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Liver disease blood serum specific protein and use thereof

A liver disease, serum protein technology, applied in the direction of serum albumin, albumin peptides, animal/human proteins, etc., can solve the problems of high sensitivity and specificity, cumbersome operation, etc.

Inactive Publication Date: 2008-12-24
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They use the detection method of western blot, which is cumbersome to operate, and the sensitivity and specificity are not as high as the enzyme-linked immunosorbent method

Method used

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  • Liver disease blood serum specific protein and use thereof
  • Liver disease blood serum specific protein and use thereof
  • Liver disease blood serum specific protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Preparation of gp73 recombinant protein

[0060] (1) Cloning the gp73cDNA sequence onto the expression vector:

[0061] Primer design: Add BamH I and Hind III restriction sites to both ends of the sense and antisense primers respectively. The primer sequences are as follows:

[0062] 5'-CATAGGGATCCCTCCAGACACGGATCATGGAGCTGGAAGGC-3'

[0063] 5'-GAGAGAAGCTTTCAGAGTGTATGATTCCGCTTTTCACGCTG-3'.

[0064] The gp73cDNA sequence was amplified by reverse transcription using mRNA as a template.

[0065] Expression vector: pTrichisA (there is a 6×his tag upstream of the BamH I restriction site of the vector)

[0066] The vector and the target fragment were double-digested with BamH I and Hind III respectively, and the digested products were ligated by T4 ligase, and transformed into TOP10 competent (purchased from TAKARA company, the transformation step was operated according to the competent instructions), and ampicillin plate 37 Cultivate overnight at ℃, pick a single clone, id...

Embodiment 2

[0070] Preparation of gp73 antibody

[0071] (1) The steps of gp73 monoclonal antibody preparation are:

[0072] Animal immunization procedure: Balb / c mice were used as immunized animals, and the immunization dose was 50-100ug / mL. At the first immunization, the immunogen was mixed with an equal amount of Freund's complete adjuvant to make an emulsifier, and injected intraperitoneally, with an interval of 2 weeks Afterwards, the same amount of immunogen was emulsified with the same amount of Freund's incomplete adjuvant, and after an interval of 10 days, the antigen was directly immunized. After an interval of one week, the spleen or tail vein was boosted once, and the spleen cells were collected 3 days later.

[0073] Cell fusion and cloning: Splenocytes from immunized mice were fused with SP2 / 0 myeloma cells at a ratio of 5-10:1, cell supernatants were measured by indirect ELISA, positive wells were screened, and antibodies were identified by westblot , using the limiting di...

Embodiment 3

[0079] The construction of gp73 detection kit:

[0080] Set up the gp73 ELISA kit to include the following components:

[0081] (1) Enzyme plate coated with gp73 monoclonal antibody;

[0082] (2) Enzyme-labeled rabbit anti-gp73 antibody;

[0083] (3) gp73 standard solution;

[0084] (4) Substrate chromogenic solution and stop solution;

[0085] (5) Concentrate the washing solution.

[0086] The preparation steps of the enzyme label plate coated with gp73 monoclonal antibody in the kit of the present invention are:

[0087](1) Dilute the gp73 monoclonal antibody to 1ug / mL with coating buffer;

[0088] (2) Add 100 μl of diluted monoclonal antibody to each well of the 96-well ELISA plate, place at 4°C for 18 hours after sealing, pour off the coating solution, wash 3 times with washing solution, and pat dry;

[0089] (3) Add 200 μl of blocking solution to each well of the 96-well ELISA plate, incubate at 37° C. for 2 hours, pour off the liquid in the well, wash and drain, an...

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Abstract

The invention relates to a novel pneumonosis serum marker, namely a Golgi protein gp73, which belongs to the biological monitoring technical field. The invention also discloses an application of the serum Golgi protein gp73 in diagnosing pneumonosis and provides a detection kit for the serum gp73.

Description

technical field [0001] The invention relates to a new liver disease serum marker-Golgi protein gp73, which belongs to the technical field of biological monitoring. The invention also discloses the use of serum Golgi protein gp73 in diagnosing liver diseases and provides a detection kit for serum gp73. Background technique [0002] The liver is the largest organ in the human body, and liver disease is also the disease with the highest morbidity and mortality. Due to the strong compensatory ability of the liver, some injuries often have no clinical manifestations, resulting in a strong latent liver disease. When clinical manifestations appear, the liver disease is already at an advanced stage and it is difficult to effectively treat it. At present, there are many kinds of liver function tests that can be carried out clinically, but each test can only detect a certain function of a certain aspect of the liver. Up to now, there are still no indicators that can reflect most of t...

Claims

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Application Information

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IPC IPC(8): C07K14/765G01N33/68G01N33/577
Inventor 彭涛古艳丽
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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