Specific marker Aire (Autoimmune Regulator) of embryonic stem cells and application thereof

A technology of embryonic stem cells and markers, applied in the fields of cell biology and immunology, can solve the problem of limited number of markers on the membrane surface of embryonic stem cells, and achieve high specificity and sensitivity

Inactive Publication Date: 2013-07-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the specificity of these markers has certain limitations: they are also expressed in some adult stem cells, norma

Method used

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  • Specific marker Aire (Autoimmune Regulator) of embryonic stem cells and application thereof
  • Specific marker Aire (Autoimmune Regulator) of embryonic stem cells and application thereof
  • Specific marker Aire (Autoimmune Regulator) of embryonic stem cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of Rabbit Polyclonal Antibody

[0030] 1.1 Materials and reagents

[0031] Carrier protein KLH was purchased from Thermo Fisher. The peptide was synthesized by Hangzhou Zhongpei Biochemical Co., Ltd.

[0032] 1.2 Preparation of rabbit polyclonal antibody

[0033] Synthesize a polypeptide consisting of 21 amino acids at the C-terminus of the Aire sequence shown in SEQ ID No.1 and a polypeptide consisting of 12 amino acids at the C-terminus of the Aire sequence shown in SEQ ID No.2; respectively couple the polypeptides to the carrier Protein KLH is the complete antigen; mix the complete antigen with complete Freund's adjuvant to immunize New Zealand white rabbits; after the first immunization, boost immunization every two weeks; after the fourth immunization, take whole blood and separate and purify it with a polypeptide-chromatographic column Aire rabbit polyclonal antibody.

Embodiment 2A

[0034] Example 2 Application of Aire as an embryonic stem cell marker

[0035] 1.1 Aire gene maintains the self-renewal and differentiation ability of embryonic stem cells

[0036]ICC staining: cultured cells were fixed with 4% formaldehyde (Sigma) at 37°C for 1.5 hours; then blocked with blocking solution (PBS containing 10% goat serum and 1% BSA) at 37°C for 1 hour; blocked cells were treated with primary antibody (Aire rabbit polyclonal antibody prepared in Example 1) was incubated at 37°C for 1 hour; washed with PBS; after washing, the cells were incubated with secondary antibody at 37°C for 45 minutes in the dark; finally, 0.5 μg / ml Hochest33258 (Sigma) was used for nuclear staining 5 Minutes; observed and photographed under a fluorescent microscope (Olympus IX-70). ICC staining results as figure 1 As shown, double staining of Aire and SSEA-1 is positive, proving the expression of Aire on undifferentiated embryonic stem cells.

[0037] RT-PCR experiment: Using TRIzol r...

Embodiment 3

[0041] Example 3 Application of Glut3 as a marker for embryonic stem cells

[0042] 1.1 Preparation of Glut3 rabbit monoclonal antibody

[0043] Using mouse embryonic (mES) stem cells as immunogen, use 1×10 8 A mES was used to immunize 3-month-old New Zealand white rabbits, rabbit spleen cells were isolated, fused with myeloma cells 240E-W2 (purchased from Epitomics), and a hybridoma cell line ZJUESRMAB39 (accession number CGMCC NO. 7301), applied to the identification and isolation of stem cells.

[0044] 1.2Glut3 rabbit monoclonal antibody recognizes natural conformational epitope and can be used for flow cytometric screening of embryonic stem cells

[0045] The statistical results of flow cytometry data showed that the expression of Glu3 decreased significantly with the differentiation of ES cells.

[0046] Such as Figure 4 As shown: 70% of the intact mES can be positively stained by the Glut3 rabbit monoclonal antibody prepared in the example, while only 3% of the mES...

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Abstract

The invention provides a novel specific marker Aire (Autoimmune Regulator) of embryonic stem cells and application thereof. Polypeptide coupling carrier protein KLH (Keyhole Limpet Hemocyanin) composed of a part of amino acid sequences at the C tail end in an Aire sequence is used as immunogen; a prepared Aire polyclonal antibody and a rabbit monoclonal antibody Glut3 (Glucose Transporter 3) and/or GM-CSFRalpha (Granulocyte Macrophage Colony-Stimulating Factor Receptor) prepared by directly taking the embryonic stem cells as the immunogen are used as stem cell identification reagents; the specificity and the sensitivity of detection are higher; furthermore, the specific marker Aire can be used for separation and activity detection of the embryonic stem cells. An embryonic stem cell detection kit containing the Aire polyclonal antibody and the rabbit monoclonal antibody Glut3 and/or GM-CSFRalpha can be used for identifying the totipotency of the embryonic stem cells and also can be used for detecting activity of stem cells simultaneously; therefore, functions of the detection kit are expanded.

Description

technical field [0001] The invention belongs to the field of cell biology and immunology, and in particular relates to the embryonic stem cell specific marker Aire and its application. Background technique [0002] Embryonic stem cells (ES cells) refer to normal diploid cells isolated from the inner cell mass of embryos, which can be cultured in vitro for a long time and maintain the ability of self-renewal and the potential to differentiate into cells of all three germ layers. The high self-renewal ability and differentiation pluripotency of embryonic stem cells are important characteristics that distinguish it from other cells. Its specific molecular markers are of great significance for the identification, isolation, self-renewal and differentiation mechanism of embryonic stem cells. Currently commonly used embryonic stem cell-specific markers include transcription factors OCT4, NANOG, REX1, etc.; surface markers SSEA-1, SSEA-3, TRA-1-60, CD9, etc. Antibodies correspondin...

Claims

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Application Information

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IPC IPC(8): C07K14/47G01N33/68G01N33/577
Inventor 顾斌张佳蓉张铭宋秀丽谭舟侯倩女
Owner ZHEJIANG UNIV
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