Small molecule recombination toxin GnRH-luffinS2 fusion albumen and its preparation and application

A fusion protein and hormone-releasing technology, applied in drug combination, peptide/protein components, recombinant DNA technology, etc., can solve the problem of difficult to accurately control the location and degree of sulfhydrylation, space hindrance of receptors and ligands, and generation of heterologous molecules problems such as low immunogenicity, small molecular weight, and high-efficiency expression

Inactive Publication Date: 2008-09-17
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are two methods for the construction of immunotoxins, one is the chemical coupling method, but this method has the following disadvantages: it is difficult to accurately control the location and degree of sulfhydrylation, which may produce heterologous molecules; the random connection between the toxin and the ligand, Prone to steric hindrance of receptors and ligands; low yield, large amounts of protein must be prepared and purified for conjugation, high cost
Whether the Linker design is correct or not is very important for the activity of the two parts of the recombinant immunotoxin. The Linker must have a certain length to ensure that the protein molecules on both sides will not affect each other in space. In addition, if the Linker amino acid is not properly selected, it is easy to carry high static electricity. It is difficult to guarantee the desired effect

Method used

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  • Small molecule recombination toxin GnRH-luffinS2 fusion albumen and its preparation and application
  • Small molecule recombination toxin GnRH-luffinS2 fusion albumen and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 PCR Amplification of GnRH-luffin S2 Recombinant Fragment

[0033] Use the Codon software package to design 6 overlapping PCR primers, use overlapping PCR to amplify the GnRH-luffin S2 fragment, and add BamHI and HindIII restriction sites to both ends of the first primer and the last primer respectively. Each primer is shown in the sequence list SEQ ID NO.1~NO.6.

Embodiment 2

[0034] Example 2 Connection of GnRH-luffin S2 amplified fragment and expression vector pQE-30

[0035] GnRH-luffin S2 and pQE-30 were digested with BamHI and HindIII respectively, recovered and ligated to obtain pQE / GnRH-luffin S2, whose sequence is shown in SEQ ID NO.7 in the sequence list.

Embodiment 3

[0036] Example 3 Expression of Fusion Gene

[0037] Transform into Escherichia coli M15 for expression, the specific conditions are: Inoculate the Escherichia coli strain containing the expression plasmid into LB medium, shake overnight at 37°C, inoculate fresh LB medium at 1:100 the next day, shake at 37°C to OD 600 =0.4, add the inducer IPTG, the concentration is 0.4mM, continue to shake at 37°C for 5 hours, induce the production of GnRH-luffin S2 fusion protein, its sequence is as SEQ ID NO.8 in the sequence listing.

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Abstract

The invention relates to an anittumor recombinant immunotoxin, preparation method and application thereof. The invention constructs a targeting toxin fusion, which contains amino acid fragment of luffin S2, human gonadotropin-releasing hormone and intermediate connecting peptide. The preparation method of the fusion includes (1) constructing plasmid containing objective gene; (2) transforming to express in Escherichia coli; (3) centrifuging to collect thallus, ultrasonic cracking, and washing to obtain crude inclusion body; and (4) dialyzing, performing affinity chromatography, excising His label and eliminating Xa factor.. The targeting toxin fusion has strong cell cytotoxic, less usage dosage and low product cost, which is effective in inhibiting tumor in early growth stage and tumor in middle growth stage; and is adaptable to the therapy of solid tumor such as breast cancer, oophoroma, endometrial carcinoma, prostatic cancer, etc.

Description

technical field [0001] The invention relates to an anti-tumor recombinant cytotoxin, in particular to human gonadotropin-releasing hormone (GnRH or LHRH), luffin S2 targeting toxin fusion protein and a preparation method thereof. Background technique [0002] The current treatment plan for tumors is mainly based on surgery, supplemented by chemotherapy and radiotherapy. However, due to the poor specificity of this treatment for tumor killing, there are relatively large toxic and side effects, such as bone marrow suppression, gastrointestinal reactions, etc. , decreased immune function, skin and mucous membrane damage, organ toxicity, etc. Therefore, tumor-guided therapy has become one of the research hotspots in tumor therapy. [0003] Tumor-directed therapy refers to the use of highly specific pro-tumor substances as carriers, cytotoxic substances as "warheads", relying on the carrier's specific affinity for tumors, to concentrate the warhead substances on tumor cells as m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/09C12P21/02A61K38/16A61P35/00
Inventor 王景林刘艳华康琳高姗
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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