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Site-specific biotinylation of recombinant IgG affinity peptide and its application in three-dimensional directional immobilization of IgG antibodies

A biotin-labeled, site-specific technology, which is applied in the field of site-specific biotin-labeled recombinant IgG affinity peptides, can solve non-uniqueness, loss of antigen binding ability, inability to effectively guarantee antibody directional fixation and high immunological Activity and other issues

Active Publication Date: 2015-12-02
WEIFANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this mode is a controllable immobilization method, the problem is that the position of the active group participating in the covalent chemical bond in the protein molecule is uncertain and not unique, and it cannot effectively guarantee the directional immobilization of the antibody and high immunological activity.
However, biotin is randomly combined with the active group in the antibody molecule. When the amino acid residue of the reactive group is located at or near the Fab of the antibody, the Fc end of the immobilized biotinylated antibody will face outward and be covered. Fab segment, such a solid-phase antibody loses its antigen-binding ability (such as figure 1 shown)

Method used

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  • Site-specific biotinylation of recombinant IgG affinity peptide and its application in three-dimensional directional immobilization of IgG antibodies
  • Site-specific biotinylation of recombinant IgG affinity peptide and its application in three-dimensional directional immobilization of IgG antibodies
  • Site-specific biotinylation of recombinant IgG affinity peptide and its application in three-dimensional directional immobilization of IgG antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 contains (Gly 4 -Ser) 3 -Construction of Avi-tag gene sequence intermediate plasmid vector

[0066] Proceed as follows:

[0067] (1) According to (Gly 4 -Ser) 3 And the Avi-tag gene sequence is designed as follows:

[0068] 5'-GCGGAGGTGGATCTGGCGGAGGTGGATCGGGCGGAGGTGGATCAGGTCTGAACGATATCTTCGAAGCTCAGAAAATCGAATGGCACGAA-3'; as shown in SEQIDNO.2.

[0069] There are PstI and HindIII restriction sites on the 5'end and 3'end respectively.

[0070] The chemical synthesis of the above gene sequence was entrusted to Dalian Bao Biological Biological Engineering Company to complete;

[0071] (2) The DNA fragments obtained by chemical synthesis are double-enzyme digested and recombined into pUC18 vector to obtain (Gly 4 -Ser) 3 -Avi-tag fusion gene intermediate vector pUC (Gly 4 -Ser) 3 -Avi-tag.

Embodiment 2I

[0072] Example 2 Preparation and purification of IgG affinity peptide-Avi-tag fusion protein (ZZ-Avi-tagfusion protein) by gene recombination technology

[0073] Proceed as follows:

[0074] (1) Amplification of IgG affinity peptide (ZZ gene) sequence, construction of vector and genetic engineering bacteria: design primers:

[0075] 5'-CCGGAATTCCATGGCGCAACACGATGAAG-3' (as shown in SEQ ID NO. 3);

[0076] And: 5'-CCCCTGCAGTTAATTCGCGTC-3' (as shown in SEQ ID NO. 4);

[0077] Using pEZZ18 as a template, the ZZ gene was amplified by PCR with the above primers, and cloned into the EcoRI and PstI sites of the intermediate plasmid vector pUC(Gly4-Ser)3-Avi-tag constructed in Example 1, to obtain the recombinant expression vector pUCZZ-(Gly4-Ser ) 3-Avi-tag / E.coli BL21 plasmid, CaCl 2 Transform E.coli BL21, ampicillin LB resistant plate (that is, the medium contains 100μg / mL ampicillin in addition to the components of conventional LB medium) to screen positive clones and obtain genetically eng...

Embodiment 3

[0091] Example 3 Directional immobilization of ZZ-Biotin to ZZ affinity peptide

[0092] Proceed as follows:

[0093] (1) Add 100μL / well of ZZ-Biotin protein solution (PBST solution, containing 1M NaCl; ZZ-Biotin protein concentration 0.05μg / mL) into the wells of 96-well polystyrene plate with avidin pre-coated with polystyrene ,Incubate at 37℃ for 2h;

[0094] (2) Wash the 96-well plate with PBST solution containing 0.2M NaCl for 3 times, 5 min each time, and pat dry the 96-well plate on absorbent paper to obtain a ZZ-Biotin fixed polystyrene microplate (such as figure 2 Shown), stored at 4°C.

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Abstract

The invention discloses a locus specificity biotin labeled IgG affine peptide. The c-terminus of the IgG affine peptide is bound with biotin in a biological manner to construct the peptide. Once the locus specificity biotin labeled IgG affine peptide is constructed, the IgG antibody can be affinely adsorbed on the avidin or streptavidin pre-enveloped solid phase carrier surface in an orientation manner without in-vitro labeling. The locus specificity biotin labeled recombinant IgG affine peptide can be used for fixing the IgG antibody on the solid phase carrier surface. After binding, the Fab terminal of the IgG antibody can be fully exposed to sufficiently retain the antigen binding activity of the antibody, so as to effectively improve flexibility, specificity and stability of solid phase immunoassay. Avi-tag and the IgG antibody affine peptide are adopted to construct fusion proteins to solve the oriented affine fixation problem of the IgG antibody affine peptide, and further obtain the uniform three-dimensional oriented fixation IgG antibody which fully exposes the Fab terminal and maintains the high antigen binding activity.

Description

Technical field [0001] The invention relates to a site-specific biotin-labeled recombinant IgG affinity peptide and its application in three-dimensional directional immobilization of IgG antibodies, belonging to the field of genetic engineering and immunoassay. Background technique [0002] Solid-phase immunoassay technology is an important part of clinical immunological testing, such as enzyme-linked immunosorbent assay and immunosensor, immunodiagnostic chip and other new technologies. Its application basis is to coat antigen or antibody on solid-phase carrier for immunoassay , Generally coated antibodies are more commonly used. For example, enzyme-linked immunosorbent assay (Enzymelinked immunosorbent assay, ELISA) is a classic solid-phase immunoassay technology, which is sensitive, simple, fast and easy to automate. It is widely used in biomedicine, clinical diagnosis and other fields. Styrene (Polystyrene, PS) microplates / tubes / beads, etc. are solid-phase carrier materials ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/70C07K1/36C07K1/34C07K1/16C07K17/08C07K17/06G01N33/532G01N33/545
Inventor 唐金宝杨洪鸣
Owner WEIFANG MEDICAL UNIV
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