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Paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha antibody as well as preparation method and application thereof

A technology of lymphocytes and surface markers, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, chemical instruments and methods, analytical materials, etc., can solve the problem of failing to prepare CD8α recombinant protein from flounder, Issues such as lack of scientific reporting

Active Publication Date: 2022-05-10
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the particularity of the flounder CD8α protein, the recombinant protein of flounder CD8α has not been prepared. There is still a lack of scientific reports on whether the CD8α and CD8β peptide chains of the flounder are co-expressed on the cell membrane surface, and whether there is a homodimer of the CD8αα in the flounder

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  • Paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha antibody as well as preparation method and application thereof
  • Paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha antibody as well as preparation method and application thereof
  • Paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Screening and synthesis of epitope peptides of flounder CD8α molecule

[0031] According to the flounder CD8α gene sequence published by NCBI, after translating into amino acid sequence, IEDB online prediction software (http: / / tools.immuneepitope.org / bcell / ) was used to analyze the molecular epitope of flounder CD8α ( figure 1 ), considering parameters such as hydrophilicity, flexibility, antigenicity and surface accessibility, the final selection of the polypeptide chain is 134 APRVIASTRSPSLT 147 , the peptide was synthesized in Nanjing KingScript Biological Co., Ltd. and coupled with hemocyanin (KLH), the purity was verified by mass spectrometry as high as 95%.

Embodiment 2

[0032] Example 2: Construction of prokaryotic and eukaryotic plasmids expressing flounder CD8α molecules

[0033](1) According to the flounder CD8α gene sequence published by NCBI, the flounder CD8α gene was synthesized and connected to the pTag-GFP plasmid.

[0034] (2) Transfect the successfully ligated pTag-GFP-CD8α plasmid into HEK293 cells, and observe the green fluorescence to judge the expression effect of the flounder CD8α molecule.

[0035] Results: The eukaryotic plasmid of the flounder CD8α molecule was successfully constructed, and after the cells were transfected, green fluorescent protein was displayed (see figure 2 ), indicating that the flounder CD8α molecule was successfully expressed in HEK293 cells.

Embodiment 3

[0036] Example 3: Preparation of rabbit polyclonal antibody against flounder CD8α molecule

[0037] (1) The synthetic flounder CD8α polypeptide-KLH complex was used as an antigen to immunize New Zealand white rabbits. The dose of each immunization was 0.6 mg. The immunization was divided into 4 times. The interval between the first two immunizations was 2 weeks, and the interval between the last two immunizations for 1 week.

[0038] (2) Basic immunization: CD8α polypeptide-KLH complex was mixed with the same amount of Freund's complete adjuvant (V / V) as the antigen, and injected subcutaneously at 6 points on the back.

[0039] (3) Booster immunization: CD8α polypeptide-KLH complex was mixed with the same amount (V / V) of Freund's incomplete adjuvant as the antigen, and injected subcutaneously at 6 o'clock on the back.

[0040] (4) Second booster immunization: CD8α polypeptide-KLH complex (600 µl) was used as antigen and injected into the ear vein.

[0041] (5) Booster immuni...

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Abstract

The invention relates to a paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha antibody as well as a preparation method and application thereof, and belongs to the technical field of fish molecular immunology. The polyclonal antibody of the paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha is prepared by immunizing a New Zealand white rabbit with a paralichthys olivaceus CD8 alpha antigen epitope peptide-KLH compound of which the amino acid sequence is APRVIASTRSPSLT, and purifying the serum of the new Zealand white rabbit. Indirect immunofluorescence and immunoblotting experiments show that the rabbit polyclonal antibody disclosed by the invention can react with HEK293 cells transfecting paralichthys olivaceus CD8 alpha eukaryotic plasmids and paralichthys olivaceus head kidney lymphocytes. According to flow cytometry detection, the quantity proportion of CD8 alpha + T lymphocytes in peripheral blood lymphocytes of paralichthys olivaceus is 5.6 + / -0.8%, the quantity proportion of CD8 alpha + T lymphocytes in spleen lymphocytes is 14.8 + / -1.6%, and the quantity proportion of CD8 alpha + T lymphocytes in head kidney lymphocytes is 19.5 + / -0.9%. The results show that the polyclonal antibody can be used as a reagent for identifying T cell subsets of paralichthys olivaceus or a reagent for researching immune response of CD8 + T lymphocytes of paralichthys olivaceus.

Description

technical field [0001] The invention belongs to the technical field of fish molecular immunology, and in particular relates to a flounder ( Paralichthys olivaceus ) T lymphocyte surface marker molecule CD8α antibody and its preparation method and application. Background technique [0002] T lymphocytes are the main immune cells of the body and participate in the body's humoral and cellular immune responses. Among them, CD8 + As one of the main functional subsets of T cells, T lymphocytes play a variety of immune functions in the immune process. For example, in related immune defenses such as resisting pathogenic invasion, CD8 + T lymphocytes play a vital role. CD8 + After the TCR on the surface of T cells recognizes the endogenous antigenic peptide-MHC class I molecular complex on the surface of APC, it activates into cytotoxic T cells, directly contacts the target cells invaded by pathogens, and produces killing factors such as granzyme and perforin or Through the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K16/28C07K16/06G01N33/569
CPCC07K7/08C07K16/2815C07K16/065G01N33/56966
Inventor 邢婧田洪飞战文斌唐小千绳秀珍迟恒
Owner OCEAN UNIV OF CHINA