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Novel apoptosis test regent and related kit

A kit and apoptosis technology, applied in the biological field, can solve the problems of high reaction system requirements, no amino derivatization, wide emission spectrum, etc., and achieve the effect of reducing costs.

Active Publication Date: 2010-03-10
井健
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of fluorescein derivatives are: high photoquenching rate, strong pH sensitivity and broad emission spectrum [Ignatius MJ, Gebicke-Haerter PJ, Skene JHP, Schilling JW, Weisgraber KH, MahleyRW, et al.Expression of apolipoprotein E during nerve degeneration and regeneration[J].Proc NatlAcad Sci USA, 1986, 83:1125-1129.]
The amino acid carboxyl group can also be used as a derivatization site, but because of the higher requirements of the reaction system, it is far less widely used than amino derivatization.

Method used

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  • Novel apoptosis test regent and related kit

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Experimental program
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Effect test

Embodiment 1

[0047] Preparation of embodiment 1.GPPAN-V recombinant protein

[0048] Experimental materials and reagents: GPPAN-V genetically engineered strains are kept by our laboratory; Tryptone and Yeast Extract are products of Oxoid; various chromatography media are products of Pharmacia, Sweden; other reagents of analytical purity required are products of Beijing Chemical Plant ; Some chromatographic grade reagents are products of Sigma Company in the United States;

[0049] Experimental equipment: desktop low-temperature centrifuge (5417R) is a product of Eppendorf Company in Germany; electrophoresis instrument and gel imaging system are products of Bio-Rad Company in the United States; high-speed refrigerated centrifuge is a product of Beckman Company in the United States; constant temperature air shaker (OSFT-HS- R-32) is a product of Teq company in Portugal; Aolilong 520A acidity meter is a product of Orion company in the United States; JY98-ultrasonic cell pulverizer is a prod...

Embodiment 2

[0061] Example 2. Preparation of FITC-GPPAN-V fluorescently labeled protein

[0062] Experimental materials and reagents: FITC, DTT, and β-mercaptoethanol are products of German Mark; Tris, SDS, glycine, acrylamide, and methylenebisacrylamide are products of American Sigma; GST affinity media are products of GE Healthcare; others The required analytical grade reagents are products of Beijing Chemical Plant; some chromatographic grade reagents are products of Sigma Company of the United States. Experimental equipment: high-speed refrigerated centrifuge is a product of Beckman Company of the United States; Aolilong 520A acidity meter is a product of Orion Company of the United States; electrophoresis instrument is a product of Bio-Rad Company of the United States; large-scale protein purification system (FPLC, UPC-900) is a product of Pharmacia Company of Sweden Products; fluorescence microscope is a product of Christ Company in Germany; Elix / RiOs pure water system is a produc...

Embodiment 3

[0072] Example 3. Application of novel fluorescent marker protein FITC-GPPAN-V to detect cell apoptosis

[0073] Experimental materials and reagents: human annexin V-FITC kit is a product of Beijing Zhongkang Zhiheng Biotechnology Co., Ltd.; PMSF, DTT, and β-mercaptoethanol are products of Mark Company of Germany; cell culture medium is a product of Invitrogen Company of the United States; glycerol, NaH 2 PO 4 、Na 2 HPO 4 , NaCl, HCl, and EGTA are domestic analytically pure products.

[0074] Experimental equipment: flow cytometer is the product of Cole-parmer Company in the United States; the desktop low-temperature centrifuge (5417R) is the product of Eppendorf Company in Germany; the high-speed refrigerated centrifuge is the product of Beckman Company in the United States; the Elix / RiOs pure water system is the product of Millipore Company in the United States.

[0075] experimental method:

[0076] 1. Induction of apoptosis: Hela cells were treated with hydrogen per...

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Abstract

The invention relates to the development of a novel apoptosis test regent and a related kit. On the basis of modern biology engineering technology, glutathione-S-transferase (GST) and human annexin V are coupled to form a novel recombinant fusion protein GPPAN-V, and the primary structure diagram of the protein is shown as attached diagram 1. Efficient expression product GPPAN-V recombinant fusion protein after being induced by colibacillus host cell containing a GPPAN-V recombinant gene expression vector can exist in a soluble form, and GPPAN-V recombinant fusion protein with higher purity can be prepared by affinity chromatography purification. After being chemically coupled, the GPPAN-V recombinant fusion protein and the fluorescein isothiocyanate (FITC) can form a novel apoptosis testregent FITC-GPPAN-V for testing apoptosis. The novel apoptosis test kit formed by taking FITC-GPPAN-V as the core has the advantages of economy, sensitivity and efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, more precisely, to prepare novel cell apoptosis detection reagents based on modern biochemical technology and modern protein chemistry technology, and to form corresponding cell apoptosis detection kits, and its preparation and application characteristics. Background technique [0002] 1. Background knowledge of human annexin V (Annexin V) [0003] Human annexin is a calcium ion-dependent membrane phospholipid-binding protein. It is widely distributed and expressed in various tissue cells (such as cardiomyocytes, vascular endothelium, skeletal muscle, liver cells, etc.). Human annexin V is a protein that exists in the human body. It consists of 319 amino acid residues, with a molecular weight of about 36kDalton and an isoelectric point of 4.71 [Rand J H. "Annexinopathies" a new class of diseases[J].Nature, 1999, 340(13): 1035-1036.]. It contains a cysteine ​​residue, located in the fourth repeat. ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12Q1/02G01N21/64
Inventor 井健
Owner 井健
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