composed of human serum albumin and interferon, its encoding gene and application. Background technique [0002] Interferon (IFN) is a class of cytokines with antiviral, antiproliferative and immunomodulatory activities. According to its activity and the type of producing cells, interferon can be divided into alpha interferon, beta interferon and gamma interferon. Among them, α-interferon is widely used clinically to treat various viral diseases and tumors. However, the half-life of natural interferon-alpha is relatively short, and only frequent administration (in clinical treatment, three injections per week are often used) can obtain satisfactory curative effect. Such frequent medication combined with a longer treatment cycle (6-12 months) makes it difficult for patients to bear. Therefore, there is an urgent need to develop recombinant human alpha interferon with a long acting time. [0003] Human serum ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/16A61P35/00A61P31/12
Inventor 刘志敏赵洪亮薛冲王洋熊向华姚学勤李献玉
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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