HGG (Human Gammaglobulin) polypeptide in combination with tissue specificity of cerebral arterial thrombosis and application thereof

An ischemic stroke, tissue-specific technology, applied in the field of protein peptides, to achieve the effect of high activity, strong penetration, and enhanced neuroprotective effect

Active Publication Date: 2015-05-06
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Currently, there are no pe...

Method used

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  • HGG (Human Gammaglobulin) polypeptide in combination with tissue specificity of cerebral arterial thrombosis and application thereof
  • HGG (Human Gammaglobulin) polypeptide in combination with tissue specificity of cerebral arterial thrombosis and application thereof
  • HGG (Human Gammaglobulin) polypeptide in combination with tissue specificity of cerebral arterial thrombosis and application thereof

Examples

Experimental program
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Embodiment 1

[0055] Screening and preparation of embodiment 1 HGG polypeptide

[0056] This example uses in vivo phage display peptide library screening technology to obtain HGG polypeptides that specifically bind to ischemic stroke tissues. The specific steps are as follows:

[0057] 1. Construction of MCAO model: C57 mice were anesthetized by intraperitoneal injection of 0.4% sodium pentobarbital (40 mg / kg), fixed in supine position. A midline incision was made in the neck, the muscle and fascia were separated along the inner edge of the sternocleidomastoid muscle, and the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were separated. Ligate the CCA at the proximal end and the ECA at the distal end, temporarily clamp the ICA with an arteriole clip, then cut a small opening at 2 mm from the bifurcation of the ECA, insert the tethered line into the ICA, and gently push the tethered line with fiber surgical forceps. Calculate the distance...

Embodiment 2

[0070] Example 2 Phage Monoclonal DNA Extraction and Sequence Determination

[0071] In this example, the DNA of 120 candidate phage monoclonals was extracted and sequenced.

[0072] After amplifying the monoclonal phage picked in Example 1, centrifuge to remove bacteria, take 500 μL of phage supernatant and add 200 μL PEG / NaCl, mix well and let stand on ice for 10 minutes, centrifuge at 14000 rpm at 4°C for 10 minutes, and resuspend the precipitate in 100 μL Add 250 μL of ethanol to iodide buffer, incubate at room temperature for 10 minutes, centrifuge for 10 minutes, discard the supernatant, wash the precipitate with 70% ethanol, briefly dry in vacuum, and resuspend the precipitate in 30 μL TE for sequencing.

[0073] All sequencing in this example was performed by BGI. The sequencing primer is -96gIII:5'- HO GTA TGG GAT TTT GCT AAA CAA C-3'.

[0074] The results of phage clone sequencing were analyzed by SeqMan software, and the base sequence was translated into amino ac...

Embodiment 3

[0075] Synthesis and fluorescence modification of embodiment 3 HGG polypeptide

[0076] The HGG polypeptide is artificially synthesized by Jill Biochemical (Shanghai) Co., Ltd. according to the amino acid sequence through chemical methods. The synthesized product was purified by high performance liquid chromatography (HPLC) and identified by mass spectrometry (MS). The purity of the polypeptide of the present invention is above 95%.

[0077] HGG polypeptide fluorescent labeling was completed by Gill Biochemical (Shanghai) Co., Ltd. The modification method is to add a lysine K at the -COOH end of the HGG peptide, which is connected to the 5-TAMRA fluorescent dye through its side chain amino group. The 5-TAMRA fluorescence-labeled peptide of the present invention is purified by high-performance liquid chromatography (HPLC), identified by mass spectrometry (MS), and the purity is above 95%.

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Abstract

The invention discloses HGG (Human Gammaglobulin) polypeptide in combination with the tissue specificity of cerebral arterial thrombosis. The invention relates to a method for obtaining the polypeptide. The method comprises the following steps: carrying out in-vivo selection of a mouse MCAO (Middle Cerebral Artery Occlusion) cerebral arterial thrombosis model by adopting an in-vivo phage display peptide library screening technology so as to obtain phage clones in combination with cerebral arterial thrombosis tissue; and randomly selecting the plurality of phage clones to sequence, and authenticating the in-vivo combination specificity of HGG peptide and coded phage clones HGG-M13 thereof. The invention further relates to application of the polypeptide in preparation of a high-sensitivity imaging molecular probe and a targeting delivery neuroprotective drug for cerebral stroke. The polypeptide can be synthesized through an artificial method; the polypeptide is low in molecular weight, high in activity and penetrating power, good in specificity and low in toxicity, and has good tissue targeting of cerebral arterial thrombosis in vivo; and therefore, the polypeptide is applicable to serving as a carrier of the high-sensitivity imaging molecular probe and the targeting delivery neuroprotective drug.

Description

technical field [0001] The invention belongs to the technical field of protein polypeptides, and relates to a peptide specifically binding to pathological tissue, in particular to a HGG polypeptide specifically binding to ischemic stroke tissue. The invention also relates to the screening and preparation method of the polypeptide, and the application of the polypeptide in the preparation of molecular imaging probes for ischemic stroke. Background technique [0002] Stroke is a sudden-onset cerebral blood circulation disorder. It is the second cause of death and the first cause of disability in the world. About half of the survivors are permanently disabled. According to data from the Chinese Center for Disease Control and Prevention, stroke is also the first cause of disability and the "number one killer" in my country. There are more than 7 million stroke patients in China, and the incidence rate increases by 8.7% every year. [0003] The treatment of stroke patients needs...

Claims

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Application Information

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IPC IPC(8): C07K7/06A61K49/00A61K49/14A61K47/42
Inventor 张建琼付波张莹龙伟
Owner SOUTHEAST UNIV
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