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Long-acting interferon fusion protein and application thereof

A technology of fusion protein and interferon, applied in the direction of interferon, prolonging plasma life fusion, cytokine/lymphokine/interferon, etc., can solve the problem of no interferon

Active Publication Date: 2011-01-12
CHENGDU ZEN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant research on interferon at present, and how to carry out effective non-covalent binding of real interferon to albumin is currently unknown

Method used

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  • Long-acting interferon fusion protein and application thereof
  • Long-acting interferon fusion protein and application thereof
  • Long-acting interferon fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Obtaining of recombinant cIFN-ABP protein coding gene and construction of high-efficiency expression engineering bacteria

[0039] The cIFN coding gene sequence was synthesized artificially and cloned into the pBAD18 plasmid vector (purchased from Invitrogen, Carlsbad, USA, its sequence is referred to J. Bacteriol. 177, 4121-4130 (1995)) to obtain pBAD-cIFN.

[0040] First, using the cIFN coding sequence in the plasmid as a template, the human albumin-specific affinity short peptide (HSA-BP, ABP for short) coding sequence and linking sequence are added to its C-terminus by PCR. The pBAD18 plasmid is a high-efficiency expression plasmid in Escherichia coli. The cIFN coding sequence was cloned into the XbaI and SacI restriction sites of this plasmid. Therefore, the coding sequence of ABP, connecting peptide and restriction site was added in 2 steps by PCR method so that the cIFN-ABP fusion protein coding sequence could be re-cloned into the pBAD18 expression ve...

Embodiment 2

[0079] Example 2. High-efficiency expression and purification of rcIFN-ABP protein

[0080] TOP10 competent bacteria were purchased from Invitrogen, USA. The pBAD-IFNm plasmid was transformed into TOP10 bacterial cells according to conventional methods, and the transformed bacteria were inoculated on LB agarose culture plates (containing 100 μg / ml ampicillin), and cultured overnight at 37°C. Take a single colony and inoculate it in 5ml LB liquid medium (containing 100μgml ampicillin), culture it in a shaker at 37°C (225-250RPM) overnight. Take 1ml of the overnight bacterial culture and add it to 100ml LB liquid medium (containing 100μg / ml ampicillin), culture it on a shaker at 37 degrees, when OD600=~0.5, add 1ml 20% L-arabinose, and continue to cultivate for 4 -8 hours. The induced bacterial cells were harvested, and the bacterial culture was centrifuged in a Sorvall centrifuge (RC-5C, Dupont, USA, rotor model SS-34) at 4° C., 6000 RPM for 15 minutes. The bacterial pellet ...

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Abstract

The invention relates to the field of biological pharmacy, in particular to a recombinant long-acting interferon fusion protein and application thereof. The technical problem to be solved is to improve internal action time of the interferon and provide a fusion protein. The fusion protein consists of an N-end interferon, C-terminal human albumin binding peptide and glycin linking peptide which links between the N-end interferon and the C-terminal human albumin binding peptide. The protein can be expressed efficiently in colon bacillus, is simple in purification process and can be used for large-scale industrial production and preparation of a pharmaceutical grade rcIFN-ABP fusion protein pure product. Compared with the recombinant cognate interferon (rcIFN) per se, the rcIFN-ABP fusion protein obtained by using the invention has the characteristic of longer blood serum half-life period. Meanwhile, the specificity antivirus ratio activity is equivalent to cIFN. The rcIFN-ABP fusion protein can be used for treating virus infectious diseases, such as AIDS, hepatitis, SARS, bird flu and the like, and also can be used for treating tumors and other cell proliferative diseases.

Description

Technical field: [0001] The invention relates to the field of biopharmaceuticals. It specifically relates to a recombinant long-acting homologous interferon (cIFN)-human protein binding peptide (ABP) fusion protein for treating viral infection diseases, tumors and cell proliferation diseases, and its application. Background technique: [0002] In the early days, people discovered a protein called interferon as an antiviral substance when they were studying virus interference. In 1957, Isaacs and Lindenmann proved that chicken cells infected with influenza virus could secrete a soluble factor that mediated cell resistance to both homologous and heterologous viruses. In 1958, Nagano and Kojima also observed and obtained similar results. All of these laid the early foundation for the later elucidation of the interferon system of biological organisms in more detail. In the 1970s, the purification of interferon protein from white blood cells made interferon truly available for...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A61K38/21A61K47/48A61P31/12A61P35/00
CPCA61K38/00C07K14/555A61K47/48284C07K2319/31A61K47/643A61P31/12A61P35/00
Inventor 吴炯白敏易兴旺
Owner CHENGDU ZEN BIOSCI
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