Monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and preparation method thereof

A technique for granulated blood cells and Portunus trituratus, which is applied in the field of monoclonal antibodies and their preparation, can solve problems such as molecular marker display, and achieves the effects of objective analysis, intuitive result judgment, and reasonable design.

Inactive Publication Date: 2013-09-11
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still no clear conclusions about the exact functions of these two types of blood cells, the occurrence process in embryonic development, and the mechanism of participating in the body's immune regulation. The main reason is that there is no suitable molecular marker to show the process of their occurrence and participation in the immune response.

Method used

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  • Monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and preparation method thereof
  • Monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and preparation method thereof
  • Monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Continuous density gradient centrifugation of Percoll medium to separate whole blood cells of Portunus trituberculatus

[0023] (1) Commercially available Percoll stock solution and 10 times concentration of 0.01M phosphate buffer (0.135M NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM K 2 HPO 4 ;pH7.2) according to the volume ratio of 9:1 to prepare the Percoll application solution, and then dilute the Percoll application solution with phosphate buffer to different gradients of 60%, 50%, 40%, 30%, 20% (v / v) , put 2.5ml of each gradient in a centrifuge tube, and let stand overnight at 4°C.

[0024] (2) Take 3 to 4 normal and healthy Portunus trituberculatus, use a sterile syringe to extract hemolymph from the pia mater at the base of the swimming foot, and mix with 4°C pre-cooled anticoagulant (0.14M NaCl; 3mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; 20mM EDTA; pH7.3) according to the volume ratio of 1:2, the mixture was centrifuged at 4°C, 1500rpm for 6min, and...

Embodiment 2

[0030] Embodiment 2: the preparation of antigen

[0031] (1) The granulated blood cells obtained after continuous density gradient centrifugation of Percoll medium were resuspended in phosphate buffer (adjusted concentration to 10 9 cells / ml), mixed with an equal volume of electrophoresis sample buffer (0.5M Tris-HCl pH6.8; 1% sodium dodecylsulfonate; 1% mercaptoethanol; 10% glycerol; 0.01% bromophenol blue) Evenly, boil for 4 to 5 minutes, and cool down.

[0032] (2) Install the electrophoresis instrument (Mini-PROTEAN II, Bio-Rad), pour the prepared 12% separation gel into the gel maker, cover with distilled water, after the separation gel is polymerized, suck out the distilled water, add 5% stacking gel, Immediately insert the spotting comb. After the stacking gel is polymerized, pour the electrophoresis buffer (0.025M Tris-Base; 0.25M glycine; 0.1% sodium dodecylsulfonate; pH8.3), and pull out the spotting comb.

[0033] (3) Add the sample into the sample well (15 μl per...

Embodiment 3

[0039] Example 3: Preparation of monoclonal antibody against 26.7kDa protein of Portunus trituberculatus granule blood cells

[0040] 1. Immunization of Mice

[0041] (1) Basic immunization: 26.7kDa protein of purified granule blood cells was mixed with an equal volume of Freund's complete adjuvant as an antigen, and injected intraperitoneally into 4-week-old female Balb / C mice.

[0042] (2) Booster immunization: 2 weeks later, the purified protein was mixed with an equal volume of Freund's incomplete adjuvant and injected intraperitoneally.

[0043] (3) Second booster immunization: 1 week later, the purified protein was injected into the tail vein.

[0044] (4) Expansion immunization three days before fusion: 1 week later, the purified protein was injected into the tail vein.

[0045] 2. Cell Fusion

[0046] (1) Execute the immunized mice by dislodging the cervical spine, take the spleen aseptically, wash and grind with RPMI-1640 solution, put the grinding liquid in a cent...

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Abstract

The invention relates to a monoclonal antibody of anti-blue crab particle hemocyte 26.7kDa protein, and the monoclonal antibody is secreted by hybridoma cell of which the name is hybridoma cell strain Crab, the preservation number is CCTCC NO: C2012196, the preservation unit is China Typical Culture Preservation Center, and the preservation data is Jan 7th, 2013. The monoclonal antibody has a characteristic of being specifically combined with blue crab particle hemocyte 26.7kDa protein. The preparation method comprises the steps of: obtaining particle hemocyte by continuous density gradient centrifugation of Percoll medium; purifying the particle hemocyte 26.7kDa protein as antigen to immunize mice by electrophoresis rubber cutting and recycling; preparing hybridoma cells by a cell fusion method; screening out the monoclonal antibody of the anti-blue crab particle hemocyte 26.7kDa protein by an immunological detection method. The monoclonal antibody disclosed by the invention provides an important tool for researching functions of particle hemocyte in nonspecific immune response of blue crab and in other physiological function aspects.

Description

technical field [0001] The invention relates to a monoclonal antibody against 26.7kDa protein of neptunus trituberculatus granule blood cells 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 immunoglobulin, lack antibody-mediated immune system, and are non-specific immunity. The immune defense response mainly depends on the completion of humoral factors in blood cells and hemolymph. Blood cells complete the process of cellular immunity through phagocytosis, encapsulation, agglutination, nodule formation, exocytosis, repair, etc., and at the same time assist in the completion of humoral immunity by synthesizing and releasing immune regulatory factors such as hemolysin and phenoloxidase. play a decisive role in the protection process. At present, crab blood cells are divided into two categories according to the shape, size and granules in the cytoplasm: clear b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18
Inventor 程顺峰吴晓春邓灯
Owner QINGDAO AGRI UNIV
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