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Preparation method and application of fusion protein with broad spectrum adsorption capacity to antibodies

A technology of fusion protein and adsorbent, which is applied in the fields of bioengineering, antibody purification and blood purification, can solve the problems that there are no reports in the field of blood purification, and achieve the effects of avoiding non-specific adsorption, good binding ability and easy expression

Active Publication Date: 2012-09-19
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These adsorbent materials are currently widely used in antibody purification, but their application in the field of blood purification has not been reported

Method used

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  • Preparation method and application of fusion protein with broad spectrum adsorption capacity to antibodies
  • Preparation method and application of fusion protein with broad spectrum adsorption capacity to antibodies
  • Preparation method and application of fusion protein with broad spectrum adsorption capacity to antibodies

Examples

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Embodiment 1

[0041] The following non-limiting examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way. Reagents, experimental instruments, etc. described in the examples herein, unless otherwise specified, were purchased from conventional means or prepared by conventional methods. In the examples, the fusion protein AG is also abbreviated as CPAG for convenience of description. Example 1: Construction and Identification of pET-23a-CPAG Recombinant Plasmid

[0042] Use pET-23a (commodity information: Biovec, USA) as the expression vector; protein A gene includes immunoglobulin binding domains E, D, A, B, C, excluding signal peptide and cell wall anchoring protein region; protein G gene Including immunoglobulin binding domains C1, C2, and C3, excluding signal peptide, albumin binding domain, and cell wall anchoring protein region; the following uses E. coli BL21 (DE3) as the expression host for a specifi...

Embodiment 2

[0114] Embodiment 2: Expression and purification of fusion protein AG

[0115] The plasmid pET-23a-CPAG was extracted using the plasmid extraction kit (Code: DV801A) produced by Treasure Bioengineering (Dalian) Co., Ltd., and transformed into Escherichia coli BL21(DE3) by heat shock method, and coated on the surface of solid LB medium. And cultured overnight at 37°C in a constant temperature incubator. In an ultra-clean workbench, use an inoculation loop to pick multiple positive clones, inoculate them into a 50 mL Erlenmeyer flask containing 20 mL of LB liquid medium, add 100 mg / L ampicillin at the same time, and culture overnight at 37°C on a shaker. The cultured shake flask seeds were inserted into a fermenter equipped with 2L of fermentation medium with a 1% inoculum amount, and cultured at 37°C. During the cultivation process, the stirring speed and ventilation rate were gradually increased to maintain the dissolved oxygen level > 30%. When the pH value and dissolved ox...

Embodiment 3

[0133] The above-mentioned purified fusion protein AG was coupled to Sepharose CL 4B. The specific method was as described in Examples 1-4 of Jia Lingyun et al.’s patent CN1367181A. After epoxy activation, diamino reagents and glutaraldehyde are connected as linking arms, and then ligand fusion protein AG is coupled, and the adsorption medium-fusion protein AG adsorbent (CPAG-Sepharose) is synthesized after blocking and reduction. By controlling the amount of the fusion protein AG, the ligand bonding amount of the adsorbent is controlled to be 2-10 mg / mL gel.

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Abstract

The invention discloses a preparation method and application of fusion protein with broad spectrum adsorption capacity to antibodies. The method includes the steps: obtaining a protein A and a protein G immune globulin binding zone gene by means of T vector connection and double digestion by the genetic engineering means and via PCR (polymerase chain reaction) amplification; then connecting onto an expression vector pET-23a to form a recombinant plasmid, converting E.coli BL21(DE3), and obtaining a great number of thalli containing the fusion protein AG after inducible expression; and finally, performing ultrasonication, high-temperature heating, precipitation of DNA (deoxyribonucleic acid) and purification of weak anion exchange chromatography and affinity chromatography so that the fusion protein AG is obtained. The fusion protein has dual advantages of the protein A and the protein B and is wider in spectrum combination and low in nonspecific adsorption of non-immune globulin substances of albumin in serum and the like. The fusion protein serving as a ligand is connected to a solid-phase vector matrix to be prepared into adsorbent so that the shortcoming of poor IgG3 binding force of protein A adsorbent is overcome, and can be applied to the fields of antibody purification, blood purification and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a preparation method of fusion protein AG with broad-spectrum adsorption capacity for antibodies and its application in the fields of antibody purification and blood purification. Background technique [0002] Autoimmune diseases (AID) are under certain circumstances, the body's own tolerance mechanism is destroyed, the immune system mistakenly recognizes its own substances as foreign harmful substances, resulting in the recognition of autoantibodies and self-reactive T lymphocytes. And damage the body's own normal cells, tissues, and organs, leading to a series of pathological changes. Since the pathogenicity of autoantibodies and immune complexes in autoimmune diseases has been confirmed, reducing the concentration of autoantibodies and immune complexes in the blood of patients through the removal of adsorbents in vitro can significantly improve symptoms and play a rol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/63C12N1/21C07K19/00C07K1/22C07K1/18B01J20/26B01D15/08C12R1/19
Inventor 贾凌云张嘉玉任军徐丽谢健
Owner DALIAN UNIV OF TECH
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