Preparation and application of GP73 C-terminal antigen

A monoclonal antibody and protein technology, applied in the direction of anti-animal/human immunoglobulin, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc.

Active Publication Date: 2018-03-02
海南中升健康发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] According to the data retrieval conducted by the inventor, there are no other reports on the detection of the double monoclonal antibody sandwich ELISA method for the C-terminus of GP73 at home and abroad

Method used

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  • Preparation and application of GP73 C-terminal antigen
  • Preparation and application of GP73 C-terminal antigen
  • Preparation and application of GP73 C-terminal antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation method of GP73 C-terminal antigen

[0052] In order to prepare a monoclonal antibody with good specificity and high affinity and then apply it to a clinical GP73 detection kit, it is first necessary to express the GP73 C-terminal antigen. The GP73 gene is located on chromosome 9, with a total length of 3042bp, contains a unique open reading frame (1200bp), and encodes 402 amino acids. GP73 is structurally distinct from serum GP73 (sGP73). GP73 consists of an N-terminus that is myristoylated in the cytoplasm, a single transmembrane domain, and a long C-terminal extracellular domain. Its N-terminus is hydrophobic, glycosylated, contains a single transmembrane region and a signal peptidase cleavage site (aa28-aa29), and contains two helix-helix domains. Its C-terminus is rich in acidic amino acids, contains myristyl (decanoyl) acylation continuous sequence (GLGNGRRS) and has 5 glycosylation sites. Immediately following the transmembrane region ther...

Embodiment 2

[0068] Example 2 Preparation of monoclonal antibody at C-terminus of GP73

[0069] 1. Animal immunization

[0070] Take 300 mg / L of recombinant GP73 C-terminal antigen protein and mix it with complete Freund's adjuvant to make an emulsifier, and immunize 5 Balb / c mice. For the first immunization, 100 μg per mouse was injected subcutaneously at multiple points. After 2 weeks, the animals were immunized for the second time with incomplete Freund's adjuvant. The dose and route were the same as the first time. After 2 weeks, blood was taken from the tail vein to measure the titer, and it was used for fusion when the titer reached 1:1000-1:5000. Three days before the fusion, the antigen was used to boost the spleen area of ​​the peritoneal cavity once.

[0071] 2. Determination of potency by conventional indirect ELISA method

[0072] The concentration of the purified recombinant GP73 C-terminal antigen coated with polystyrene plate was 5 mg / L, the enzyme-labeled antibody was go...

Embodiment 3

[0090] The preparation of the monoclonal antibody (enzyme-labeled antibody) of the GP73C terminal of embodiment 3 HRP labeling

[0091] The HRP-labeled monoclonal antibody was prepared by the improved sodium periodate method, and the specific steps were as follows:

[0092] 1. Take 5mg HRP and dissolve it in 0.5ml double distilled water, add the newly prepared 0.06Mol / L NaIO 4 Aqueous solution (10ml double distilled water+128mg NaIO 4 ) 0.5ml, mix well, and place at 4°C for 30min;

[0093] 2. Add 0.16Mol / L ethylene glycol aqueous solution (10mlH 2 (0+0.09ml ethylene glycol) 0.5ml, place at room temperature for 30min;

[0094] 3. Add 1ml of an aqueous solution containing 5mg of purified GP73C-terminal monoclonal antibody, mix well, put it into a dialysis bag, and slowly stir and dialyze 0.05Mol / L pH 9.5 carbonate buffer for 6h (or overnight) to allow it to bind;

[0095] 4. Add NaBH 4 Solution (5mg / ml) 0.2ml, mix well, set at 4°C for 2h;

[0096] 5. Slowly add an equal vo...

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Abstract

The invention provides a GP73 C-terminal protein and an anti GP73 C-terminal monoclonal antibody prepared thereby; for solving the deficiency of lack of GP73 quantitative detection kits as a liver cirrhosis early diagnosis detection means in clinic currently, another objective of the invention is to provide a test kit having the advantages of being simple in operation, accurate, sensitive, stablein quality and capable of mass production and a determination method thereof, so as to observe the expression content of GP73 at a protein level, take effective integrated treatment schemes as soon aspossible and effectively prevent further malignancy of cirrhosis.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to the preparation and application of GP73 C-terminal antigen. Background technique [0002] Liver cirrhosis is the advanced stage of the development of various chronic liver diseases, and it is diffuse liver damage caused by long-term or repeated effects of one or more etiologies. In my country, most of them are post-hepatitis cirrhosis, and a small part are alcoholic cirrhosis and schistosome cirrhosis. Histopathologically, extensive liver cell necrosis, nodular regeneration of residual liver cells, connective tissue hyperplasia, and fibrous septum formation lead to structural destruction of hepatic lobules and formation of pseudolobules. The liver gradually deforms and hardens and develops into cirrhosis. In the early stage, there are no obvious symptoms due to the strong compensatory function of the liver. In the later stage, liver function damage and portal hype...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/70C07K16/18C12N5/20G01N33/577
CPCC07K14/435C07K16/18C12N15/70G01N33/577G01N2800/085
Inventor 马茂森孟超关素梅张旭李保芬毛茹倩杜晓丹周晶金
Owner 海南中升健康发展有限公司
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