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Cell-penetrating peptide-pre-B cell leukemia transcription factor 1 fusion protein and preparation method and application thereof

A fusion protein and transcription factor technology, applied in the field of biomedicine, can solve problems such as tissue and organ dysfunction and limit the scope of application, and achieve the effects of promoting proliferation, delaying aging, and maintaining self-renewal ability

Pending Publication Date: 2020-02-28
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the one hand, the senescence of stem cells is considered to be an important driving force for the aging of the body, which can cause dysfunction of various tissues and organs in the body; The scope of application in

Method used

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  • Cell-penetrating peptide-pre-B cell leukemia transcription factor 1 fusion protein and preparation method and application thereof
  • Cell-penetrating peptide-pre-B cell leukemia transcription factor 1 fusion protein and preparation method and application thereof
  • Cell-penetrating peptide-pre-B cell leukemia transcription factor 1 fusion protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Expression of fusion protein TAT-PBX1 of TAT membrane-penetrating peptide and PBX1 using prokaryotic expression system

[0042] 1. The coding gene of PBX1 is amplified by PCR method, and EcoR1 and Kpn1 restriction endonuclease sites are added to its two ends respectively. Using pLVX-PBX1-IRES-mCherry (purchased from Changsha Youbao Biology) as a template, the template contains the complete coding sequence of the above PBX1, and the primer sequence is as follows: forward primer: 5'-GGTACCATGGACGAGCAGCCCAGGCTG (SEQ ID NO.15), reverse primer : 5'-GAATTCTTATCAGTTGGAGGTATCAGAGTGAACACTGCC (SEQID NO.16); the PCR product was connected to the cloning vector pT-EASY by T-A cloning, and then the white clones that were correctly connected were picked by blue-white screening, and were identified by bacterial liquid PCR, enzyme digestion and gene expression. After being identified by conventional molecular biology methods such as sequencing, the PBX1-encoding gene fragment in the pos...

Embodiment 2

[0046] 1. Use TAT-PBX1 fusion protein to transduce hHF-MSCs

[0047] (1) Dilute TAT-PBX1 to 1 μg / mL with complete cell culture medium;

[0048] (2) Incubate hHF-MSCs with diluted medium, and perform immunofluorescence staining after 6h;

[0049] The result is as Figure 5 As shown, TAT-PBX1 can efficiently penetrate cells and enter the nucleus.

[0050] 2. Using TAT-PBX1 fusion protein to promote the proliferation of hHF-MSCs

[0051](1) Dilute TAT-PBX1 to 0.0, 5.0, 7.5, 10.0, 12.5 μg / mL with complete cell culture medium;

[0052] (2) Incubate the hHF-MSCs with the diluted medium, and detect the cell proliferation level with the CCK-8 kit at 24h and 96h after incubation, and the results are as follows: Figure 6 As shown, after incubation for 24h, there was no significant difference in the cell proliferation rate of each dose group, and after incubation for 96h, as the dose increased, the cell proliferation rate of each group gradually increased, compared with the 0.0μg / mL...

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Abstract

The present invention provides a cell-penetrating peptide-pre-B cell leukemia transcription factor 1 fusion protein and preparation method and application thereof, and relates to the technical field of biomedicine; PBX1 transcription factor is efficiently introduced into stem cells by means of a CPP and PBX1 fusion expression mode by making use of the cell-penetrating characteristic that CPP can carry exogenous proteins to enter cells, so that self-renewing capacity of the stem cells is retained, the proliferation of the stem cells is promoted, the senescence of the stem cells is delayed, anda new solution is provided for the in-vitro large-scale preparation of the stem cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a fusion protein of penetrating peptide-pre-B-cell leukemia transcription factor 1 and its preparation method and application. Background technique [0002] Stem cells have the potential of self-renewal and multi-directional differentiation, and are the core component of regenerative medicine. They have been used in the treatment of various diseases such as hematopoietic system diseases, central nervous system diseases and immune system diseases. Like other body cells, stem cells will also age, affecting their ability to proliferate, differentiate and repair tissues. On the one hand, the senescence of stem cells is considered to be an important driving force for the aging of the body, which can cause dysfunction of various tissues and organs in the body; in the scope of application. Stem cells are divided into embryonic stem cells, adult stem cells and induced p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61P39/06
CPCA61P39/06C07K14/4702C07K2319/10C07K2319/50
Inventor 刘晋宇王勃刘菲琳
Owner JILIN UNIV
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