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Artificial binding protein capable of specifically recognizing allophycocyanin and acquisition method

An allophycocyanin and protein-binding technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc. Problems such as large molecular weight

Active Publication Date: 2020-09-22
INST OF AQUATIC LIFE ACAD SINICA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the structure of allophycocyanin and phycocyanin is very similar, and it is difficult to completely separate them in the process of antigen preparation and purification, it is difficult to prepare polyclonal antiserum or hybridoma monoclonal antibodies by immunizing animals with traditional antigens. achieve higher specificity
Currently commercial allophycocyanin antibodies are not very specific and have more or less cross-reactivity with phycocyanin
In addition, the antibody molecule itself has a large molecular weight and poor tolerance to acid, alkali and temperature, which greatly limits its application in the detection and purification of antigen molecules.

Method used

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  • Artificial binding protein capable of specifically recognizing allophycocyanin and acquisition method
  • Artificial binding protein capable of specifically recognizing allophycocyanin and acquisition method
  • Artificial binding protein capable of specifically recognizing allophycocyanin and acquisition method

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Embodiment Construction

[0080] In order to better explain the present invention and facilitate understanding, the present invention will be described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0081]The overall concept of the present invention is to select highly stable, highly soluble protein A dimer as the skeleton protein, and use genetic engineering technology to treat 8-24 protein A dimers exposed on the molecular surface Randomly mutate amino acid positions to form a variable protein-binding region on the surface of the molecule, thereby constructing a backbone protein library. Then, clones that can specifically bind to target molecules are screened from the protein library by surface display technology.

[0082] Further, the method of the present invention is divided into the following steps:

[0083] S1: Phage preparation of phage display protein A dimer mutant library to obtain protein A dimer mutant library expressed on the surface of phag...

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Abstract

The invention discloses an artificial binding protein capable of specifically recognizing allophycocyanin. The amino acid sequence is as follows: HMVDNKFNKEIVNAITEIHHLPNLNLEQRWAFIFSLFDDPSQSANLLAEAKKLNDAQAPKSGGGGSGGGGIGVDNKFNKEWQNAHNEIIWLPNLNWEQKWAFINSLYDDPSQSANLLAEAKKLNDAQAPKAAASDYKDDDDKLEHHHHHH. The protein has the characteristics of strong specificity, high affinity, strong thermal stability, strong acid-base tolerance and the like, and can be applied to the fields of detection, purification and the like of allophycocyanin. In addition, the invention also relates to a DNA fragment for encoding the artificial binding protein and an acquisition method of the artificial binding protein. According to the invention, a protein A dimer with high stability and high solubility is selected as a skeleton protein, and on the basis of the skeleton protein, the protein with antigen binding activity is finally obtained through the processes of genetic engineering modification, mutant library construction, affinity screening and the like.

Description

technical field [0001] The invention relates to the technical field of artificial binding proteins, in particular to an artificial binding protein capable of specifically recognizing allophycocyanin and a method for obtaining it. Background technique [0002] Phycobiliprotein is an important light-harvesting pigment protein present in Cyanophyceae, Phodophyceae, Cryptophyceae and a few dinoflagellates (Pyrrophyceae). According to literature records, phycobiliproteins are generally divided into three categories: Phycocyanin (Phycocyanin, referred to as PC, with a maximum absorption peak of 620nm, which is azure blue), phycoerythrin (Phycoerythrin, referred to as PE, with a maximum absorption peak of 562nm, which is purple) red) and Allophycocyanin (APC for short, the maximum absorption peak is 652nm, appearing sky blue). These proteins can not only be used as natural pigments in the fields of food, cosmetics and dyes, but also have special effects on cancer, diabetes, cardio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C12N15/31G01N33/68
CPCC07K14/31G01N33/68G01N2333/31G01N2333/795
Inventor 袁丽杨苗韩丹翔胡强
Owner INST OF AQUATIC LIFE ACAD SINICA
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