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High-efficiency expression and application of human recombinant interferon B

A high-efficiency expression, interferon technology, applied in the application, recombinant DNA technology, drug combination and other directions, can solve the problems of lack of glycosylation function, lack of glycosylation in recombinant protein, etc., to achieve reduced preparation cost, large amount of protein, and cheap preparation. the effect of

Inactive Publication Date: 2007-07-11
JINWEI GENE TECHN JINWEI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, prokaryotic cells such as E.coli lack glycosylation function, and the recombinant protein expressed by it lacks glycosyl groups, which has certain limitations in biological activity and biological function.

Method used

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  • High-efficiency expression and application of human recombinant interferon B
  • High-efficiency expression and application of human recombinant interferon B
  • High-efficiency expression and application of human recombinant interferon B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, clone human interferon beta gene:

[0019] Figure 1 is a flowchart of the expression of recombinant human beta-interferon in insect cells and Figure 2 is a flowchart of the expression of recombinant human beta-interferon in yeast. Viral RNA was extracted from human fibroblasts obtained from ATCC as described above, and purified total RNA or m RNA (5 μg) was used as a template to synthesize cDNA. The primer used for reverse transcription into cDNA is the synthetic oligonucleotide dT primer provided in the kit. The β-IFN gene was amplified from the cDNA using standard polymerase chain reaction (PCR) procedures. The PCR reaction mixture (50 μL) contained 20 pmol of primers specific to the 5' and 3' ends of the beta-interferon gene, and the gene sequence was determined by the consensus sequence found in the GenBank DNA file. Amplification was performed for 30 cycles, each cycle including denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, and...

Embodiment 2

[0022] Embodiment 2 Expression of recombinant human interferon beta in insect cells:

[0023] Figure 1 is a flowchart of the expression of recombinant human beta-interferon in insect cells. After the above-mentioned mature beta-interferon gene is digested by the restriction endonuclease ECOR I, it is inserted in the pM / bac insect baculovirus vector containing the mellitin secretion signal peptide, and the resulting recombinant vector contains the polyhedrin promoter, A chimeric recombinant plasmid of mellitin secretion signal peptide and human beta interferon gene. Then, the chimeric transfer plasmid DNA of purified human beta interferon gene was mixed with AcNPV wild-type DNA, and transfected into S. Frugiperda insect cells by calcium co-precipitation. Recombinant baculoviruses were selected on the basis of plaque morphology and further purification by several rounds of plaques. Screened recombinant baculovirus clones are used to express beta-interferon, and a single baculo...

Embodiment 3

[0025] Embodiment 3 Expression of recombinant human interferon beta in yeast:

[0026] Figure 2 is a flow chart of the expression of recombinant human beta-interferon in yeast. Yeast has many advantages of higher eukaryotic cells, such as protein folding and post-translational modification, but it is as easy to operate as prokaryotic cells. In terms of producing heterogeneous proteins, it is faster, more convenient and cheaper than mammalian cells, and Yields are ten to a hundred times higher. The expression of yeast alcohol oxidase promoter gene can reach a very high level under methanol induction and strict control. Since yeast itself produces very few secreted proteins, the vast majority of secreted proteins produced are beta-interferons.

[0027]The mature beta-interferon gene obtained in Example 1 can also be inserted into a yeast vector after being digested with a specific restriction endonuclease ECOR I. Controlled AOX1 promoter, alpha factor secretion signal peptide...

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Abstract

The present invention relates to high-effective expression of human recombinant interferon B and its application, further relates to adoption of biological gene engineering technique to make expression and produces a recombinant human interferon B, and utilizes eukaryocyte expression vector to convert host cell and produces a recombinant human interferon B. The recombinant human human interferon B which is high in yield and is obtained by means of expression and production of eukaryocyte system is a glycosylated protein modified after protein translation, and is similar to natural human interferon B in structure and function. The amount of protein produced by this invented expression system is large, and its purity is high, it can extensively be used for clinical curing autoimmune diseases.

Description

technical field [0001] The high-efficiency expression of human recombinant beta interferon of the present invention involves the use of biological genetic engineering methods to express and produce a recombinant human beta interferon in a eukaryotic cell system. The eukaryotic cell expression system used includes insect baculovirus Expression system and yeast expression system, and the use of recombinant human beta interferon. Background technique [0002] Immune interferons are a family of natural proteins induced by viruses and other biological primers, which have antiviral, antidifferentiation and immune regulation functions during pathogenic infection. There are three types of interferon: type A, type B, and type C interferon. The three interferons have common characteristics, but have significantly different biological effects. The main function of type A interferon is antiviral infection, which is used to treat viral diseases such as hepatitis A, B and C; type C inte...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/67C12N15/20A61P29/00A61P19/02A61P17/06A61P3/10A61P35/00A61P1/16
Inventor 秦莹
Owner JINWEI GENE TECHN JINWEI
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