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Application of asialoglycoprotein receptor fragment sh2a as a marker

A sialic acid glycoprotein and receptor technology, applied in the field of liver disease, can solve the problem of unclear distinction and identification

Active Publication Date: 2022-04-01
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, how to quickly and easily distinguish active liver disease from inactive liver disease and other types of liver disease (drug-induced hepatitis, fatty liver, etc.) is still unclear

Method used

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  • Application of asialoglycoprotein receptor fragment sh2a as a marker
  • Application of asialoglycoprotein receptor fragment sh2a as a marker
  • Application of asialoglycoprotein receptor fragment sh2a as a marker

Examples

Experimental program
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Effect test

preparation example Construction

[0045] In a specific example, the method for preparing the asialoglycoprotein receptor fragment sH2a includes the following steps: loading the sH2a gene fragment into an expression vector to obtain a recombinant plasmid, transferring the recombinant plasmid into eukaryotic cells, and transfecting for 18-22 hours Add a transfection enhancer, collect the supernatant after 5-7 days, filter the supernatant with a filter membrane, and then purify to obtain the sH2a recombinant protein.

[0046] In a specific example, the above purification includes the following steps: performing Ni-NTA affinity chromatography on the filtrate obtained by filtering the filter membrane under non-denaturing conditions, the equilibration buffer is 50mM PBS, 10mM imidazole, 150mM NaCl, pH 7.2~7.4, After sample loading, wash 10mL; elute with 50mM PBS, 250mM imidazole, 150mM NaCl, pH7.2~7.4, collect the eluate; use a 10kDa ultrafiltration tube to concentrate the protein solution, and store it in 50mM PBS (...

Embodiment 1

[0048] Embodiment 1sH2a recombinant protein expression and purification identification

[0049] Protein expression: NCBI searched the human ASGPR2 sequence, selected the extracellular segment, namely sH2a amino acid sequence, to synthesize the gene and optimized the codons for mammalian expression, and loaded it into the pcDNA3.1 vector at the same time. Transform the obtained sH2a recombinant plasmid (pcDNA3.1-sH2a-His) into eukaryotic mammalian cells, and use the pcDNA3.1 empty vector as a negative control, add a transfection enhancer after 18-22 hours of transfection, 5-7 days Collect the supernatant and filter the supernatant with a 0.22 µm filter membrane.

[0050] Protein purification: The resulting filtrate was subjected to Ni-NTA affinity chromatography under non-denaturing conditions, and the equilibrium buffer was 50mMPBS, 10mM imidazole, 150mM NaCl, pH7.2~7.4. After sample loading, wash 10mL; elute with 50mM PBS, 250mM imidazole, 150mM NaCl, pH7.2~7.4, and collect ...

Embodiment 2

[0054] Example 2 sH2a antibody pairing

[0055] Use 3 μg / mL capture antibody to coat the microtiter plate, add different concentrations (6.25~200ng / mL) of sH2a calibrator, incubate at 37°C for 1 hour, add 100 μL of HPR-labeled detection antibody with a concentration of 10ng / mL after washing, and Incubate at 37°C for 1 hour, add TMB substrate chromogenic solution after washing, and measure the OD value of each well. From the results, the capture antibody (manufacturer: SinoBiological, product number: 13908-R002) and the detection antibody (manufacturer: Thermo, product number: MA5-29046) are well matched and can be used for the construction of a double-antibody sandwich system, as shown in Table 2.

[0056] Table 2

[0057]

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Abstract

The invention relates to the application of an asialoglycoprotein receptor fragment sH2a as a marker in the preparation of products for diagnosing active liver diseases. The present invention finds through research that by measuring the content of the asialoglycoprotein receptor fragment sH2a in biological samples, it can be well differentially diagnosed with active liver disease, thereby being applied to the early diagnosis and differential diagnosis of active liver disease, and Provide a strong basis for disease treatment. Active liver disease can be efficiently distinguished from inactive liver disease and other types of liver disease (drug-induced hepatitis, fatty liver, etc.) by detecting sH2a. sH2a has the value as a diagnostic marker for active liver disease and has a high diagnostic value . The discovery of this marker provides a new experimental theoretical basis and a new direction for further research on the mechanism of active liver disease and the treatment of active liver disease, and enriches the diagnostic and detection methods of active liver disease.

Description

technical field [0001] The invention relates to the field of liver diseases, in particular to the application of an asialoglycoprotein receptor fragment sH2a as a marker in the preparation of products for diagnosing active liver diseases. Background technique [0002] The liver is the largest digestive gland in the human body and the central station of material and energy metabolism in the body. It is estimated that there are more than 500 chemical reactions taking place in the liver. First of all, it secretes bile to help digest food; it synthesizes protein from absorbed amino acids to provide energy for the body; it can store and burn fat in the body and control body shape; it is a storage organ for fat-soluble vitamins; it can also oxidize, reduce, and decompose in the body It is the largest detoxification organ of the human body. Experiments have shown that animals can only survive for more than 50 hours after the liver is completely removed, even if given correspondin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/6893
Inventor 姜芳曹丽娟王晨颖张鹤耀孙玉龙王弢
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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