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Method of producing glycoprotein composition

A protein and sugar chain technology, applied in the field of cells

Inactive Publication Date: 2007-08-22
KYOWA HAKKO KIRIN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, RNA molecules designed by this method are not necessarily molecules that can effectively inhibit the function of target genes (non-patent reference 18), and designing RNA molecules that exhibit potent functional inhibitory effects on specific genes is still trial and error

Method used

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  • Method of producing glycoprotein composition
  • Method of producing glycoprotein composition
  • Method of producing glycoprotein composition

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Experimental program
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preparation example Construction

[0527] 1. Preparation of cells of the present invention

[0528] (1) Preparation of cells into which RNA capable of inhibiting the function of GDP-mannose converting enzyme has been introduced

[0529] In the present invention, cells introduced with RNA capable of inhibiting the function of GDP-mannose converting enzyme can be prepared, for example, by the following method.

[0530] Preparation of cDNA or genomic DNA of GDP-mannose converting enzyme.

[0531] Determine the nucleotide sequence of the prepared cDNA or genomic DNA.

[0532] According to the determined DNA sequence, an RNAi gene construct with a suitable length including the GDP-mannose converting enzyme coding region or non-coding region is designed.

[0533] In order to express the RNAi gene in cells, the recombinant vector is prepared by inserting the fragment or full length of the prepared DNA into the downstream of the promoter of the appropriate expression vector.

[0534] Transformants are obtained by in...

Embodiment 1

[0780] Prepare lectin-resistant CHO / DG44 cells by introducing GMD-targeting small interfering RNA (siRNA) expression plasmids:

[0781] 1. Construction of GM D-targeted siRNA expression vector

[0782] (1) Clone the "human U6 promoter-cloning site-terminator" sequence expression cassette (cassette)

[0783] The "human U6 promoter-cloning site-terminator" sequence expression cassette was obtained according to the following method (Figure 1).

[0784] First, design forward primers and reverse primers, the recognition sequences of restriction enzymes HindIII and EcoRV in the forward primers are added to the 5'- (hereinafter referred to as "hU6p-F-HindIII / EcoRV", expressed as SEQ ID NO: 59), the recognition sequence of the restriction enzyme XbaI and EcoRV in the reverse primer (6 consecutive adenines corresponding to the terminator sequence base) and recognition sequences of restriction enzymes KpnI and SacI for inserting different synthetic oligonucleotide DNAs were added to t...

Embodiment 2

[0830] Antibody compositions were produced using lectin-resistant CHO / DG44 cells into which a GMD-targeting siRNA expression plasmid was introduced:

[0831] 1. Obtaining an antibody composition produced by a lectin-resistant clone into which a GMD-targeting siRNA expression plasmid has been introduced

[0832] Anti-CCR4 chimeric antibodies produced by the lectin-resistant clone 12-GMDB-2 and clone 12-GMDB-5 obtained in Example 1 into which the GMD-targeting siRNA expression plasmid was introduced were obtained according to the following method.

[0833] Take 3×10 5 Density of cells / mL, clone 32-05-12 suspended in basal medium, clones 12-GMDB-2 and 12-GMDB-5 suspended in basal medium supplemented with 12 μg / mL of puromycin (manufactured by SIGMA) , they were inoculated at 15 mL in culture flasks for T75 adherent cells (manufactured by Greiner). at 5% CO 2 After culturing at 37°C for 6 days, the culture supernatant was removed, washed twice with 10 mL of Dulbecco's PBS (manu...

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Abstract

The present invention relates to a cell into which an RNA capable of suppressing the function of an enzyme catalyzing a reaction which converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is introduced; a process for producing a glycoprotein using the cell; a cell into which an RNA capable of suppressing the function of an enzyme relating to modification of a sugar chain in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through alpha-bond in the complex type N-glycoside-linked sugar chain, and an RNA capable of suppressing the function of an enzyme relating to synthesis of an intracellular sugar nucleotide, GDP-fucose, or an RNA capable of suppressing the function of a protein relating to transport of an intracellular sugar nucleotide, GDP-fucose, to the Golgi body are introduced; a process for producing a glycoprotein composition using the cell; and the like.It is intended to provide a cell having an RNA, which inhibits the function of an enzyme catalyzing the reaction of converting GDP-mannose into GDP-4-keto,6-deoxy-GDP mannose, transferred thereinto; a method of producing a glycoprotein composition by using this cell; a cell having an RNA which inhibits the function of an enzyme participating in the sugar chain modification of the binding, via a-bond, of the 1-position of fucose to the 6-position of N-acetylglucosamine at the reducing end of an N-glycoside bond type complex sugar chain, an RNA which inhibits the function of an enzyme protein participating in the intracellular synthesis of a sugar nucleotide GDP-fucose, or an RNA inhibiting the function of a protein participating in the intracellular transport of a sugar nucleotide GDP-fucose into the Golgi body, transferred thereinto; a method of producing a glycoprotein composition by using this cell; and so on.

Description

technical field [0001] The present invention relates to a cell into which is introduced RNA capable of inhibiting the function of an enzyme that catalyzes the conversion of GDP-mannose into GDP-4-ketone, 6-deoxy-GDP-mannose; a method for producing glycoprotein, The method includes using the cell; RNA used to prepare the cell; DNA corresponding to the RNA; and a vector comprising the DNA and its complement. In addition, the present invention also relates to a cell into which N-acetylglucosamine capable of inhibiting sugar chains (wherein the 1-position of fucose and the reducing terminal in complex N-glycoside-linked sugar chains The 6-position is connected by α-bond) to modify the function of the relevant enzyme RNA, and the RNA that can inhibit the enzyme protein function related to the synthesis of intracellular sugar nucleotide GDP-fucose, or can inhibit the intracellular sugar nucleoside Acid GDP-fucose transported to Golgi-related protein function RNA; and a method for p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N5/10C12P21/02C12P21/08C07K16/00C07K2/00A61K39/395C12N1/19C12N15/113C12N15/63
Inventor 西谷春江佐藤光男森胜弘
Owner KYOWA HAKKO KIRIN CO LTD