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Recombined diphtheria toxin, preparation method, and application

A diphtheria toxin and recombinant plasmid technology, applied in the field of immune recombinant toxin and its preparation, can solve the problem of no effect on malignant melanoma, and achieve the effect of high hydrophilicity and good extensibility

Inactive Publication Date: 2009-07-29
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Application No. 01138705.X ​​and 01138706.8 Chinese patent applications have constructed a receptor-directed chimeric toxin with a linker sequence and a diphtheria toxin-gonadotropin-releasing chimeric protein. Tests have shown that the above two recombinant toxins have good target targets. It has good therapeutic effect on some cancers, but it has no effect on malignant melanoma. Therefore, it is necessary to construct a recombinant toxin with target specificity for melanoma

Method used

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  • Recombined diphtheria toxin, preparation method, and application
  • Recombined diphtheria toxin, preparation method, and application
  • Recombined diphtheria toxin, preparation method, and application

Examples

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Effect test

Embodiment 1

[0031] Embodiment 1: recombinant plasmid pET28a / prefix body+DAB 389 (Gly 4 Ser) 2 - Construction of αMSH.

[0032] This example describes the recombinant expression plasmid pET28a / prefix+DAB used to express the recombinant toxin of the present invention 389 (Gly 4 Ser) 2 - For the construction strategy and basic method of αMSH, please refer to the attached figure 1 ;

[0033] In order to complete the present invention, the recombinant toxin protein of the present invention can be prepared using gene fusion techniques known to those skilled in the art. For this purpose, various DNA manipulations can be performed according to known recombinant DNA techniques (see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989). pET28a / DAB 389 (Gly 4 Ser) 2 EGF was provided by Zhang Guoli, plasmid pET28a(+) (Novagen), recipient bacteria JM109, BL21 (DE3) were purchased from Novagen. PCR kit, DNA Marker DL-2000, DNA recovery kit...

Embodiment 2

[0054] Example 2: Expression and Identification of Proteins Expressed by Recombinant Plasmids

[0055] This example describes the induced expression of recombinant toxins of the present invention in E. coli host cells.

[0056] With the recombinant plasmid pET28a / prefix body+DAB that makes in embodiment 1 389 (Gly 4 Ser) 2 - Transform competent Escherichia coli BL21 (λDE3) strain (Novagen) with αMSH, pick positive colonies, and culture them at 37°C according to conventional methods. When OD600nm=0.6, lower the culture temperature to the optimal induction temperature of 30°C and add Induced expression with IPTG inducer, the optimal amount of inducer IPTG is 1.4mmol / L, induced for 6 hours; collect BL21 (λDE3) bacterial cells and suspend in 10 times volume containing 20mmol / LTris-HCL solution, after ultrasonic lysis The supernatant and precipitate were taken separately for SDS-PAGE electrophoresis analysis; the analysis results showed that there was a band with a molecular wei...

Embodiment 3

[0058] Embodiment 3: the purification of fusion protein

[0059] The fusion protein was purified by salting out, Cu Chelating Sepharose Fast Flow affinity chromatography, desalting, and factor Xa digestion;

[0060] Add ammonium sulfate to the bacterial lysate supernatant to make the concentration reach 55%, place at 4°C for 1 hour and then centrifuge, dissolve the target protein precipitate with 20mmol / LTris-Cl (containing 2mmol / LEDTA, pH 8.0), and collect the supernatant sample by centrifugation , The samples were analyzed and detected by SDS-PAGE. The protein was purified by Cu Chelating Sepharose Fast Flow chromatography, the sample was loaded, and the gradient elution was carried out with A (20mmol / L Tris-Cl, 0.5mol / L NaCl, pH 7.5) → B (A solution + 50mmol / L imidazole) ( Flow rate is 3mL / min, time 90min), collects each component peak component and carries out SDS-PAGE analysis; The target protein solution is desalted through Superdex G-25 column, collects solution, ultra...

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Abstract

The invention discloses recombinant diphtheria toxin and its preparation method and application. A connecting peptide composed of (Gly4Ser)2 is added between diphtheria toxin DAB389 and α-melanocytostimulating hormone. For the convenience of recombinant toxin purification, add Prefix body, amplify the prefix body + DAB389(Gly4Ser) 2-αMSH gene fragment, construct the pET28a / prefix body + DAB389(Gly4Ser) 2-αMSH expression plasmid, transform it into E. coli for expression, induce with IPTG, and obtain the prefix body + The expression protein of DAB389(Gly4Ser)2-αMSH is purified, digested with factor Xa, and separated to obtain the fusion protein of DAB389(Gly4Ser)2-αMSH. The present invention proves through experiments that the recombinant diphtheria toxin has the advantages of convenient purification and reduced cost , high yield, and the advantages of improving the activity of the recombinant toxin; at the same time, it has the characteristics of specific attack and killing on malignant melanoma, but has no damage to normal cells, and achieves the purpose of effectively treating malignant melanoma.

Description

technical field [0001] The invention relates to an immune recombinant toxin and its preparation method and application, more specifically a recombinant diphtheria toxin, its preparation method and application. Background technique [0002] Malignant tumors are one of the major diseases that seriously threaten human health and survival. Currently, 12% of the world's deaths are caused by cancer. The incidence of malignant tumors in the world is generally on the rise. According to statistics, by the end of the 1990s, cancer mortality had become the number one killer of human health. Among urban residents, cancer has risen from 11.2% of the causes of death in the 1970s to 18.14% now, ranking first among the causes of death and posing an increasing threat to human health and life. [0003] At present, the traditional tumor treatment methods mainly include surgery, radiotherapy, and chemotherapy, but they are still far from satisfactory. Various countries have been actively com...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/195C07K19/00C12N15/31C12N15/62C12N15/09A61K38/16A61P35/00
Inventor 李泽鸿张国利岳玉环朱平陈萍张林波赵福广吴广谋郑艳军张玲
Owner JILIN AGRICULTURAL UNIV
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