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Fused protein restructuring target lethal leukemia cells and preparation method and use thereof

A leukemic cell and fusion protein technology, applied in the direction of drug combination, peptide/protein components, recombinant DNA technology, etc., to achieve the effect of broad application prospects

Inactive Publication Date: 2008-03-12
THE SECOND AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the targeted therapy of AML with recombinant IL3 and PE fusion protein, which has important research value

Method used

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  • Fused protein restructuring target lethal leukemia cells and preparation method and use thereof
  • Fused protein restructuring target lethal leukemia cells and preparation method and use thereof
  • Fused protein restructuring target lethal leukemia cells and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Preparation method of plasmid pGEX-4T-1-PE38KDEL

[0031] 1. Amplification of the PEA gene

[0032] Chromosomal DNA of Pseudomonas aeruginosa PA103 was extracted according to conventional methods. A pair of primers located on both sides of the PEA structural gene were designed according to the sequence published by Gray et al.

[0033] 5' Primer: 5'-GAT CAG CCT CAT CCT TCA C-3',

[0034] 3' primer: 5'-GCT CGC GGC AGT TAC TT-3'.

[0035] Reaction system: 5 μl DNA template (0.5 μg), 5 μl 10×PCR amplification buffer, 2 μl (100 ng) each of 5’ primer and 3’ primer, 1 μl of 10 mmol / L dNTP mixture, 5 μl of 50% glycerol, 2.5 μl of DMSO, Bacteria water 27μl, TaqDNA polymerase 0.5μl (2.5u), total volume 50μl. Reaction conditions: Denaturation at 94°C for 4 wins, followed by cycling, denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 3 min, and a total of 30 cycles. Final extension at 72°C for 30 min.

[0036] 2. PE40 gene fragment...

Embodiment 2

[0052] Example 2: Expression and Activity Identification of Recombinant Protein IL3-PE38KDEL

[0053] 1. Materials and methods

[0054] 1.1 Reagents

[0055] Various restriction endonucleases, TaqDNA polymerase, PrimeSTARTM HS DNA Polymerase, DL2000Marker, 500bpMarker, protein marker, T4 ligase, plasmid mini-pumping kit, and gel recovery reagents were all purchased from Takara Company, and the primers were synthesized by Shanghai Ying Jun company completed.

[0056] IPTG, Coomassie Brilliant Blue R250 were purchased from Takara Company.

[0057] Nickel ion affinity chromatography column (Ni2+-NTA) was purchased from Novagen.

[0058] RPMI1640 was purchased from GIBCO Company.

[0059] Calf serum was purchased from Hangzhou Sijiqing Biomaterials Co., Ltd.

[0060] MTT is a product of Amresco.

[0061] 1.2 Construction of fusion gene PQE30-IL3-Linker-PE38KDEL

[0062] 1.2.1 Construction of recombinant plasmid pQE30-Linker

[0063]In order to ensure that the protein exp...

Embodiment 3

[0094] Example 3: Expression and Activity Identification of Recombinant Protein IL3-Linker-PE38KDEL

[0095] 1. Materials and methods

[0096] 1.1 Reagents

[0097] Various restriction enzymes, TaqDNA polymerase, PrimeSTAR TM HS DNA Polymerase enzyme, DL2000Marker, 500bpMarker, protein marker, T4 ligase, plasmid mini-pump kit, and gel recovery reagent were all purchased from Takara Company.

[0098] The synthesis of primers was completed by Shanghai Yingjun Company.

[0099] IPTG, Coomassie Brilliant Blue R250 were purchased from Takara Company.

[0100] Nickel ion affinity chromatography column (Ni 2+ -NTA) was purchased from Novagen.

[0101] RPMI1640 was purchased from GIBCO Company.

[0102] Calf serum was purchased from Hangzhou Sijiqing Biomaterials Co., Ltd.

[0103] MTT is a product of Amresco.

[0104] 1.2 Synthetic primers:

[0105] Primerl (upstream) 5'-TCGC GCATGC GCTCCCATGACCCAG-3' (the underline is the sphI restriction site),

[0106] Primer2 (down...

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Abstract

The present invention provides recombinant inosculated albumen which can kill and wound leucocythemia cells. The albumen comprises human leucocyte interleukin IL3 segment and pseudomonas exotoxin. The human leucocyte interleukin IL3 segment is better to be located at the N-end of the inosculated albumen, and the pseudomonas exotoxin is better to be located at the C-end of the inosculated albumen. A bridge peptide is arranged between the human leucocyte interleukin IL3 segment and the pseudomonas exotoxin.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a fusion protein for targeting and killing leukemia cells, a preparation method and application thereof. Background technique [0002] Acute myeloid leukemia (AML) is the main type of acute leukemia in my country. In recent years, bone marrow stem cell transplantation and high-dose chemotherapy have significantly improved the prognosis of AML, but a considerable proportion of patients still relapse and die. Refractory and drug resistance are also important obstacles to the cure of AML. In addition, standard treatment measures are less selective for leukemia cells, often leading to significant toxic side effects and increased treatment-related mortality. [0003] It is now believed that the root cause of leukemia relapse is the natural resistance of leukemia blast cells (leukemic stem / progenitor cells) in the quiescent phase to chemotherapy / radiotherapy. Acute myelo...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/16A61P35/02
Inventor 娄世锋邓建川陈林陈姝张萍沈燕陈妤曾瀚庆白凤霞廖乐乐周慷罗云
Owner THE SECOND AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIV
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