Recombined protein having phenotypic knock-out effect on chemotropism factor receptor CXCR4 and its construction method
A chemokine receptor, recombinant protein technology, applied in the biological field, achieves the effect of high technology content, environmental protection requirements, and large investment benefits
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Embodiment 1
[0049] The establishment of the prokaryotic expression system of embodiment 1 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 Ta...
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 800u1LB culture solution and shake and culture at 37°C for 45min, take 200ul and spread it on the LB plate containing 50ug / ml kanamycin, culture at 37°C Overnight (about 12hr), randomly pick 5 positive colonies and inoculate them into 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 LB liquid medium (containing 50ug / ml kanamycin) was shaken at 37°C for about 3hrs, when OD600=0.5, IPTG with a fin...
Embodiment 354
[0068] Example 354R / Tat / KDEL in vitro CXCR4 phenotype knockout activity research
[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 membrane surfac...
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