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High-endophilicity targeting amalgamation protein

A fusion protein and genetic engineering technology, applied in the field of high-affinity targeted fusion proteins, can solve the problems of strong non-specific toxicity, low affinity, and poor specificity

Inactive Publication Date: 2008-06-11
HUNAN KANGDU PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, targeted therapeutic agents using GnRH or its derivatives as the guiding part and PE40 and its derivatives as the toxin part have been reported (Chinese Patent: Method for Diagnosing Cancer Using Chimeric Toxin, 99806580.3; Genetic engineering recombinant protein of dead tumor cells, 03137587.1; Chinese patent: a series of functional proteins with high efficiency and low toxicity, 200410033621.6), but it has the disadvantages of weak affinity with GnRH receptors and poor stability, which leads to its potential as a tumor Therapeutic agents have the characteristics of poor specificity and strong non-specific toxicity, so this type of molecule cannot be used as an effective tumor treatment drug at present

Method used

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  • High-endophilicity targeting amalgamation protein

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Effect test

example 1

[0041] According to the principle of codon preference in Escherichia coli, the gene sequence of SEQ ID NO: 1 was synthesized by the method of total gene synthesis combined with PCR, and then SEQ ID NO: 2-8 and the currently known GnRH- PE derivatives, gene sequences of three control test fusion proteins GnRH-PE40 (SEQ ID NO: 9), (Ala6) GnRH-PE40 (SEQ ID NO: 10) and (Ala6) GnRH-PE40KDEL (SEQ ID NO: 11) . After sequencing verification, connect the target gene into the expression vector, transfer the verified recombinant plasmid into the host bacteria, obtain the target bacteria after expression and fermentation, and then break the bacteria by osmotic pressure method, centrifuge to take the supernatant, pass The methods of hydrophobic chromatography and ion exchange chromatography were purified to obtain 8 targeted fusion proteins of the present invention with a purity greater than 95% and three kinds of control test fusion proteins GnRH-PE40, (Ala6) GnRH-PE40 and (Ala6) GnRH- P...

example 2

[0043] Take eight kinds of targeted fusion proteins in the present invention and three kinds of control fusion proteins to carry out cell activity test: use MTT method, first digest and collect Hela cells, and dilute the cells to 6-8×10 with RPMI 1640 culture medium 4 cell suspension per milliliter, seeded in 96-well plate, 100ul / well, cultured at 37°C for 4 hours. Add 100ul culture solution to the control. The 11 kinds of samples were first diluted with RPMI 1640 culture medium to 100ul / mL as the initial well, and then the samples were diluted according to the relationship of 2.5 times between each well and the upper well, and each diluted sample was added to each well with 100ul, 37 degrees, and incubated for 24 hours. Take it out, stain it with MTT method, measure it with a microplate reader at 570nm and calculate the IC50 value. The results of the activity assay are shown in the table below,

[0044]

example 3

[0046] Get eight kinds of targeted fusion proteins in the present invention and three kinds of control test fusion proteins to carry out affinity test (affinity measurement of target protein and GnRH receptor): prepare placental plasma membrane, get placental villi, place in ice-cold 25mM PB, pH7.4, 1mM MgCl 2 Homogenize in medium, filter, and centrifuge at 10000g for 20min, remove the supernatant, wash the precipitate once with the above buffer, then centrifuge at 10000g for 15min, suspend the precipitate in the above buffer at a wet weight ratio of 60mg / mL for later use, and use the Lowry method protein concentration. Iodine labeling of targeted fusion proteins: add 3.8 x 10 to a 1.5 mL doff tube 7 Bq Na 125 I, 20uL 0.5M PB, pH7.5, 500ug targeting fusion protein, 2.3mU lactate catalase β-D(+)-glucose to start the reaction, react at 20°C for 10min, then add 50uL 0.1M boric acid buffer pH9. 2 Terminate the reaction. Chromatographic purification was then performed using 1 m...

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Abstract

The invention mainly relates to fusion protein which is composed of a hormone releasing factor derivative produced through human promoting corpus luteum (GnRH), a lacunae body PE40 or PE38 of pseudomonad exotoxin A, and the mutant thereof which are connected in series, the invention has the key points that the 5, 7, and 8-position amino acid of the N tail end of LHRH is abruptly changed, to ensure the abruptly changed fusion protein to have the stronger tomour cell affinity and distinguishing destruction compared with the known target-oriented fusion protein.

Description

1. Technical field [0001] The invention belongs to the field of guiding fusion proteins in biological genetic engineering. At present, cancer is still the disease with the second highest mortality rate. At present, people do not have a particularly effective means to treat it. Chemotherapy is still the most commonly used treatment method at present. Although there are It has a certain curative effect, but its side effects are large, the therapeutic effect is only partially ideal, and the disadvantages such as easy generation of drug-resistant cancer cell lines during treatment have affected the curative effect and efficiency of this method. [0002] Guided therapy is a new treatment method that is expected to replace chemotherapy. There are many documents and patents (Chinese patent: method for diagnosing cancer with chimeric toxin, 99806580.3; Chinese patent: a genetic engineering recombinant that can specifically kill tumor cells protein, 03137587.1; Chinese patent: a series...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/62C07K19/00C12N1/19A61K38/16A61P35/00
Inventor 王桂有赵自育陈海军陆学山
Owner HUNAN KANGDU PHARMA
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