Bioactivity detection method and application of recombined humanized neuroglobin

A technology for cerebroglobin and a detection method, applied in the field of biomedicine, can solve the problems of insufficient sensitivity of detection technology, unproven, unsuitable for detection of rhNgb biological activity, etc., and achieves high repeatability of detection data, reliable measurement results, and detection method. stable effect

Inactive Publication Date: 2011-09-07
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above studies can only show that Ngb has a protective effect on cell damage caused by free radicals, but cannot prove that Ngb itself has a scavenging effect on free radicals, nor can it prove its antioxidant capacity, so the content reported in the literature is not suitable for rhNgb Detection of biological activity; at the same time, the only research on scavenging free radicals directly from Ngb protein (Neuro-and cytoglobin are found to show no appreciable superoxide dismutase, catalase, and peroxidase activities. (Gene, Neuroglobin and cytoglobin as potential enzyme or substrate, 2007, 398(1-2): 103-13)), but did not find that Ngb has superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities, It has not been proved that it has the ability to scavenge free radicals, resulting in conflicting determinations of the biological activity of Ngb
This may be due to insufficient sensitivity of the detection technique
Therefore, there is no report on the direct detection of the free radical scavenging activity of Ngb protein from the protein level.

Method used

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  • Bioactivity detection method and application of recombined humanized neuroglobin
  • Bioactivity detection method and application of recombined humanized neuroglobin
  • Bioactivity detection method and application of recombined humanized neuroglobin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: detection of rhNgb scavenging superoxide anion activity

[0042] Nitro blue tetrazolium chloride (NBT)-photoreduction method was adopted to measure, VB in this reaction 2 It reacts with EDTA under light to produce superoxide anion, which reacts with NBT and has a maximum absorption peak at 560nm. In the reaction, VB2 and EDTA react under light to produce superoxide anion, which reacts with NBT. After adding rhNgb, rhNgb reacts with superoxide anion, which further prevents the reaction between superoxide anion and NBT. The value of the absorption peak at will change. According to the reaction degree of rhNgb and superoxide anion, the ability of rhNgb to scavenge superoxide anion can be calculated.

[0043] In the embodiment, the assay method for removing superoxide anion activity is divided into reagent preparation, sample / reference substance preparation, measuring sample and reference substance absorbance, and calculating the activity four steps of removing...

Embodiment 2

[0059] Example 2: detection of rhNgb scavenging hydrogen peroxide activity

[0060] In the case of relatively sufficient hydrogen peroxide, catalase can catalyze hydrogen peroxide to produce water and oxygen. Residual hydrogen peroxide can produce red product N-(4-antipyrinyl)-3-chloro-5-sulfonic acid group-p-benzoquinone imine (N-(4- antipyryl)-3-chloro-5-sulfonate-p-benzoquinonemonoimine), its maximum absorption wavelength is 520nm.

[0061] The hydrogen peroxide removal activity of rhNgb was detected using the catalase detection kit of Beyond Biotechnology Research Institute.

[0062] 1. Reagent preparation

[0063] (1) Preparation of rhNgb sample solution: On the basis of the prepared rhNgb sample, rhNgb was prepared into a 1 mg / ml sample solution with double distilled water.

[0064] (2) Preparation of the reference substance vitamin C (Vc), reduced glutathione (GSH), N-acetylcysteine ​​(NAC) and hemoglobin (Hb) solution: prepare with rhNgb, weigh 10 mg of the referenc...

Embodiment 3

[0083] Example 3: Detection of rhNgb scavenging hydroxyl radical activity

[0084] o-phenanthroline-Fe 2+ The activity of rhNgb to scavenge hydroxyl radicals was determined by colorimetry. Hydroxyl radicals generate Fe through the Fenton reaction 2+ and H 2 o 2 .

[0085] 1) Use double distilled water to prepare rhNgb into sample solutions with different concentrations of 0.1, 0.5, and 1 mg / ml, while hemoglobin (Hb), Vc, reduced glutathione (GSH), N-acetylcysteine ​​(NAC ) solution was prepared in the same way as rhNgb, and stored in a 4°C refrigerator for later use;

[0086] 2) Add 100 μl of 0.75 mM o-phenanthroline solution, 200 μl of PBS (pH7.2), and 0.75 mM FeSO to the microtube (centrifugal Eppendorf tube) 4 100μl and 50μl double distilled water, shake well;

[0087] 3) Add 0.01% H 2 o 2 50μl, start the reaction, and place in a water bath at 37°C for 60min;

[0088] 4) After taking the mixed solution out of the water bath, measure its absorbance at 536nm, and ...

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Abstract

The invention discloses a bioactivity detection method and application of recombined humanized neuroglobin. The detection method disclosed by the invention comprises the following steps: 1) preparing the recombined humanized neuroglobin to be detected into a sample solution of the set concentration; 2) detecting the capability of the sample solution in eliminating radicals, and using the superoxide anion clearance rate through rhNgb, the hydrogen oxide clearance amount through rhNgb and/or the hydroxyl radical clearance rate through rhNgb as detection indexes; and 3) computing the detection data and comparing the detection data with the bioactivity standard data of the recombined humanized neuroglobin to obtain the detection result. The invention also provides the application of the neuroglobin Ngb in the preparation of a medicament with a function of eliminating the radicals, which provides the train of thoughts for the development of a new medicament. The detection method disclosed by the invention is stable, has the advantages of reliable detection result, high repeatability of the detection data and the like and can be widely applied to tracking the activity of the rhNgb and biological products containing rhNgb, and the detection result can better reflect the bioactivity of rhNgb proteins.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for detecting biological activity of recombinant human brain globin and its application, in particular to a method for detecting free radical scavenging activity of recombinant human brain globin and its use in the preparation of drugs with free radical scavenging functions. use in . Background technique [0002] Neuroglobin (Neuroglobin, Ngb) is the third type of oxygen-carrying globin protein mainly expressed in the nervous system newly discovered in 2000. It is similar to myoglobin in terms of function and properties, and has a high affinity with oxygen (P 50 ≈2 torr). For the detection of Ngb protein, most of the current methods focus on the detection of protein immunoactivity (Western blot detection) and protein content. For the biological activity of Ngb protein, especially the activity of scavenging free radicals, there is no suitable detection method repor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78G01N21/31A61K38/42A61P9/10A61P25/00A61P39/06
Inventor 张成岗李伟光吴永红高艳
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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