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Recombinant beta-lactamase-RGD-fusion protein and application thereof in medicine

A fusion protein, lactamase technology, applied in recombinant DNA technology, drug combinations, peptide/protein components, etc., can solve problems such as toxic side effects

Inactive Publication Date: 2012-05-30
天津天颐科苑科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Highly selective tumor chemotherapy is one of the important topics in current cancer research. Most chemotherapy drugs lack high selectivity to tumor tissue, and often produce toxic and side effects on normal tissues of the body while exerting effects on tumor tissue.

Method used

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  • Recombinant beta-lactamase-RGD-fusion protein and application thereof in medicine
  • Recombinant beta-lactamase-RGD-fusion protein and application thereof in medicine
  • Recombinant beta-lactamase-RGD-fusion protein and application thereof in medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Construction of β-lactamase-RGD4C fusion gene:

[0047] (1) According to the amino acid sequence of the fusion protein, use OptimumGene TM The gene optimization software optimizes the DNA sequence of the protein, and the obtained DNA sequence is as follows:

[0048] TGTGACTGTCGTGGCGATTGTTTCTGTGGCGGTGGCGGCAGCACACCAGTTAGTGAAAAACAACTGGCAGAGGT

[0049] GGTGGCGAACACGATTACCCCACTGATGGCTGCTCAATCCGTTCCAGGTATGGCAGTGGCAGTTATTTTATCAGG

[0050] GCAAACCGCATTACTATAACTTTCGGGAAAGCGGATATCGCGGCCAACAAACCAGTCACTCCACAAACCCTGTTC

[0051] GAACTGGGGAGTATTTCCAAAACGTTTACGGGTGTACTGGGCGGCGACGCTATTGCCCGCGGGGAAATTAGTCT

[0052] GGACGATGCTGTAACCCGTTACTGGCCACAACTGACTGGGAAACAGTGGCAGGGTATCCGCATGCTGGACCTGG

[0053] CGACATACACAGCCGGGGGGCTGCCACTGCAGGTCCCAGATGAGGTTACTGACAACGCCTCTCTGCTGCGTTTT

[0054] TATCAGAACTGGCAACCACAATGGAAACCAGGCACAACACGCCTGTACGCAAATGCATCGATTGGTCTGTTTGG

[0055] CGCTCTGGCTGTTAAACCAAGCGGTATGCCGTATGAACAGGCAATGACGACCCGCGTTCTGAAACCACTGAAAC

[0056] TGGACCATACGTGGATCAACGTACCAAAAAGCCGAA...

Embodiment 2

[0065] Expression of β-lactamase-RGD4C fusion protein:

[0066] The β-lactamase-RGD4C fusion gene verified by sequencing was cloned into the vector pET30a(+) to transform the competent cell BL21(DE3) bacteria, and a single bacterium was picked and inoculated in LB liquid medium, cultured with shaking at 37°C overnight, and then 1 : 100 inoculated in liquid medium containing 25-170 μg / ml chloramphenicol, when OD 600 When reaching about 0.5-1.0, add inducer IPTG to induce protein expression, and the concentration of IPTG is 0.5mM. After continuing to cultivate for 2 hours, centrifuge at 15000 g for 10 min. After being purified by nickel column affinity chromatography, the fusion protein β-lactamase-RGD4C was obtained by freeze-drying, and the induced expression fusion protein was subjected to SDS-polyacrylamide gel electrophoresis.

[0067] The special feature of the present invention is that the time and temperature for inducing expression can be adjusted according to the act...

Embodiment 3

[0069] Enzyme activity and cell adhesion activity assay of β-lactamase-RGD4C fusion protein:

[0070] Dilute the purified β-lactamase-RGD4C fusion protein with coating solution so that the final concentrations are 30,000, 20,000, 10,000, 6,000, 2,000, 500, and 200 ng / ml, and add to 96 wells at a volume of 100 μl per well Cell culture plates were coated overnight at 4°C. After washing the plate three times with 0.9% NaCl, add 200 μl of blocking solution to each well, block at 37° C. for 2 hours, and then wash the plate three times with 0.9% NaCl. After the MCF-7 breast cancer cells were washed three times with 0.9% NaCl, the cells were resuspended with incomplete medium. Add 100 μl of cell suspension to each well, incubate for 1 hr, wash the plate, and remove non-adherent cells. Cells adhering to the culture wells were fixed with fixative solution, stained with 0.5% crystal violet, observed cell adhesion with an inverted microscope and photographed. The results are shown in ...

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Abstract

The invention discloses a beta-lactamase-RGD-fusion protein and a preparation method thereof. The amino acid sequence of the recombinant beta-lactamase-RGD-fusion protein comprises a protein sequence which is derived from beta-lactamase and a sequence which comprises short peptide RGD. The recombinant beta-lactamase-RGD-fusion protein not only has the structure and the function of the beta-lactamase, but also has cell adhesion activity, so that the fusion protein can be positioned at the place of the tumor, thereby having better target. The beta-lactamase-RGD-fusion protein can be used for the treatment of the antibody guide enzyme prodrug, can be used for treating a series of solid tumors such as breast cancer and the like jointly with the cephalosporins prodrug, and has better effect than single melphalan and low toxicity.

Description

technical field [0001] The invention belongs to the technical field of antibody-directed enzyme-prodrug therapy (ADEPT), in particular to a recombinant β-lactamase and RGD fusion protein and its application in treating breast cancer. Background technique [0002] Highly selective tumor chemotherapy is one of the important topics in cancer research at present. Most chemotherapy drugs lack high selectivity to tumor tissue, and often produce toxic and side effects on normal tissues of the body while exerting effects on tumor tissue. Since the advent of monoclonal antibody technology, it has provided a basis for tumor-guided therapy. Prodrugs are a class of drugs that are inactive or have very low activity, but can be converted into active substances in vivo. Using antibodies as carriers to carry specific activating enzymes of prodrugs can selectively bind to tumor sites, so that prodrugs can be converted into active cytotoxic molecules in tumor tissues with regional specificit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/96C12N15/70C12P21/02A61K38/50A61P35/00A61P15/14C12R1/19
Inventor 周晓靓吕茜茜王浩施培基王荣先
Owner 天津天颐科苑科技有限公司
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