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Recombined protein having phenotypic knock-out effect on chemotropism factor receptor CXCR4

A technology of chemokine receptors and recombinant proteins, which is applied in the biological field to achieve the effects of high technological content, environmental protection requirements, and large investment returns

Inactive Publication Date: 2007-10-31
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, considering that there are many limitations in the overall application of the current transgenic method, how to ensure the effective introduction of the protein with CXCR4 antagonistic effect into cells is the key to realize this new idea

Method used

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  • Recombined protein having phenotypic knock-out effect on chemotropism factor receptor CXCR4
  • Recombined protein having phenotypic knock-out effect on chemotropism factor receptor CXCR4
  • Recombined protein having phenotypic knock-out effect on chemotropism factor receptor CXCR4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Establishment of the prokaryotic expression system of recombinant protein 54R / Tat / KDEL

[0050] 1) Synthesis of Tat (47-57) membrane-penetrating peptide and KDEL fragment: search for the nucleic acid sequence encoding Tat (47-57) and KDEL fragment on the www.pubmed.com website, and connect them sequentially according to BamHI-TAT-KDEL-XhoI Two single-stranded oligonucleotides are artificially synthesized sequentially, and the two strands are reversely complementary. After denaturation at 95°C for 5 minutes, the artificially synthesized single-stranded oligonucleotides are naturally cooled to room temperature, that is, annealed to form double-stranded oligonucleotides acid. The two single-stranded oligonucleotides are as follows:

[0051] A: 5'-GATCCTACGGCCGTAAGAAGCGCCGCCAGCGCCGTCGTAAGGACGAGCTCC-3';

[0052] B: 5'-TCGAGGAGCTCGTCCTTACGACGGCGCTGGCGGCGCTTCTTACGGCCGTAG-3'.

[0053] Among them, the N-terminal of the high-efficiency transmembrane carrier Tat (47-5...

Embodiment 2

[0055] Example 2 Transformation of Escherichia coli BL21 with 54R / Tat / KDEL / pET-28a(+) recombinant plasmid and IPTG induced expression and purification

[0056] Take about 200ng of the correct 54R / Tat / KDEL / pET-28a(+) recombinant plasmid identified by sequencing and add it to 200ul competent BL21(DE3) bacteria (prepared by calcium chloride method), mix gently, and place on ice for 30min. Then heat shock at 42°C for 90 sec, immediately transfer to ice and place for 2min, then add 800ul LB culture solution and shake at 37°C for 45min, take 200ul and spread it on the LB plate containing 50ug / ml kanamycin, 37°C Cultivate overnight (about 12hr), randomly pick 5 positive colonies and inoculate them in 5ml of LB liquid medium (containing 50ug / ml kanamycin) and shake at 37°C for about 12hr, then take 50ul of the above culture solution and transfer them to 50ml of LB liquid medium (containing 50ug / ml kanamycin) was shaken at 37°C for about 3hrs, when OD600=0.5, IPTG with a final concentr...

Embodiment 3

[0068] Example 3 Study on the phenotype knockout activity of CXCR4 in vitro by 54R / Tat / KDEL

[0069] 1) Study on the activity of 54R / Tat / KDEL entering cells

[0070] MOLT-4 cells were treated with 54R / Tat / KDEL for 24 hours, stained with SDF-1-fluorescent antibody, and the efficiency of cell invasion was identified by fluorescence microscope and flow cytometry. 54R / Tat / KDEL increased.

[0071] 2) Subcellular localization research

[0072] MOLT-4 cells were treated with 54R / Tat / KDEL for 24 hours, stained with anti-SDF-1 and CXCR4-fluorescent antibodies, and then labeled with ConA. Confocal microscopy showed that 54R / Tat / KDEL and CXCR4 co-localized in the endoplasm in the net.

[0073] 3) Study on phenotype knockout activity

[0074] After MOLT-4 cells were treated with 54R / Tat / KDEL for 24 hours, they were washed twice with PBS, incubated with anti-CXCR4-PE fluorescent antibody on ice for 30 minutes, and then washed twice with PBS, and the expression of CXCR4 on the cell memb...

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Abstract

The invention discloses a restructuring protein with phenotypic knock-up effect for chemotactic factor acceptor CXCR4 and constructing method, which is characterized by the following: proceeding twice genetic reform for restructuring muton SDF-1 / 54R of SDF-1 alpha with CXCR4 antagonistic active; constructing the restructuring protein 54R / Tat / KDEL with SDF-1 / 54R, high effective wearing film carrier Tat(47-57) and endoplasmic reticulum locating fragment KDEL; utilizing high effective wearing film function of Tat(47-57) and endoplasmic reticulum locating function of KDEL; leading CXCR4 specific antagonist SDF-1 / 54R into breast cancer cell; reaching the goal of phenotype knock up film surface CXCR4; laying necessary work foundation for blocking-up breast cancer transition.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a recombinant protein (named 54R / Tat / KDEL) having a phenotype knockout effect on chemokine receptor CXCR4 and a construction method thereof. Background technique [0002] The high metastasis of breast cancer and other malignant tumors is an important reason for the development, refractory, deterioration and even death of patients. Effectively preventing, delaying or blocking tumor cell metastasis is the key to improving the survival rate of patients with related tumors. It has been noticed that the metastasis of many malignant tumors is not random migration, but has its own migration path and specific organs. There are various theories attempting to explain this phenomenon. Among them, the "soil theory" assumes that different organs provide a special microenvironment suitable for the growth of specific cancer cells; the "homing theory" believes th...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435A61K38/17A61P35/00
Inventor 蔡绍晖杜军马伟峰陈宏远蔡绍皙谭毅郭芝刚
Owner JINAN UNIVERSITY
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