Immunogen by using mutant CRM197 of diphtheria toxin as carrier, preparation method, and application

A technology of CRM197, G17CRM197, applied in the field of immunogen G17CRM197 and its preparation, can solve the problems of not reaching the theoretical cross-linking rate, the difference in the cross-linking rate of sugar and the like

Active Publication Date: 2009-11-11
QILU PHARMA HAINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

From the experimental results, although the chemical cross-linking rate is high, it has not reached the theoretical cross-linking rate, and the cross-linking rate of different sugars is also different, which shows that the cross-linking rate is also related to the cross-linking product, see: Francisco Betty, Paul Crestanti, Mike Fletcher, Croceo Ruchiraz. Water Solubility, Sedimentability and Kinetic Characteristics of Polysaccharide-Protein Crosslinked Vaccines

Method used

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  • Immunogen by using mutant CRM197 of diphtheria toxin as carrier, preparation method, and application
  • Immunogen by using mutant CRM197 of diphtheria toxin as carrier, preparation method, and application
  • Immunogen by using mutant CRM197 of diphtheria toxin as carrier, preparation method, and application

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Experimental program
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Effect test

Embodiment 1

[0100] 1. Materials used

[0101] 1.1 Main reagents

[0102] Monohydrate cysteine ​​hydrochloride (Cysteine ​​Hydrochloride Monohydrate), sulfhydryl group determination reagent Ellman reagent, cross-linking agent EMCS: product of Pierce (Pierce) company; fluorescamine: product of Sigma company; others are domestic reagents.

[0103] 1.2 Main instruments and equipment

[0104]TGL-16 high-speed desktop centrifuge: product of Shanghai No. 6 Medical Device Factory.

[0105] Micropipettes: products of Eppendof, Germany.

[0106] HD95-1 protein detector and 3057 recorder: (Shanghai Kanghua Biochemical Instrument Factory). Peristaltic pump: Lange pump, YZ1515, Baoding Lange Constant Flow Pump Co., Ltd. BHW-IV electric heating constant temperature water temperature box: Beijing Medical Equipment Factory. J2-MC high-speed low-temperature centrifuge: Beckman company products. Ice machine (GRANT), 1-15 centrifuge (Sigma), Spectrumlad54 UV-Vis spectrophotometer (Shanghai Prism Techn...

Embodiment 2

[0132] 1. In vitro tumor inhibition experiment

[0133] 1.1 Detection of gastrin produced during tumor cell culture

[0134] The invention uses a gastrin detection kit (ELISA method) to measure the production of gastrin in the process of tumor cell culture.

[0135] The cultured tumor cell lines were taken, and the cells were observed under an inverted microscope as a single layer covering the entire growth surface, indicating good growth. Digest and make a single cell suspension in a sterile room. Take the cell suspension and count on the hemocytometer, adjust the amount of RPMI 1640 solution, so that the final cell number is 1×10 9 cells / L, use a pipette to inoculate 1ml of the cell suspension into a T25 culture flask, and add about 4ml of RPMI1640 solution. The liquid was changed once a day, and 5 ml of the replaced RPMI 1640 liquid was collected and reserved for ELISA determination. When changing the medium, take 2 bottles at random, make a single cell suspension accor...

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Abstract

The invention relates to an immunogen and a preparation method thereof. The diphtheria toxin mutant CRM197 is used as a carrier protein, and the heterotype bifunctional cross-linking agent ε-maleimidyl caproic acid N-hydroxysuccinimidyl ester (EMCS ) G17CRM197 obtained by cross-linking gastrin G17 molecules. The carrier protein used in the invention has the advantages of high recovery rate, easy purification, and is suitable for large-scale industrial production. At the same time, the cross-linked product has high cross-linking rate, short preparation time, and reduces production cost. The present invention also relates to the application of the immunogen prepared by the method as the active ingredient of the therapeutic vaccine and the drug prepared with the appropriate immune adjuvant as the auxiliary material in the treatment of tumors.

Description

(1) Technical field [0001] The invention relates to an immunogen G17CRM197 with diphtheria toxin mutant CRM197 as a carrier and a preparation method thereof, as well as the application of the immunogen in the treatment of digestive tract tumors, belonging to the technical field of medicine. (2) Background technology [0002] CRM197 (cross-reacting materials 197) is a mutant that loses the toxicity of diphtheria toxin, see Wu Qida 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.,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K17/02C07K19/00C07K14/595C07K1/10A61K38/16A61K38/22A61P35/00
Inventor 王晶翼王栋海李模孝张薛王庆民刘禄娟卫立辛
Owner QILU PHARMA HAINAN
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