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Eukaryon expression vector and application thereof to preparing medicament for inhibiting leukemia cell proliferation

A technology of eukaryotic expression vectors and leukemia cells, applied in the field of genetic engineering, can solve the problems of time-consuming, labor-intensive, poor economy, and no biological activity

Inactive Publication Date: 2011-06-01
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above techniques are practical, mature, and have sufficient protein yields, they are time-consuming and labor-intensive, and economically poor [Barka T, Gresik ES, Henderson SC (2004) Production of celllines secreting tat fusion proteins. J Histochem Cytochem 52(4): 469-477]
Moreover, the fusion protein obtained by the prokaryotic expression system lacks post-transcriptional cleavage and translation modifications, and there is a possibility that it has no biological activity to a large extent.

Method used

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  • Eukaryon expression vector and application thereof to preparing medicament for inhibiting leukemia cell proliferation
  • Eukaryon expression vector and application thereof to preparing medicament for inhibiting leukemia cell proliferation
  • Eukaryon expression vector and application thereof to preparing medicament for inhibiting leukemia cell proliferation

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Construction of pcDNA3.0-190CT3 eukaryotic expression vector

[0029] 1.1 Required reagents

[0030] Restriction enzymes Sal I, Nhe I, Xho I and BamH I were purchased from Shanghai Yingjun Company (Invitrogen, Shanghai). DNA polymerase, PCR product purification kit, and gel recovery kit were purchased from Takara Bioengineering Co., Ltd. (Takara, Dalian). The eukaryotic expression vector pcDNA3.0 was purchased from Invitrogen (Carlsbad, CA, USA). Mouse anti-human cMyc epitope (tag) monoclonal antibody was purchased from Santa Cruz Company (Santa Cruz, CA, USA); FITC-labeled fluorescent goat anti-mouse secondary antibody was purchased from Shanghai Kangcheng Company (Kangcheng, Shanghai). All the following primers were synthesized by Shanghai Yingjun (Invitrogen, Shanghai).

[0031] 1.2 Construction preparation of eukaryotic vector pcDNA3.0-190CT3

[0032] Using the eukaryotic expression vector pcDNA3.0-gp190CT3 as a template (see Chinese Patent ZL20041001...

Embodiment 2

[0045] Example 2: Establishing a stable pcDNA3.0-190CT3 recombinant CHO cell line

[0046] First determine the appropriate concentration of G418 (Invitrogen) in the screening medium. The method is as follows: 1000 CHO cells per well are inoculated in a 24-well cell culture plate, and G418 is added to make the concentration respectively 500, 1000, 2000, and 3000 μg / ml. The wells in which the cells were completely killed in 10-14 days were used as the screening concentration of G418.

[0047] Transfect pcDNA3.0-190CT3 into CHO cells according to the instruction manual of the transfection reagent Fugene 6. 24 hours after transfection, the cells were subcultured at a ratio of 1:10, and the cultured cells were screened with pressure selection medium containing 3000 μg / ml G418. After 10 to 14 days, the neomycin-resistant cell clone colonies were surrounded by cloning rings. , Digested with trypsin and expanded in 96-well, 24-well and 6-well cell culture plates in turn. Different c...

Embodiment 3

[0054] Example 3: Isolation and purification of fusion protein 190CT3

[0055] The above CHO-190CT3 cell line according to the following medium composition, in 75cm 2 Expand culture in a culture flask: 90% RPMI 1640 medium, 9% fetal bovine serum (FBS), 1% penicillin and streptomycin, and 3000 μg / ml G418 neomycin was added after passaged cells adhered to the wall. The culture condition is 37℃+5%CO 2 . When the cells are fused to 70-80%, replace the medium with 15ml of 100% RPMI 1640 medium. After 72 hours, the supernatant was collected, centrifuged at 10,000 rpm for 20 minutes to remove cell debris, and the remaining supernatant was immediately stored at -20°C. Repeat the above supernatant recovery procedure until the recovered volume reaches 1L. Before using the protein chromatography column to purify related proteins, put the supernatant at 4°C to let it melt slowly, and then pass through a 0.45 μm filter to remove possible impurities, dead cells and cell debris, ready to...

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Abstract

The invention relates to the technical field of gene engineering and aims to provide a eukaryon expression vector combining technologies of LIFRalpha-CT3 (Leukemia Inhibitory Factor Receptor alpha)-(Cell Therapy 3) and TAT-PTD (Transactivator of Transcription-Protein Transduction Domain) and application thereof to preparing a medicament for inhibiting leukemia cell proliferation. The eukaryon expression vector provided by the invention comprises nucleotide sequences shown as SEQ ID NO: 1. The invention also provides a CHO (Chinese Hamster Ovary) cell line transfected by the eukaryon expression vector. The invention also relates to a polypeptide 190CT3 produced by utilizing the CHO cell line. The polypeptide 190CT3 has very high stability and transmembrane property, and can remarkably improve the problems of poor targeting property, easy resistance to medicaments and difficulty of endocytosis / endoeukaryon of medicaments, which are caused by all-trans retinoic acid and routine chemotherapy, in the clinical promyelocytic leukemia treatment. Vitro tests prove that the 190CT3 can penetrate through a cell membrane and a nuclear membrane quickly when the 190CT3 directly acts on acute promyelocytic leukemia cells, and further inhibit the proliferation of the cells and promote the cells to differentiate into mature granular cells.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a eukaryotic expression vector and the application of the eukaryotic expression vector in preparing medicines for inhibiting leukemia cell proliferation. Background technique [0002] Leukemia inhibitory factor (Leukemia inhibitory factor, LIF), as a member of the IL-6 cytokine family, can inhibit the proliferation of leukemia cells, such as human HL-60, U937, and mouse M1 lines. However, the characteristics of rapid growth of leukemia cells and poor sensitivity to LIF make the limited LIF in the body unable to effectively inhibit the growth of leukemia cells[T.Maekawa, D.Metcalf, Clonalsuppression of HL60 and U937 cells by recombinant human leukemia inhibitoryfactor in combination with GM-CSF or G-CSF, Leukemia 3(1989) 270-276; D.P.Gearing, N.M.Gough, J.A.King, D.J.Hilton, N.A.Nicola, R.J.Simpson, E.C.Nice, A.Kelso, D.Metcalf, Molecular cloning and expre...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N5/10C07K19/00C12P21/02A61K38/00A61K48/00A61P35/02
Inventor 刘厚奇孙擎杨玲
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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