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Oligonucleotide for targeted activation of chronic granulocyte leukaemia protein kinase PKR and application thereof

A chronic myeloid and oligonucleotide technology, applied in the field of nucleotide sequences for the treatment of chronic myeloid leukemia, to achieve the effect of easy tracking and observation

Inactive Publication Date: 2007-10-17
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inducing the expression and activity of PKR in tumor cells is a new trend in the field of tumor biotherapy research. At present, there is no report on the activation of PKR to induce the apoptosis of CML cancer cells in the blast phase, so as to achieve the therapeutic target gene.

Method used

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  • Oligonucleotide for targeted activation of chronic granulocyte leukaemia protein kinase PKR and application thereof
  • Oligonucleotide for targeted activation of chronic granulocyte leukaemia protein kinase PKR and application thereof
  • Oligonucleotide for targeted activation of chronic granulocyte leukaemia protein kinase PKR and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Construction and Identification of pH1-BCR-ABL40as Recombinant Plasmid

[0088] 1.1 Obtaining of pH1-BCR-ABL40as insert fragment

[0089] Design SEQ ID NO 1 and SEQ ID NO 2 containing restriction endonuclease BamHI and HindIII sites at both ends respectively. Dissolve each fragment of 1OD in 50μL annealing buffer (10mM Tris, pH7.5~pH8.0, 50mM NaCl, 1mM EDTA), and mix well by inverting, and take 10μL each of SEQ ID NO 1 and SEQ ID NO 2 to 40as tube, placed in boiling water, naturally cooled and annealed into double strands, placed at 4°C for ligation reaction.

[0090] 1.2. Digestion and recovery

[0091] pSilencer3.1-H1

BamHI

Hind III

10×K buffer

wxya 2 o

Totol

40μL

3μL

3μL

6μL

8μL

20 μL

[0092] The reactants were mixed and centrifuged and placed in a 37°C water bath overnight. After the enzyme digestion was completed, all the samples were subjected to 1% agarose gel electrophoresis, and the...

Embodiment 2

[0131] Example 2 Construction and identification of pMSCVeGFP retroviral vector

[0132] 2.1 PCR amplification of IRES2-EGFP vector eGFP fragment with primers Ec-GFP and Xh-GFP (Table 7)

[0133] ① PCR reaction system

[0134] 10×Buffer 3μL

[0135] 2mM dNTP 3μL

[0136] 25mM MgCl 2 1.8μL

[0137] Ec-GFP (10pmol / μL) 3μL

[0138] Xh-GFP (10pmol / μL) 3μL

[0139] IRES2-EGFP 0.2 μL

[0140] pfu Taq (5u / μL) 0.2μL

[0141] Add ddH 2 O up to 30μL

[0142] All the reagents were added into a 100 μL PCR tube, vortexed, centrifuged at 12,000 rpm for 30 sec, and put into a PCR machine for PCR reaction.

[0143] ② PCR thermal cycle reaction conditions

[0144] 94℃ 5min 1cycle

[0145] 94°C 1min

[0146] 55℃ 50sec 30cycles

[0147] 72℃ 40sec

[0148] 72℃ 5min 1cycle

[0149] After the thermal cycle is completed, centrifuge briefly at 12,000 rpm for 30 sec, take 5 μL of the reaction product, and perform electrophoresis analysis on 1.5% agarose gel.

[0150...

Embodiment 3

[0157] Example 3 Construction and identification of pMSCVeGFP-H1-BCR-ABL40AS recombinant retrovirus double expression vector

[0158] 3.1 Acquisition of insert fragments

[0159] The short fragment RNA expression cassette: H1-40as was amplified by PCR from the recombinant plasmid pH1-BCR-ABL40as, and the primers sa and Hd are shown in Table 7.

[0160] ① PCR reaction system

[0161] 10×Buffer 3μL

[0162] 2mM dNTP 3μL

[0163] 25mM MgCl 2 1.8μL

[0164] sa (10pmol / μL) 3μL

[0165] Hd (10pmol / μL) 3μL

[0166] 0.2 μL of each plasmid template

[0167] Taq (5u / μL) 0.2μL

[0168] ddH 2 0 to 30 μL

[0169] All the reagents were added into a 100 μL PCR tube, vortexed, centrifuged at 12,000 rpm for 30 sec, and put into a PCR machine for PCR reaction.

[0170] ② PCR thermal cycle reaction conditions

[0171] 94℃ 5min 1cycle

[0172] 94°C 1min

[0173] 55℃ 50sec 30cycles

[0174] 72℃ 40sec

[0175] 72℃ 5min 1cycle

[0176] After the thermal cycl...

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Abstract

The present invention is aimed to provide oligonucleotide sequence of target activated chronic myeloid leukemia protein kinase PKR and retrovirus dual expression vector possessing the oligonucleotide sequence. The complementary sequence of 20bp SEQ1 and SEQ2 are designed according to BCR-ABL b3a2 type mRNA fusion point upper and down stream, enzyme-cutting point is also designed, a pair of nucleotides T-A is changed to C-G for preventing early stop of transcription.The recombinant retrovirus vector is transformed into K562 cell line of chronic granulocytic leukemia of blast period. The transferred gene is transcribed and hybridizes with BCR-ABL mRNA to form double-chain RNA, activates PKR targetedly, results in K562 cell apoptosis and have no effect on normal cell. The effective ingredient nucleotides sequence is prepared for clinical drug and can treat chronic granulocytic leukemia.

Description

technical field [0001] The invention relates to a nucleotide sequence for treating chronic myeloid leukemia, in particular to an oligonucleotide targeting and activating chronic myelogenous leukemia protein kinase PKR and its application in preparing a medicine for treating chronic myeloid leukemia. Background technique [0002] The occurrence of chronic myeloid leukemia (CML) is due to the BCR-ABL fusion gene encoded by t(9;22)(q34;q11), which produces a BCR-ABL fusion protein with strong tyrosine kinase activity , the fusion protein activates PI3K (Phosphatidylinositol-3kinase), Ras (Renin-angiotensin system), STAT (Signal transducer and activator of transcription) and other signal transduction pathways, leading to malignant transformation of cells. Currently, the drugs for treating chronic myelogenous leukemia mainly include various chemotherapeutic drugs. These drugs have relatively large toxic and side effects on various systems of the whole body, and it is difficult fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/65C12N15/867A61K48/00A61P35/02
Inventor 冯文莉曾建明王小中
Owner CHONGQING MEDICAL UNIVERSITY
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