Human interferon alpha 2a gene cDNA code modification recombination sequence

An interferon α, encoding technology, applied in the field of high-efficiency expression of recombinant proteins, can solve the problems of incorrect formation of double disulfide bonds, hindering interferon molecules, etc.

Inactive Publication Date: 2003-06-11
武汉柏傲生物工程有限公司
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Problems solved by technology

Although most studies believe that the soluble form of the expression product will be beneficial to the harvest and purification of the expression product, however, the reducing condition in the cytoplasm of Escherichia coli makes the double disulfide bond of the soluble interferon molecule in the cytoplasm unable to form correctly , preventing the correct folding of the interferon molecule into its native structure

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  • Human interferon alpha 2a gene cDNA code modification recombination sequence
  • Human interferon alpha 2a gene cDNA code modification recombination sequence
  • Human interferon alpha 2a gene cDNA code modification recombination sequence

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Embodiment approach ： Embodiment 1

[0021] The following examples are used to specifically illustrate the embodiments of the present invention: Example 1 Human interferon alpha 2a gene coding modification recombination and high-efficiency expression

[0022] The starting point of this study is to artificially modify and recombine the amino acid code in the cDNA sequence of natural human interferon α2a. According to the published natural human interferon cDNA sequence (Manual et. al. Gene 10: 110, 1980, Streuli et al. Science 209: 1343-1347, 1980, Goeddle et al., Natrue 290: 20-26 1981, GENEBANK) , compared the distribution of amino acid codes in each cDNA sequence, and determined that the artificial modification involved 23 nucleotide residues in the DNA code that coded 20 amino acid residues. See Table 1.

[0023]Table 1: Human interferon α2a gene DNA modification site site coding amino acid original code modified coding 124~126 Pro (proline) CCT CCG163~165 Leu (leucine) CTC CTG178~180 Arg (arginine ) AGG CGC...

Embodiment 2

[0024] The modified recombinant human interferon α2a cDNA sequence has 23 differences from its natural cDNA sequence (Figure 1). However, the amino acid residue sequence of the interferon α2a protein polypeptide encoded by it is the same as the amino acid residue sequence of the natural interferon α2a protein polypeptide. Example 2 Construction of plasmid vectors for cloning and expressing human interferon α2a 1. Construction of expression vectors:

[0025] 1 The original plasmid is pBR322 (4361bp).

[0026] 2 Insert the artificially synthesized DNA fragment (102bp) containing the T-7 promoter, the Lac promoter regulatory sequence and the restriction site sequence for inserting the target gene into the HindIII restriction site at position 29 of pBR322. 3 Use BamH1 and Bsp681 endonucleases to excise part of the TeT gene (about 600bp) contained in pBR322, and insert the Lac gene (1079bp) in this region.

[0027] 4 The reconstructed plasmid is about 5000bp, including the basic ...

Embodiment 3

[0032] The N-terminal 18 amino acid sequences of the interferon protein expressed by the human interferon α2a gene after gene recombination and modification were determined, and the result was: 18K-CDLPQ THSLG SRRTL MLL (identification unit MIDWESTANALYTICAL, INC., 11141E SOUTH TOWNE SQUARE St.Louis, MO 63123), and the C-terminus is glutamic acid (E) (identification unit COMMONWEALTH BIOTECHNOLOGIES, INC., 601 BiotechDrive, Richmond, VA 23235), confirming that the amino acid sequence of recombinant modified human interferon α2a is completely consistent with the amino acid sequence of natural interferon α2a. Example 4 Results and protein product activity identification

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Abstract

The invention refers to the embellishment of aminophenol coding which is unsuitable to be translated in core expressing system of human interferon alpha 2b natural gene sequence, it can forms high-expressing new cDNA sequence. The core expressing plant which contains new cDNA sequence can express 1000mg or more interferon albumen per liter fungus liquid under normal cultivating condition, and the aminophenol sequence structure is completely consistent with natural one, its activity can reach 5.0*10 to the power 7 -1.2*10 to the power 8 U / mg.

Description

technical field [0001] The present invention relates to high-efficiency expression of recombinant protein, in particular to high-efficiency expression of recombinant human interferon alpha 2a. Background technique [0002] Interferon (IFN) is a class of cytokines induced by viruses in human monocytes and B lymphocytes. Interferons produced by vertebrate cells have broad-spectrum anti-viral proliferation, inhibit tumor growth, and immune regulation. a biologically active function. Interferon was discovered and named by Isaacs and Lindenmann in 1957. According to the regulations of the International Interferon Nomenclature Committee in 1980, interferon is divided into human interferon, mouse interferon, rabbit interferon, etc. according to its source, and then according to its molecular structure and antigenicity, it is further divided into α, β, and γ and other different types. Early interferons were collected from cells. For example, in 1975, Strander et al. used the meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C12N15/21C12N15/63C12N15/70C12P21/02
Inventor 白宪鹤林雨霖
Owner 武汉柏傲生物工程有限公司
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