Gastric cancer targeted recombinant toxin and preparation method thereof

A gastric cancer, targeted technology, applied in recombinant DNA technology, chemical instruments and methods, drug combinations, etc., can solve problems such as large side effects, limited application of DT, and damage to the patient's immune system.

Inactive Publication Date: 2013-05-08
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Surgery is currently the only way to cure gastric cancer, but less than 1/3 of the patients can be treated with surgery. In addition to surgery, chemotherapy and radiotherapy are often used for advanced patients. However, the side effects of chemotherapy and radiotherapy ar...

Method used

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  • Gastric cancer targeted recombinant toxin and preparation method thereof
  • Gastric cancer targeted recombinant toxin and preparation method thereof
  • Gastric cancer targeted recombinant toxin and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of a recombinant strain highly expressing the recombinant toxin of the present invention (hereinafter referred to as rG7PE38)

[0040] Comparison and analysis of existing PEA toxin sequences published by GenBank: U78761, RFT5 (scFv) ETA immunotoxin gene sequence; AY585869, H22 (scFv) PE40 recombinant toxin gene sequence; K01397, Pseudomonas aeruginosa exotoxin A gene; EU595736 green Pseudomonas aeruginosa PACS458 strain gene; EU595734, Pseudomonas aeruginosa PACS181 strain gene; CP002496, Pseudomonas aeruginosa M18 strain gene; AE004091, Pseudomonas aeruginosa PAO1 strain gene; AP012280, Pseudomonas aeruginosa NCGM2 strain S1 gene; FM209186, Pseudomonas aeruginosa LESB58 strain Gene: CP000438, gene of Pseudomonas aeruginosa UCBPP-PA14 strain; according to the comparison result and the sequence optimized by Jcat software, artificial gene synthesis is carried out, and its nucleotide sequence is as described in SEQ No.2.

[0041] The artificial g...

Embodiment 2

[0042] Example 2 Expression condition optimization

[0043] (1) Optimization of induction temperature

[0044] Inoculate the Rosetta (DE3) (pET-rG17PE38) bacterial solution that was shaken overnight into 5ml LB liquid medium (containing 20μg / ml ampicillin) at 1% inoculum, and cultivate to OD at 160rpm at 37°C 600 was 0.4, added isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 1.0 mM, and continued at 180 rpm at 20°C, 25°C, 30°C, 37°C, and 42°C. Cultivate for 6h.

[0045] (2) Optimization of induction timing

[0046] Rosetta (DE3) (pET-rG17PE38) cultured by overnight shaking culture was inoculated into 5ml LB liquid medium (containing 20μg / ml ampicillin) with 1% concentration, and cultured at 160rpm at 37℃ for 0.5h, 1.0h, 1.5h, Isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 1.0 mM was added at 2h, 2.5h, 3.0h, and 3.5h, respectively, and culture was continued at 37°C and 180rpm for 6h.

[0047] (3) Optimization of the amount...

Embodiment 3

[0050] Example 3 protein purification

[0051] (1) Ammonium sulfate concentration selection

[0052] Recombinant bacteria were induced by isopropyl-β-D-thiogalactopyranoside (IPTG), crushed by ultrasound at 300W for 5min, and centrifuged to get the supernatant; divided into 6 equal parts, and added to the final saturation of 10% and 20% respectively , 30%, 40%, 50%, 60% ammonium sulfate, mix thoroughly and centrifuge to collect the supernatant, continue to add ammonium sulfate to saturation of 20%, 30%, 40%, 50%, 60% and 70% , and the supernatant was discarded by centrifugation; protein samples were prepared after the pellet was resuspended, and analyzed by 12% SDS-PAGE. Screening for optimum ammonium sulfate concentration.

[0053] (2) Screening of hydrophobic chromatography conditions

[0054] Phenyl FF (high sub), Phenyl HP, Butyl FF, Octyl FF, etc. (purchased from General Electric Healthcare, USA), collected the eluate from each peak, and added trichloroacetic acid at a...

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Abstract

The invention relates to a gastric cancer targeted recombinant toxin and a preparation method thereof, and belongs to a targeted recombinant toxin and a preparation method thereof. The gastric cancer targeted recombinant toxin is formed by connecting human gastrin G17 with recombinant toxin PE38KDEL derived from pseudomonas exotoxin A, and particularly the recombinant toxin is prepared by using G17 expressed by a prokaryotic expression system and translated reversely as a guiding unit through gene engineering expression; and a unique fermentation condition for producing a soluble protein in a large scale is established, and the recombinant toxin targeting gastric cancer and having high selective killing activity is provided.

Description

technical field [0001] The invention relates to the preparation of gastric cancer targeting recombinant toxin by genetic engineering. Background technique [0002] Gastric cancer is one of the most common cancers worldwide and the second most common cancer in China. Surgery is currently the only way to cure gastric cancer, but less than 1 / 3 of the patients can be treated with surgery. In addition to surgery, chemotherapy and radiotherapy are often used for advanced patients. However, the side effects of chemotherapy and radiotherapy are too large and often serious. Destroy the patient's immune system. 40 years ago, the concept of immunotoxin proposed by Moolten FL et al. brought a bright light to the treatment of cancer; the strategy of immunotoxin is to use the specific combination of antigen-antibody, cytokine-receptor, based on the special marker or tumor cell surface Specific receptors or huge differences in the number of receptors from normal cells are used as targets...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/16C12N15/62C12N15/70A61K38/16A61K47/48A61P35/00A61K47/64
Inventor 柳增善宋杰任洪林卢士英李岩松周玉张茂林高世奇
Owner JILIN UNIV
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