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Grass carp IgM monoclonal antibody, and preparation method and applications thereof

A monoclonal antibody, grass carp technology, applied in the field of bioengineering, can solve problems such as low resolution, and achieve the effect of improving sensitivity and sensitivity

Active Publication Date: 2018-08-17
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, for the detection of fish serum antibodies in the current market, the existing detection method is an indirect ELISA detection method established with viral recombinant protein as a solid-phase coating and HRP-labeled IgM antibody as a detection antibody. Apply this method Repeated tests found that the P / N value of ELISA detection is small, and the resolution of sample detection is low

Method used

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  • Grass carp IgM monoclonal antibody, and preparation method and applications thereof
  • Grass carp IgM monoclonal antibody, and preparation method and applications thereof
  • Grass carp IgM monoclonal antibody, and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Obtaining process of hybridoma cell line:

[0035] 1) Polypeptide synthesis and coupling:

[0036] Based on the amino acid sequence of grass carp IgM heavy chain region ABD76396 (sequence shown in SEQ ID NO.1), three polypeptide sequences were screened out: CIgM-HC1, whose sequence is shown in SEQ ID NO.2; CIgM-HC2, whose sequence The sequence is shown in SEQ ID NO.3; CIgM-HC3, its sequence is shown in SEQ ID NO.4;

[0037] Carry out artificial synthesis and purification according to the sequences of the three polypeptides; during the artificial synthesis process, a cysteine ​​(Cys) is added to the carboxyl terminus of CIgM-HC2 and the amino terminus of CIgM-HC3; cysteine ​​is added to different positions of the polypeptide The amino acid is the coupling site for KLH without affecting the structure of the polypeptide;

[0038] The three polypeptides were coupled to the carrier protein KLH with a polypeptide coupling kit.

[0039] 2) Obtaining of hybrido...

Embodiment 2

[0042] Embodiment 2: Obtaining process of grass carp IgM monoclonal antibody:

[0043] The hybridoma cells obtained in Example 1 were injected into mice, the mouse ascites was collected, immunoassay verification was performed, and the antibodies secreted by the hybridoma cells were purified using Protein A / G, and the purified monoclonal antibody was grass carp IgM monoclonal antibody (2E7).

Embodiment 3

[0044] Example 3: Grass carp IgM monoclonal antibody immunoassay

[0045] Grass carp, crucian carp (Carassius auratus), tilapia (Oreochromis spp), largemouth bass, carp (Cyprinus carpio) and bighead silver carp (Hypophthalmichthys molitrix) were collected from whole blood, and the serum was separated and prepared by saturated ammonium sulfate precipitation method. 6 kinds of fish IgM: prepare 4.1mol / L ammonium sulfate solution, add an equal volume of centrifuged fish serum under stirring conditions, stir at 4°C for 6-8h, centrifuge at 8000-10000Xg for 10min at 4°C, discard the supernatant; Dissolve the precipitate in PBS of 1 / 2 the original serum volume, dialyze in a dialysis card for 12-24 hours at 4°C, take the dissolved and dialyzed solution, and measure the IgM concentration of various fish by BCA method.

[0046] 1) Western-Blot detection:

[0047] The 6 kinds of fish sera were diluted 20 times respectively, and each dilution was used as a sample for SDS-PAGE. The grass ...

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Abstract

The invention discloses a grass carp IgM monoclonal antibody, and a preparation method thereof and applications thereof. The grass carp IgM monoclonal antibody is produced by a hybridoma cell strain having a preservation number of C2017130; and the applications of the grass carp IgM monoclonal antibody comprise an application of the grass carp IgM monoclonal antibody in the preparation of a reagent or a kit for detecting the Ig level of fish, an application in the preparation of a drug for treating viral infections in the fish, an application in the preparation of a reagent for evaluating immune effects of fish vaccines, and an application in the evaluation of the immune function of the fish. The invention also provides the hybridoma cell strain. The grass carp IgM monoclonal antibody canimprove the sensitivity of the serum antibody detection process, and has a good application prospect.

Description

technical field [0001] The invention relates to a grass carp IgM monoclonal antibody, its preparation method and application, and belongs to the technical field of bioengineering. Background technique [0002] Immunoglobulin (Ig) in teleost serum mainly exists in the form of tetrameric IgM. The molecular weight of IgM is about 100KDa, the size of the heavy chain is 75KDa, and the size of the light chain is 25KDa. In the preparation of IgM antibody, when commercial Protein A / G affinity chromatography is used, the binding efficiency of Protein A / G to IgM is low, and it is difficult to purify IgM, so it is necessary to obtain IgM monoclonal antibody artificially . Based on the IgM monoclonal antibody of fish, the immune function of fish can be evaluated through analysis, the method of evaluating the immune effect of vaccines by using the change rule of antibody levels in fish blood, immune organs and tissues, and the research on the effect of therapeutic drugs Wait. In addi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/68G01N33/569
CPCG01N33/56983G01N33/577G01N33/6854
Inventor 李中圣张杰伍建敏赵玉林王凤求王贵平
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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