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JAML-receptor-targeted single-chain antibody, and preparation method and application thereof

A single-chain antibody and receptor technology, applied in the biological field, can solve problems such as single-chain antibodies that have not yet been discovered, and achieve the effect of high affinity activity

Active Publication Date: 2017-01-04
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports of related single-chain antibodies targeting JAML receptors

Method used

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  • JAML-receptor-targeted single-chain antibody, and preparation method and application thereof
  • JAML-receptor-targeted single-chain antibody, and preparation method and application thereof
  • JAML-receptor-targeted single-chain antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Sequence design of single-chain antibody targeting JAML receptor and construction of expression vector pET-20b(+) / HL-scFv and acquisition of target protein

[0041] 1. Design of HL-ScFv gene sequence

[0042] 2. Preparation of vector pET-20b(+) and target fragment HL-ScFv

[0043] (1) Plasmids were extracted from DH5α competent cells as vectors, and the pET-20b(+) vector (entrusted to Nanjing GenScript Biotechnology Co., Ltd. to synthesize the whole gene) and the pUC57 / HL-scFv whole gene were respectively subjected to EcoR V / HindIII Double digestion reaction. Add the reactants according to the amount of the double enzyme digestion system, and carry out the enzyme digestion reaction for 1 hour at 37 degrees.

[0044] (2) 1% agarose gel electrophoresis to separate the digested products. Such as figure 1 As shown, the constructed vector was digested with HindIII and EcoRV to obtain two fragments, the size of which was consistent with the target gene sequence ...

Embodiment 2

[0059] Example 2 Affinity Detection of Single Chain Antibody HL-scFv and JAML Overexpressed Cells

[0060] The human peripheral blood monocyte cell line THP-1 overexpressing JAML and the mouse macrophage Raw264.7 overexpressing JAML were selected as cell lines for affinity detection.

[0061] Detecting the combination of HL-ScFv and positive cells by flow cytometry

[0062] 1) Cell count: positive cells, 5*10^5 cells / tube.

[0063] 2) Incubate HL-ScFv: wash 2 times with Staining Buffer, add HL-scFv sample, 5ug / tube, incubate at 4°C for 1h.

[0064] 3) Secondary antibody incubation: wash with Staining Buffer 3 times, add PE-His Tag antibody, 1ug / tube, and incubate at 4°C for 30min.

[0065] 4) Detection: wash 3 times with Staining Buffer, check the binding efficiency by flow cytometry, count 10,000 cells under the PE channel and analyze the results with Flowjo 6.0, the results are as follows Figure 7 shown.

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PUM

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Abstract

The invention provides a JAML-receptor-targeted single-chain antibody. The amino acid sequence of the heavy chain variable region is disclosed as SEQ ID NO:1 or conserved variant sequence thereof, and the amino acid sequence of the light chain variable region is disclosed as SEQ ID NO:2 or conserved variant sequence thereof. The single-chain antibody is free of the FC segment, is not combined with the non-cell-targeted FC receptor, and can be quickly cleared away in vivo. The single-chain antibody can be easily established and expressed, and can easily implement mass preparation.

Description

technical field [0001] The invention relates to a single-chain antibody targeting JAML receptor and its preparation method and application, belonging to the field of biotechnology. Background technique [0002] JAML (junctional adhesion molecule–like protein), as a member of the JAMs (Junctional adhesion molecules) protein family, can promote the adhesion of neutrophils and monocytes to endothelial cells, and mediate the inflammation of leukocytes in blood circulation. The process of site aggregation; JAML can also mediate the activation and proliferation of γδT cells, prompting the cells to secrete cytokines IL-2, TNFα, and IFN-g. Therefore, JAML can be used as a target for immunotherapy, such as acute or chronic wound repair. [0003] At present, it has been found in the literature that HL4E10 is an exogenous stimulatory antibody targeting the JAML receptor target. HL4E10 has the same effect as the JAML endogenous receptor CAR, and the combination of the two can induce γδ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/70A61K39/395A61P7/00A61P35/00A61P31/04A61P9/00A61P29/00
Inventor 徐宇虹刘冬妮
Owner SHANGHAI JIAO TONG UNIV
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