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Broad-spectrum secondary antibody based on ZZ domain and preparation method and application thereof

A broad-spectrum, antibody technology, applied in the field of biological/medical/food testing, can solve problems such as increased experimental costs, and achieve the effect of reducing laboratory costs, reducing usage, and enriching choices

Active Publication Date: 2019-08-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In these tests, due to the different sources of primary antibodies, it is often necessary to purchase different secondary antibodies, for example, it is necessary to purchase multiple secondary antibodies against mouse, anti-goat, anti-rabbit, etc., which directly leads to an increase in the cost of related experiments

Method used

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  • Broad-spectrum secondary antibody based on ZZ domain and preparation method and application thereof
  • Broad-spectrum secondary antibody based on ZZ domain and preparation method and application thereof
  • Broad-spectrum secondary antibody based on ZZ domain and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Expression and Characterization of Example 1ZZ-Suc2

[0038] In this embodiment, the reporter group is sucrase Suc2 with biocatalytic function, and the reporter group is fused and expressed at the N-terminus of the protein. A flexible fragment GGGGSGGGGS (G: glycine, S: serine) is inserted between the ZZ domain and the reporter group.

[0039] 1) ZZ-Suc2 expression strain construction

[0040] In order to construct the fusion protein ZZ-Suc2 expression strain, according to the base sequence of ZZ (its amino acid sequence is shown in SEQ ID NO.1), primers ZZ-Suc2-F1 and ZZ-Suc2-R1 were designed with Primer Premier 5.0, and pET22b- ZZ was amplified as a template, and the amplified fragments were recovered by a gel recovery kit to obtain ZZ ( figure 1 -A, lane 2). At the same time, according to the base sequence of Suc2 (its amino acid sequence is shown in SEQ ID NO.2), primers ZZ-Suc2-F2 and ZZ-Suc2-R2 were designed with Primer Premier 5.0, and the plasmid pET22b-Suc2 ...

Embodiment 2

[0052] Expression and characterization of embodiment 2EGFP-ZZ

[0053] In this embodiment, the reporter group is EGFP with fluorescent function, and the reporter group is fused and expressed at the C-terminus of the protein. A flexible fragment GGSGGSGGSGGS (G: glycine, S: serine) is inserted between the ZZ domain and the reporter group

[0054] 1) Construction of EGFP-ZZ expression strain

[0055] In order to construct the fusion protein EGFP-ZZ expression strain, according to the base sequence of ZZ, primers EGFP-ZZ-F2 and EGFP-ZZ-R2 were designed with Primer Premier 5.0, and pET22b-ZZ was used as a template for amplification, and the amplified fragment Recover the ZZ fragment ( Figure 5 -A, lane 1). At the same time, according to the base sequence of EGFP (its amino acid sequence is shown in SEQ ID NO.4), primers EGFP-ZZ-F1 and EGFP-ZZ-R1 were designed with Primer Premier 5.0, and the plasmid pET28a-EGFP synthesized by a commercial company was used as The template was ...

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Abstract

The invention discloses a broad-spectrum secondary antibody based on ZZ domain and a preparation method and application thereof, and belongs to the technical field of antibodies. The broad-spectrum secondary antibody has two function domains which are respectively used for exerting an IgG antibody binding function and a reporter group function; the domain for exerting the IgG antibody binding function is the ZZ domain which is derived from staphylococcus aureus Protein A, and can bind IgG from various sources; the domain for exerting the reporter group function is an enzyme with a catalyzing function or protein with a light emitting function; the broad-spectrum secondary antibody has the two function domains which are respectively used for exerting the IgG antibody (primary antibody) binding function and the reporter group function. The broad-spectrum secondary antibody provided by the invention has the advantages that the broad-spectrum secondary antibody is fusion protein; after fusion expression, the realizing of the IgG antibody binding ability and the reporter group function is not influenced; the broad-spectrum secondary antibody can bind the IgG primary antibodies of varioussubstances, such as the IgG of a mouse source, a rat source, a human source, a rabbit source and the like.

Description

technical field [0001] The invention belongs to the technical field of biology / medicine / food detection, and specifically relates to a ZZ domain-based broad-spectrum secondary antibody. Background technique [0002] In clinical testing and research on life science-related issues, it often involves the use of antibodies to detect various antigens and indicators, such as ELISA, FACS, Western Blot, etc. In these tests, due to the different sources of primary antibodies, it is often necessary to purchase different secondary antibodies, for example, it is necessary to purchase multiple secondary antibodies against mouse, anti-goat, anti-rabbit, etc., which directly leads to an increase in the cost of related experiments . In order to develop a simple and low-cost analytical method for the determination of related indicators, it is necessary to develop a broad-spectrum secondary antibody to reduce the use of related secondary antibodies. Contents of the invention [0003] Purpo...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K16/18C12N15/70G01N33/68
CPCC07K16/468C07K16/18C12N15/70G01N33/68C07K2317/626C07K2319/60C07K2319/61
Inventor 吴志猛周志昉董笑飞洪皓飞鲍奕恺胡苗苗叶可嘉李舜岩俞杭艳
Owner JIANGNAN UNIV
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