CXCR4 antagonist recombination protein SDF-1 beta P2G, preparation method and application thereof

A recombinant protein, SDF-1 technology, applied in the biological field, can solve problems such as inability to continuously promote blood flow recovery, achieve the effects of inhibiting growth and metastasis, restoring function, and high bioavailability

Inactive Publication Date: 2008-12-31
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, long-term injection of AMD3100 cannot continue to promote blood flow recovery (once a day, inj...

Method used

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  • CXCR4 antagonist recombination protein SDF-1 beta P2G, preparation method and application thereof
  • CXCR4 antagonist recombination protein SDF-1 beta P2G, preparation method and application thereof
  • CXCR4 antagonist recombination protein SDF-1 beta P2G, preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Cloning of SDF-1βP2G cDNA and Construction of Prokaryotic Expression System

[0054] 1. Primer design: According to the cDNA sequence of human SDF-1β provided by the Genebank database, according to the general primer design principles and the requirements of PET30a(+) cloning, use primer5.0 to design RT-PCR primer SDF-1βP2G-F with the coding region as a template / SDF-1βP2G-R, directly amplifies the mutant SDF-1βP2G in which the second amino acid residue at the N-terminus of SDF-1β is mutated from proline to glycine.

[0055] Primer SDF-1βP2G-F is: 5'-ggGGTACCgacgacgacgacaagaagggcgtcagcctgagctacagat-3';

[0056] The primer SDF-1β-R is: 5'-acgGAGCTCacatcttgaacctcttgtt-3'.

[0057] 2. Extraction of total RNA: Take out about 0.25ml of bone marrow sample from liquid nitrogen, immediately add 0.75ml TRIZOL LSReagent, after the sample is thawed, blow and beat the bone marrow cells repeatedly with a pipette. The lysed cell homogenate was incubated at 15-30°C for 5 minutes to ...

Embodiment 2

[0085] Prokaryotic expression and purification of recombinant protein SDF-1βP2G

[0086] Add about 200ng of the confirmed SDF-1βP2G / pET-30a(+) recombinant plasmid into 200μl competent Escherichia coli BL21(DE3)(NOVAGEN) cells (prepared by calcium chloride method), mix gently, and place on ice for 30min , then heat-shocked at 42°C for 90s, immediately transferred to ice and placed on ice for 2min, then added 800μl LB culture solution and cultured with shaking at 37°C for 45min, took 2001 and spread it on LB plates containing 50μg / ml kanamycin, at 37°C Cultivate overnight (about 12hr), randomly pick 5 positive colonies and inoculate them in 50ml LB liquid medium (containing 50μg / ml kanamycin) for about 12hr at 37°C, then transfer 50μl of the above culture solution to 50ml of LB liquid medium (containing 50μg / ml kanamycin) was shaken at 37°C for about 3hrs, when OD600=0.5, IPTG with a final concentration of 0.75mM was added to induce expression at 37°C for 3hrs, centrifuged at 30...

Embodiment 3

[0103] SDF-1βP2G antagonizes the activity of CXCR4

[0104] 1. Chemotactic activity

[0105] Take a 24-well plate, add predetermined final concentrations (0, 0.001, 0.01, 0.1, 0.5, 1, 10, 100nmol / L equal concentration gradient) of RPMI1640 dilution of SDF-1β, SDF-1βP2G, and then gently Use tweezers to place the Chemotaxic chamber with pore size of 5μm at the bottom, pay attention to avoid air bubbles, then add 1×10 7Cells / ml MOLT-4 cell suspension 200μL; Incubate in a 5% CO2 incubator at 37°C for 2-3hrs, then gently remove the chemotaxis chamber at one time, and count the number of cells in each well of the cell culture plate under a microscope . Relative Chemotaxic Activity = number of transmembrane cells / total number of uploaded cells × 100%. Each treatment was repeated three times, and the data were processed with DPSv5.02. The results were expressed as Mean±S, and the multiple comparisons were performed by the new multiple range method of DUNCAN.

[0106] The results s...

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Abstract

The invention relates to a CXCR4 antagonist recombinant protein SDF-1 Beta P2G as well as a method for the production thereof and applications thereof, comprising: using a specific primer to obtain the SDF-1 Beta P2G cDNA by taking a human SDF-1 Beta cDNA as a template through the site-directed mutagenesis PCR amplification, directly inserting the SDF-1 Beta P2G cDNA into a cloning expression vector pET-30a(+) to construct an SDF-1 Beta P2G/pET-30a(+) recombinant, transforming an Escherichia coli BL21 (DE3), screening positive clones, and preparing the SDF-1 Beta P2G recombinant protein through IPTG induced expression, purification, renaturation, restriction enzyme and high efficiency liquid chromatography separation. The SDF-1 Beta P2G recombinant protein can antagonize a chemotactic factor receptor CXCR4, promote stem cells, angioblasts, etc. to enter peripheral blood from bone marrow, and promote the revascularization and recovering of blood flow of ischemia organs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to the preparation and application of a recombinant protein SDF-1βP2G capable of antagonizing chemokine receptor CXCR4 and promoting blood flow recovery and angiogenesis after ischemia. Background technique [0002] Promoting angiogenesis and neovascularization is an effective therapeutic strategy to rescue severe ischemic tissue or organ failure (Urbich and Dimmeler, Circ Res, 2004, 95:343-353). Using pro-angiogenic factors such as VEGF (Schratzberger et al., Nat Med, 2000, 6: 405-413), FGF-2 (Cao et al., Nat Med, 2003, 9: 604-613; Khurana and Simons, Trends Cardiovasc Med, 2003, 13: 116-122) and SDF-1 (Jin et al., Nat Med, 2006, 12: 557-567) have achieved short-term efficacy in the treatment of patients with severe lower extremity ischemia, but their long-term efficacy has not yet been obtained. Clinically proven (Jin et al., Nat Med, 2006, 12:557-567). Tissue damage is o...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N15/70C07K14/47A61K38/17A61P7/00A61P9/10A61P35/00A61P35/04A61P19/02
Inventor 谭毅李校堃蔡露
Owner WENZHOU MEDICAL UNIV
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