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Glycosylated polypeptide, conjugate of glycosylated polypeptide and carrier protein, as well as preparation method and application of glycosylated polypeptide

A carrier protein and glycosylated hemoglobin technology, which is applied to glycosylated polypeptides and their conjugates with carrier proteins and their preparation and application fields, and can solve the problems of limited application and expensive pure products of human glycosylated hemoglobin antigen.

Inactive Publication Date: 2015-04-29
BEIJING YICHENG BIOELECTRONICS TECHNOLOGY COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pure product of human glycosylated hemoglobin antigen is very expensive, which limits the application of this detection technology to a certain extent

Method used

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  • Glycosylated polypeptide, conjugate of glycosylated polypeptide and carrier protein, as well as preparation method and application of glycosylated polypeptide
  • Glycosylated polypeptide, conjugate of glycosylated polypeptide and carrier protein, as well as preparation method and application of glycosylated polypeptide
  • Glycosylated polypeptide, conjugate of glycosylated polypeptide and carrier protein, as well as preparation method and application of glycosylated polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 Design and preparation of glycosylated hemoglobin analogs

[0061] The glycosylated polypeptide is Glyco-Val-His-Leu-Thr-Pro-Tyr-Tyr-Cys with a purity of over 98%, and the immunological activity of the synthetic product is detected. First synthesize glycosylated valine, and then follow the conventional peptide synthesis method (see "Peptide Synthesis", Huang Weide, Chen Changqing, Science Press), sequentially couple the amino acids of the above sequence, and finally separate and purify by high performance liquid chromatography , freeze-dried, stored at 4°C, and its purity and molecular weight were identified by mass spectrometry analysis, and it was confirmed that it was the glycosylated polypeptide, and its purity was above 98%.

[0062] The synthesis of the above-mentioned glycosylated valine can be carried out with reference to the prior art. Concrete synthetic method among the present embodiment is:

[0063] Take L-valine (0.16mol, 18.8g), add it into...

Embodiment 2

[0082] Example 2 Preparation of colored nanospheres-glycosylated hemoglobin antibody

[0083] a) Suspend 500 μl of 400 nm blue microspheres (Bangs Company) in 10 ml of 50 mM MES buffer (pH 6.5), centrifuge at 13,000 rpm for 5 minutes, and wash the microspheres twice by centrifugation.

[0084] b) For the third wash, suspend in 10ml of 50mM MES buffer (pH6.5) to disperse the microspheres evenly.

[0085] c) Dissolve 0.5mg of EDAC (MW=191.7) and 0.3mg of NHS (N-Hydroxysuccimimide, MW=115.09) in 1ml of MES solution and adjust the pH to 5.42 with 1N HCl.

[0086]d) Under vigorous stirring, the EDAC aqueous solution was slowly dropped into the microsphere solution. Stir at room temperature for 15 minutes. Centrifuge at 13000 rpm for 5 minutes, wash once with 10ml of 0.05M borate buffer (pH8.5). Centrifuge again and suspend the microspheres with 10ml of 0.05M borate buffer (pH 8.5).

[0087] e) Coupling under slight stirring, add the microspheres dropwise to 6ml of 1mg / ml glycos...

Embodiment 3

[0093] The construction of embodiment 3 glycosylated hemoglobin detection test strip

[0094] 1. The polypeptide-carrier protein (glycosylated hemoglobin analogue) solution prepared in Example 1 was dissolved in 10 mM PB at pH 7.0 in advance to prepare a concentration of 2 mg / ml.

[0095] 2. If Figure 4 As shown, on the reaction membrane, the 2 mg / ml glycosylated hemoglobin analogue prepared in the above step was spotted on the nitrocellulose (NC) membrane in a strip-shaped detection area 401 with a spotting instrument at an amount of 1 μl / mm.

[0096] 3. In the same way, coat the goat anti-mouse secondary antibody (Changsha Boyou Biotechnology Co., Ltd.) on the control area 402 of the test strip.

[0097] 4. The coated nitrocellulose membranes were dried and stored in a dry, airtight oven to obtain the reaction membrane 4.

[0098] 5. Spray the colored nanosphere-glycosylated hemoglobin antibody prepared in Example 2 evenly on the glass cellulose membrane with a spotting i...

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Abstract

The invention provides glycosylated polypeptide, a conjugate of the glycosylated polypeptide and carrier protein, as well as a preparation method and application of the glycosylated polypeptide. The glycosylated polypeptide has a sequential structure of Glyco-Val-His-Leu-Thr-Pro-Tyr-Ryr-Cys; the conjugate of the glycosylated polypeptide and the carrier protein is a product generated after the tail end Crs of the glycosylated polypeptide is coupled with the carrier protein (such as bovine serum albumin, myohemoglobin or hemocyanin); the glycosylated polypeptide or the conjugate of the glycosylated polypeptide and the carrier protein can be used for replacing an expensive pure human glycosylated hemoglobin antigen product to prepare a chromatographic testing bar (testing paper bar); the chromatographic testing b bar can be used for rapidly, accurately, quantitatively, simply and conveniently detecting the glycosylated hemoglobin in the whole blood.

Description

technical field [0001] The present invention relates to a glycosylated polypeptide and its conjugate with carrier protein as well as its preparation method and application. The glycosylated polypeptide or its conjugate with carrier protein can replace the expensive human glycosylated hemoglobin antigen pure Product preparation Chromatography test strips (test strips), which can quickly, accurately, quantitatively and easily detect glycosylated hemoglobin in whole blood. Background technique [0002] Diabetes mellitus is a group of endocrine and metabolic diseases whose etiology and pathogenesis are not yet fully understood, and its incidence rate is second only to cardiovascular diseases and tumors. According to the medical journal "The New England Journal of Medicine" reported in March 2010, the number of diabetic patients in my country has reached 92 million. At present, the blood glucose detection of patients has been widely carried out clinically, and a three-dimensiona...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K19/00G01N33/72
Inventor 段学军杨彬胡昕芳郭振刚钟啸李元光何伟
Owner BEIJING YICHENG BIOELECTRONICS TECHNOLOGY COMPANY
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