Monoclonal antibody against 30.9kda protein of hemocyte cell membrane of Chinese mitten crab granule and preparation method thereof

A monoclonal antibody and granule blood cell technology, applied in biochemical equipment and methods, anti-animal/human immunoglobulin, microorganisms, etc., can solve problems such as inconclusive function and occurrence process, so as to avoid uncertainty and Experimental artifacts, fast and sensitive response, and time-saving effects

Inactive Publication Date: 2016-08-24
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because there are no suitable molecular markers to track the occurrence of the two types of blood cells in aquatic crabs and the process of participating in the immune response, there is no definite conclusion on the function and occurrence process of the two types of blood cells in crabs.

Method used

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  • Monoclonal antibody against 30.9kda protein of hemocyte cell membrane of Chinese mitten crab granule and preparation method thereof
  • Monoclonal antibody against 30.9kda protein of hemocyte cell membrane of Chinese mitten crab granule and preparation method thereof
  • Monoclonal antibody against 30.9kda protein of hemocyte cell membrane of Chinese mitten crab granule and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Sorting Eriocheir sinensis Whole Blood Cells by Flow Cytometry

[0026] (1) Take 5-6 Chinese mitten crabs with clean body surface, vigorous vitality, healthy and disease-free, and use a 5mL sterile syringe to draw 4°C pre-cooled anticoagulant (0.14M NaCl; 3mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; 20mM EDTA; pH 7.3), draw hemolymph from the pia mater of the swimming foot at a ratio of 1:2 (v / v), mix well; centrifuge the mixture at 4°C, 1500rpm for 5min, and resuspend the blood cell pellet in phosphate buffer , and adjust the suspension concentration to 10 8 cells / mL.

[0027] (2) Sorting the whole blood cell suspension by flow cytometry to recover various types of cells.

[0028] (3) Centrifuge whole blood cells and sorted cells at 4°C, 1200 rpm for 5 minutes, resuspend various blood cell pellets in phosphate buffer, and adjust the concentration of the suspension to 10 7 cells / mL.

[0029] (4) Take 30 μL of various cell suspensions and drop them on the glass ...

Embodiment 2

[0032] Embodiment 2: the preparation of antigen

[0033] Extraction of Hemocyte Membrane Proteins from Eriocheir sinensis Granules:

[0034] (1) Resuspend the granular blood cells obtained after cell sorting by flow cytometry in phosphate buffer (adjust the concentration to 10 8 cells / mL), at 4°C, 1000rpm, centrifuge for 10min;

[0035] (2) Discard the supernatant, add NP-40 cell lysate (1% NP-40, 10% glycerol, 0.25M sucrose, 137mM NaCl, 20mM Tris-HCl, pH 8.0) and various protease inhibitors to the pelleted blood cell pellet (2mM EDTA, 10mM NaF, 5μg / mL Leupetin, 2μg / mL Aprotinin, 1mM PMSF), blood cells were ultrasonically disrupted in an ice bath, 39% amplitude, set time 4min, on 3s, off 7s.

[0036] (3) After ultrasonication, gradient centrifugation at 4°C (1000g, 10min; supernatant, 10000g, 10min; supernatant, 100000g, 20min) to remove cell debris, nuclei, organelles, after the last centrifugation, take the precipitate, phosphate Resuspended in the buffer, it is the Chine...

Embodiment 3

[0047] Example 3: Preparation of monoclonal antibody against 30.9kDa protein monoclonal antibody of Eriocheir sinensis granule hemocyte cell membrane

[0048] 1. Immunization of Mice

[0049] (1) Adjust the concentration of the purified granule blood cell membrane 30.9kDa protein to 1mg / mL with sterile phosphate buffer, mix it with Freund's complete adjuvant in equal volume, and intraperitoneally inject 4-week-old female Balb / C mice (0.1mL ).

[0050] (2) Two weeks later, the protein suspension was mixed with Freund's incomplete adjuvant in equal volume, and 0.1 mL was injected intraperitoneally.

[0051] (3) One week later, 0.1 mL of protein suspension was directly injected into the tail vein.

[0052] (4) One week later, inject 0.1 mL of protein suspension into the tail vein.

[0053] 2. Cell Fusion

[0054] (1) Three days after the last immunization, the mice were killed by dislocation of the cervical spine, blood was drawn from the heart (stored at 4°C for later use), ...

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Abstract

The invention relates to a monoclonal antibody against the 30.9kDa protein of the blood cell membrane of mitten crab granules, which is characterized in that the monoclonal antibody is named: hybridoma cell line ES, and the preservation number is: CCTCC NO: C201414, The preservation unit is: China Center for Type Culture Collection, and the preservation date is: secreted by hybridoma cells on January 07, 2014. The monoclonal antibody can specifically bind to the cell membrane protein of mitten crab granulosa hemocytes. In the present invention, the mitten crab granule blood cells obtained by sorting blood cells by flow cytometry are lysed to obtain the granule blood cell membrane protein, and the 30.9kDa protein of the granule blood cell membrane is recovered and purified by electrophoresis and gel cutting as an antigen to immunize mice, and the cell fusion method is adopted. The hybridoma cells were prepared, and the monoclonal antibody against the 30.9kDa protein of the blood cell membrane of the mitten crab granulosa was screened out by an immunological detection method, and then its characteristics were identified by an immunological identification method.

Description

technical field [0001] The invention relates to a monoclonal antibody against the 30.9kDa protein of Chinese mitten crab (Eriocheir sinensis) granular hemocyte cell membrane secreted by hybridoma cells and a preparation method thereof, belonging to the technical field of crab cell immunity. Background technique [0002] Crabs do not have the complete specific immune system of higher animals, lack of immunoglobulin, and their immune defense response mainly depends on the enzymes and immune factors in blood cells and hemolymph. Studies have shown that blood cells can use phagocytosis, embedding, wound repair, osmotic adjustment, exocytosis, autolysis, etc. to identify and remove foreign bodies, and through the synthesis and release of lectin, lysin, hydrolase, oxidase, etc., assist in the completion of humoral immunity. Crabs play a key role in the process of resisting external environmental stimuli and invasion of foreign pathogens. Crab hemocytes are currently divided into ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N5/20
Inventor 程顺峰邓灯吴晓春张敏
Owner QINGDAO AGRI UNIV
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