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Ferritin nanometer material for displaying antibody and preparing method and application of ferrtin nanometer material

A technology of nanomaterials and ferritin, which is applied in the field of nanomaterials to achieve the effects of improving timeliness, efficiently displaying antibodies, and reducing costs

Active Publication Date: 2019-09-24
INST OF GEOLOGY & GEOPHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] The present invention provides a ferritin nanomaterial displaying antibodies, using protein A or protein G combined with antibody Fc domains as a bridge to solve the structural changes of antibodies caused by direct fusion expression of traditional ferritin and antibodies, and because the ferritin nanomaterials The material contains a metal oxide core and has its own label, which eliminates the need to carry a label on the antibody to be bound, simplifying the subsequent steps of protein purification, quantification, and localization

Method used

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  • Ferritin nanometer material for displaying antibody and preparing method and application of ferrtin nanometer material
  • Ferritin nanometer material for displaying antibody and preparing method and application of ferrtin nanometer material
  • Ferritin nanometer material for displaying antibody and preparing method and application of ferrtin nanometer material

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Experimental program
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Embodiment 1

[0194] Construction of embodiment 1 fusion protein expression strain

[0195] 1.1 Edit and design the fusion sequence of the gene encoding the B domain of Protein A (SEQ ID NO: 1) and the gene encoding ferritin (SEQ ID NO: 2): the related gene sequence of protein A is at the 5' end, and the gene sequence of ferritin The gene sequence is at the 3' end, a 30-60bp connection sequence is added between the two, an NdeI restriction site is added at the 5' end, and an XhoI restriction site is added at the 3' end;

[0196] 1.2 Perform codon optimization (SEQ ID NO: 4) on the designed fusion sequence (SEQ ID NO: 3) to make the sequence more suitable for the expression system of Escherichia coli;

[0197] 1.3 The codon-optimized fusion sequence was synthesized by Huada Gene, and the synthesized fusion sequence was cloned into the pUC57 vector (pUC57-PA-HFn) by Huada Gene, and introduced into E. coli Top10 strain;

[0198] 1.4 Extract the pUC57-PA-HFn plasmid from the Top10 strain provi...

Embodiment 2

[0208] Expression and purification of embodiment 2 fusion protein

[0209] 2.1 Streak on LB solid plate (containing ampicillin) to activate the heterologous expression strain obtained in Example 1, pick a single clone into liquid LB (adding ampicillin), culture for 8-10h to logarithmic phase, and transfer according to 1% inoculum Connect a large bottle of liquid LB (with ampicillin), and when the expression strain grows to an OD value of 0.6-1.0, add a final concentration of 1mM IPTG to activate the T7 promoter, induce the expression of the fusion protein, and induce for 8-10 hours at 30°C;

[0210] 2.2 Collect the cells by centrifugation at 4°C, wash the cells twice with PBS, and finally resuspend the cells in PBS solution, break the cells by ultrasonic, and collect the supernatant by centrifugation;

[0211] 2.3 Heat the supernatant at 75°C for 20 minutes to remove impurities, and collect the supernatant by centrifugation;

[0212] 2.4 The supernatant was further purified b...

Embodiment 3

[0213] Embodiment 3 fusion protein thermostability analysis

[0214] 3.1 Replace the purified fusion protein solution with 0.1M NaCl solution from PBS solution, measure the protein concentration, dilute or concentrate the protein concentration to 0.15mg / mL.

[0215] 3.2 Treat the diluted fusion protein solution at 37, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100°C for 30 minutes, centrifuge at 20,000g for 30 minutes, and measure the protein concentration and CD spectrum. The results showed that the protein concentration began to decrease rapidly when the temperature exceeded 75°C (see figure 2 A), the CD spectral curve begins to deform (see figure 2 C), indicating that the secondary structure of the fusion protein begins to change at a temperature greater than 75°C, and aggregates and precipitates. The maximum temperature tolerance of the fusion protein does not exceed 75°C.

[0216] 3.3 Heat the diluted fusion protein solution at 65°C for 0.5, 1, 2, 4, 6, 8, ...

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Abstract

The invention provides a ferritin nanometer material for displaying an antibody. The ferritin nanometer material comprises a ferritin cage covering a metal oxide inner core and protein connected to a binding antibody Fc structure domain of the outer surface of the ferritin cage. The ferritin nanometer material has very high antibody display efficiency, the problem that the structure of the antibody is changed due to direct fusion expression of traditional ferritin and the antibody is solved; moreover, the ferritin nanometer material has a metal oxide label, a to-be-bound antibody does not need to carry a label anymore, and the subsequent steps of protein purification, quantifying and positioning are simplified.

Description

technical field [0001] The present invention relates to the technical field of nanomaterials, in particular to a ferritin nanomaterial displaying a general-purpose antibody, the ferritin nanomaterial comprising a metal oxide core with peroxidase-like activity, and the ferritin nanomaterial Preparation methods and applications. Background technique [0002] Antibodies are immunoglobulins produced by B lymphocytes under the stimulation of antigens against antigens. Antibodies can recognize antigens and react with them. The binding between antibodies and antigens is highly specific. Antibody-antigen specific binding reactions are widely used in scientific research, medicine and other fields. For different application scenarios, different technologies and methods have been developed, such as: enzyme-linked immunosorbent assay (ELISA), which can not only quantitatively analyze specific proteins, but also study the interaction or other characteristics between molecules; Western b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/00C12N15/62C12N15/70C12N1/21C12R1/19
CPCC07K14/00C12N15/70
Inventor 张同伟曹长乾潘永信
Owner INST OF GEOLOGY & GEOPHYSICS CHINESE ACAD OF SCI
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