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Diphtheria toxin muton CRM197 and its preparation process

A technology of CRM197 and diphtheria toxin, which is applied in the field of preparation of immune protein carrier, can solve the problems of insufficient industrial preparation

Active Publication Date: 2007-07-18
QILU PHARMA HAINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the soluble expression of CRM197 made by Bishai et al., the expression of CRM197 only accounts for 7.7% of the total protein, which is far from enough for industrial production

Method used

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  • Diphtheria toxin muton CRM197 and its preparation process
  • Diphtheria toxin muton CRM197 and its preparation process

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Experimental program
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Embodiment 1

[0164] 1. Materials used

[0165] 1.1 Plasmids and strains: plasmid pUC18-CRM197, vectors pET25b, pET32a, pET43.1a. Escherichia coli JM109, DH5α, BL21 strains.

[0166]1.2 Tool enzymes: NdeI, HindIII, T4DNA ligase, Pfu enzyme: products of Promega.

[0167] 1.3 DNA molecular weight marker: product of Dalian Bao Biological Engineering Co., Ltd. DL-2000 consists of DNA fragments of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp; DL-15000 consists of DNA fragments of 15000bp, 10000bp, 7500bp, 5000bp, 1000bp, and 250bp.

[0168] 1.4 Protein molecular weight marker: product of Dalian Bao Biological Engineering Co., Ltd. Molecular weight: 97400D, 66200D, 43000D, 31000D, 20100D, 14400D.

[0169] 1.5 Reagents:

[0170] Bacterial broth (LB): 10g refined peptone; 5g yeast extract; 10g sodium chloride; add distilled water to 1000ml, adjust pH to 7.5 with sodium hydroxide after dissolving, and autoclave.

[0171] Bacterial liquid medium (LA): add ampicillin to the above LB medium to ...

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Abstract

The present invention is diphtheria toxin mutant CRM197 and its preparation process, and belongs to the field of immune protein carrier technology. The diphtheria toxin mutant CRM197 is expressed in colibacillus in the form of inclusion body, and the target product has sequence length of 536 amino acids, the immunogenicity the same as that of diphtheria toxin and no toxicity similar to that of diphtheria toxin. The present invention clones gene coding CRM197, constructs recombinant expression plasmid expressing CRM197 and transforms to colibacillus host for expression in the form of inclusion body. The present invention has target protein accounting for over 24 % of total protein content, high CRM197 purity over 95 % and high target protein recovering rate. The preparation process is simple, low in cost and suitable for industrial production.

Description

(1) Technical field [0001] The invention relates to the preparation of an immune protein carrier, in particular to a diphtheria toxin mutant CRM197 and a preparation method thereof. (2) Background technology [0002] CRM197 (cross-reacting materials 197) is a mutant of diphtheria toxin that has lost its toxicity, see Uchida et al. Diphtheria toxin and related proteins I. Isolation and description of diphtheria toxin serotype-related mutant strains, Biochemistry, 1973.248: 3838-3844. (Uchida, T., A.M.Pappenheimer, Jr.and R.Gregory.al., Diphtheria toxin and related proteins I.Isolation and properties of mutantproteins serologically related to diphtheria toxin.J.Biol.Chem.1973.248:3838 -3844.), which is mutated from a base G to A in the base sequence of the wild-type diphtheria toxin, thus resulting in the mutation of the 52nd amino acid GLY to GLU, see Giannini et al., two diphtheria toxins without Amino acid sequences of toxic mutants CRM45 and CRM197, Nucleic Acid Research,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/005C12N15/31C12N1/21
Inventor 李模孝张薛王栋海孙英侠郭俊贤王晶翼王庆民
Owner QILU PHARMA HAINAN
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