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Zinc finger protein and its antibody preparation and use

A protein, antibody technology, applied in the direction of peptide/protein composition, application, antibody, etc.

Inactive Publication Date: 2003-06-11
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, many proteins related to the development of human hematopoietic system have been discovered and isolated, but before this application, no one has disclosed or published the human krüppel type C of the present invention 2 h 2 Zinc finger protein ZNF268 and its antibody

Method used

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  • Zinc finger protein and its antibody preparation and use
  • Zinc finger protein and its antibody preparation and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Preparation of ZNF268 protein and preparation of anti-ZNF268 antibody

[0071] The 653bp fragment of ZNF268 was inserted into the prokaryotic expression vector pRSET-B to construct the recombinant prokaryotic expression vector pRSET-BH 2 , which encodes a ZNF268 protein fused to 6His at the N-terminus. pRSET-BH 2 The transformed E.coli BL21(DE3) was cultured to A 600 At 0.8, add 0.4mM IPTG and induce at 42°C for 3 hours, lyse the bacterial cells to obtain the crude cell extract containing soluble 6his-ZNF268 fusion protein, use XPress TM After the purification system was purified, Freund's adjuvant was added, and rabbits were immunized with standard methods to obtain anti-ZNF268 antibodies. figure 1 It shows that the ZNF268 protein is highly expressed under the experimental conditions. figure 2 It is shown in that the anti-ZNF268 antibody can specifically bind to the expressed ZNF268 protein.

Embodiment 2

[0072] Example 2 Protein hybridization analysis of ZNF268 protein expression

[0073] Total protein was extracted from different tissues of 4-month-old aborted human embryos with lysis buffer, separated by 8% SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with TBST buffer containing 10% skimmed milk for 60 minutes, then incubated with TBST containing 1:640 diluted anti-ZNF268 antibody for 1 hour, and washed with TBST. Then the membrane was incubated in TBST containing appropriate goat anti-rabbit IgG antibody for 30 minutes. After TBST was eluted, the color reaction was carried out with DAB. image 3 It showed that ZNF268 was significantly expressed in fetal liver and intestine, but not expressed in fetal heart, kidney, brain and spleen.

Embodiment 3

[0074] Example 3 Immunohistochemical analysis of ZNF268 protein in the hematopoietic system

[0075] Different tissue materials were fixed with Bouin's solution, dehydrated with graded alcohol, transparent in xylene, and embedded in paraffin. The material was routinely sectioned and spread on glass slides previously treated with poly-L-lysine. Slides were blocked with normal goat serum and then reacted with anti-ZNF268 antibody. Biotin-conjugated anti-rabbit IgG was used as the secondary antibody, horseradish peroxidase-conjugated streptavidin was used as the third antibody, and DAB was used for color reaction. Finally, counterstaining was performed with hematoxylin. To identify cell types, monoclonal antibody anti-CD 34 , anti-CD 38 As the primary antibody, the corresponding biotin-conjugated anti-mouse IgG was used as the secondary antibody. PBS solution was used instead of the primary antibody as a control for non-specific staining of the experiment.

[0076] Figure...

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PUM

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Abstract

The invention refers to a zinc protein and the antibody, relative to human hematogenic system growth. The ZNF 268 protein is a typical krIIppel-type C2H2 zinc protein; the preparation mainly structures ZNF268 expression carrier, transfers into host cells to express and separate to purify ZNF268 protein; the protein can diagnose and treat the disease because of protein abnormity. The antibody of ZNF268 protein can exceptionally combine with ZNF268 protein. The antibody preparation; mix the ZNF268 protein and assist agent to immunize animal, then separate the immune antimal blood to purify the antibody; the antibody; the antibody can diagnose, treat or prevent the disease relative to the protein abnormity of ZNF268 protein.

Description

technical field [0001] The present invention relates to a protein and its antibody; specifically, it relates to a protein and its antibody related to the development of human hematopoietic system. Background technique [0002] The realization of genetic information is inseparable from the regulation of gene expression. Many of the transcriptional regulators discovered so far contain zinc-finger motif structures. Zinc finger proteins are proteins that bind Zn 2+ A class of short, stable proteins that can fold themselves into "finger" structures. Due to its own structural characteristics, it can selectively bind to specific target structures, making zinc finger proteins play an important role in life processes such as gene expression regulation, cell differentiation, and embryonic development. [0003] According to the sequence characteristics of zinc finger motifs, zinc finger proteins can be divided into 6 categories: ①C 2 h 2 type, ②(C 2 ) 2 type, ③C 4 type, ④C 6 Ty...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K39/395A61P7/00C07K14/435C07K16/18C12N15/12G01N33/68
Inventor 李文鑫孙燕苟德明高立
Owner WUHAN UNIV
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