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Anti-IBRV single-chain antibody and preparation method and application thereof

A single-chain antibody and light chain technology, applied in the field of anti-IBRV single-chain antibody, can solve the problems of low sensitivity of detection reagents, prone to false positive results, unsuitable for clinical practice, etc., achieve small molecular weight, good application value, easy to use Effects on construction and mass expression

Inactive Publication Date: 2017-11-03
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Among many therapeutic biological agents, antibodies are the most direct and effective tools for the treatment of animal diseases. However, antibodies prepared by traditional methods, such as serum from recovered animals, yolk antibodies, and monoclonal antibodies, are complex in composition and immune to heterologous animals. Rejection is becoming less and less suitable for clinical practice
Moreover, when the antibody prepared by the traditional method is used in the detection reagent, the prepared detection reagent has low sensitivity, serious cross-reactivity, and false positive results are prone to occur.

Method used

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  • Anti-IBRV single-chain antibody and preparation method and application thereof
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  • Anti-IBRV single-chain antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1 single-chain antibody VH-VL protein

[0036]In the past, single-chain antibody genes were obtained by constructing single-chain antibody phage display libraries, which was laborious and time-consuming. In the present invention, after extracting total cellular RNA from hybridoma cells secreting monoclonal antibodies, reverse transcribe cDNA as a template, and use PCR After primers were used to amplify the heavy chain variable region (VH) and light chain variable region (VL) of the antibody, 100 clones were selected for sequencing and comparison, and 3-4 genes with the highest repeatability were screened out , after linking VH and VL with a connecting peptide to form a complete ScFv gene, after constructing a PET28a expression vector, transforming the expression bacteria to prepare a recombinant ScFv protein.

[0037] The specific operation is as follows:

[0038] Among them, the experimental materials used in this example can be materials...

Embodiment 2

[0057] Example 2 Single-chain antibody is used for Western-blot to detect the expression of gD protein

[0058] The single-chain antibody prepared in Example 1 was used for Western-blot detection of IBRV gD protein. Specifically, the lysate of E. coli cells expressing gD protein was subjected to SDS-PAGE electrophoresis to separate the protein and transferred to a PVDF membrane. After blocking with 5% skim milk, the purified HRP-labeled single-chain antibody was used for immunoblotting, and the ECL chemiluminescence kit was used for color development. And set the Escherichia coli lysate expressing pET28a empty vector as the control.

[0059] The result is as Figure 4 As shown, it shows that the single chain antibody prepared by the present invention can specifically bind to the gD protein expressed in Escherichia coli. in, Figure 4 Middle M: ​​Pre-stained protein molecular mass standard; 1: E. coli lysate expressing pET 32a empty vector; 2: E. coli cell lysate expressing ...

Embodiment 3

[0060] Example 3 Single-chain antibody is used for IFA detection of IBR virus infected in MDBK

[0061] The purified single-chain antibody prepared in Example 1 is used for indirect immunofluorescence assay (IFA) to detect IBRV in MDBK cells, specifically, MDBK cells grown on glass slides in 6-well cell plates are infected with IBRV, and when pathological changes occur After fixing the cells with 4% paraformaldehyde for 1 h, permeabilize with 0.1% Triton X-100 for 2 h, block with 0.5% BSA at 37°C for 1 h, use the recombinant single-chain antibody purified in Example 1 as the primary antibody, and use 1:100 Diluted FITC-conjugated mouse anti-His monoclonal antibody was used as the secondary antibody for immunoreaction. Observe under a fluorescent microscope, and set the gD monoclonal antibody binding to the IBRV control infected MDBK cells.

[0062] The result is as Figure 5As shown, DAPI is the blue fluorescent dye used in the figure: 4', 6-diamidino-2-phenylindole, and FIT...

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Abstract

The invention provides an anti-IBRV single-chain antibody which is protein consisting of a heavy chain variable region shown as SEQ ID NO.1, a light chain variable region shown as SEQ ID NO.2 and a connecting peptide for connecting the two regions and having the characteristic of resisting an infectious bovine rhinotracheitis peplos gD protein antibody. The invention also provides a preparation method of the single-chain antibody. The single-chain antibody can be specifically combined with IBRV and can be also specifically combined with IBV gD protein in an escherichia coli expression product. An indirect immunofluorescence assay shows that the single-chain antibody can identify the IBRV infected in the bovine kidney cell MDBK, and the single-chain antibody has the good application value when applied to infectious bovine rhinotracheitis detection and development of treatment preparations.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-IBRV single-chain antibody, its preparation method and application. Background technique [0002] Infectious bovine rhinotracheitis (IBR) is a major infectious disease harmful to the cattle industry caused by infectious bovine rhinotracheitis virus (IBRV), and is widely prevalent in cattle farms at home and abroad. IBRV is a member of αherpes virus, which can be latent in the ganglion cells of cattle and cause persistent infection, so that even highly effective vaccines cannot eradicate the virus latent in cattle, so antiviral agents can only be used to kill and clear cells virus inside. Currently commonly used anti-herpes virus agents include chemical drugs and biological agents. The toxic side effects and drug residues of chemical drugs are not only harmful to animals themselves, but also to the safety of animal products. [0003] Among many therapeutic biolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13G01N33/569A61K39/42A61P31/22
CPCC07K16/085C07K2317/622C07K2317/76
Inventor 李永清许健吴靖黄秀芬
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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