Fusion protein of horseradish peroxidase and antibody fragment and application

A technology of horseradish peroxidase and fusion protein, which is applied in the field of fusion protein and its immunology application, and can solve the problems of decreased biological activity of enzymes and antibodies and low labeling efficiency

Inactive Publication Date: 2016-10-12
深圳市国创纳米抗体技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Based on the technical problems of low labeling efficiency and the decline of the biological activity of enzymes and antibodies due to the influence of chemical reagent labels in the existing technology, an immunolabeling that can maintain the biological activity of enzymes and antibodies is invented. Technology has become a real need in the field of technology to achieve efficient and rapid immunoassay

Method used

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  • Fusion protein of horseradish peroxidase and antibody fragment and application
  • Fusion protein of horseradish peroxidase and antibody fragment and application
  • Fusion protein of horseradish peroxidase and antibody fragment and application

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: preparation embodiment

[0036] 1. According to the amino acid sequence of the lysozyme nanobody of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 3 horseradish peroxidase, optimize the cDNA of lysozyme and horseradish peroxidase expressed in mammalian cells Sequences SEQ ID NO:2 and SEQ ID NO:4.

[0037] 2. Adding a flexible linker peptide (GGGGS) to the lysozyme nanobody and horseradish peroxidase sequences 3 , to obtain the DNA sequences SEQ ID NO: 5 and SEQ ID NO: 6 expressing NHP-1 and NHP-2, respectively. Two DNA sequences are artificially synthesized. figure 1 Three connection modes of the fusion protein are shown, and there may also be a signal peptide in the amino acid of the fusion protein, so as to facilitate the secretory expression of the fusion protein.

[0038] 3. Cloning NHP-1 and NHP-2 genes into the EcoRI and HindIII restriction sites of the eukaryotic expression vector pCDNA3.1 respectively to obtain expression vectors pCDNA-N...

Embodiment 2

[0046] Example 2. Detection of fusion protein immune activity

[0047] Take chicken egg white lysozyme, prepare a 10ug / ml solution with PBS, take a 96-well microtiter plate, and coat 100 microliters per well overnight. The next day, wash the wells with PBST, block with 0.5% BSA solution for 1 hour, take 100 microliters of transfected 293T cell culture medium supernatant, add to the wells, place in the dark at room temperature for 1 hour, wash with PBST 3 times, Add 100 microliters of DAB chromogenic substrate, react in the dark for 15 minutes at room temperature, and visually observe the color development (see Figure 4 ). Figure 4Middle, 1. Blank control NHP-1 transfected 293T cell supernatant; 2. Lysozyme-coated 4× diluted NHP-1 transfected 293T cell supernatant; 3. Lysozyme-coated 10× diluted NHP-1 transfected 293T Cell supernatant; 4. Lysozyme-coated 4× diluted NHP-2 transfected 293T cell supernatant; 5. Lysozyme-coated 10× diluted NHP-2 transfected 293 cell supernatant...

Embodiment 3

[0049] Example 3. Horseradish peroxidase chemical labeling of Nanobodies

[0050] Dissolve 5mg horseradish peroxidase in 0.5ml 0.1mol / L NaHCO 3 solution; add 0.5ml 10mmol / L NaIO 4 solution, mix well, tightly cap the cork, and protect from light at room temperature for 2 hours. Add 0.75ml 0.1mol / L Na 2 CO 3 Mix well, then add 0.75ml of purified anti-lysozyme nanobody (15mg / ml), and mix well. Weigh 0.3g of Sephadex G25 dry powder and add it into the outer cylinder of a 5ml syringe with a glass wool pad on the lower mouth; then transfer the above-mentioned cross-linked product into the outer casing of the syringe; cover tightly and act at room temperature (protected from light) for 3 hours or overnight at 4°C. Use a little PBS to wash out all the cross-linked products, collect the eluate, add 1 / 20 volume of freshly prepared 5mg / ml NaBH4 solution, mix well, and act for 30 minutes at room temperature; then add 3 / 20NaBH 4 solution, mix well, and act at room temperature for 1 ho...

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Abstract

The invention provides a fusion protein. The fusion protein is formed by connecting an anti-iysozyme antibody or a variable region fragment of the anti-iysozyme antibody with a horseradish peroxidase reporter gene protein in series, the invention further provides a preparation method and an application of the fusion protein. The fusion protein is used for immunoassay which takes an immunolabelling technique as a basis, so that good biological activities of enzymes and the antibody are kept, and the detection efficiency is higher.

Description

technical field [0001] The invention discloses a fusion protein and its immunological application, belonging to the fields of genetic engineering and immunology. Background technique [0002] Antibody (Ab) refers to a type of immunoglobulin formed after the antigenic substance stimulates the immune system of the body and has a specific binding reaction with the corresponding antigenic substance. Antibodies are an important product of the immune response and mainly exist in blood, interstitial fluid, and exocrine fluid. Therefore, antibody-mediated immunity is called humoral immunity. Antibody molecules are produced by B lymphocytes, which consist of two identical heavy chains and two identical light chains that are highly conserved in mammals. The molecular weight of IgG is about 160 kDa, which is a complex molecule, and the H chain and the L chain are unfolded to form 4 and 2 structural domains, respectively. The two light chains of immunoglobulin and the two heavy chains...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/85G01N33/573
CPCC07K14/00C07K16/40C07K2319/00C12N9/0006C12Y111/01007
Inventor 宋海鹏李敏王庆东
Owner 深圳市国创纳米抗体技术有限公司
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