Precise antibody nucleic acid directional connection method

A connection method and directional connection technology, applied in the field of molecular biology, can solve the problems of low cross-linking efficiency, uncontrollable stoichiometric ratio, loss of molecular activity, etc.

Pending Publication Date: 2021-05-11
深圳伯生生物传感技术有限公司 +1
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Problems solved by technology

[0004] The purpose of the present invention is to provide an efficient, stable and precise stoichiometric ratio of antibody-nucleic acid linkage for the problems of low cross-linking efficiency, obvious loss of molecular activity, and uncontrollable stoichiometric ratio in the existing connection technology of antibodies and nucleic acids. technology

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  • Precise antibody nucleic acid directional connection method
  • Precise antibody nucleic acid directional connection method
  • Precise antibody nucleic acid directional connection method

Examples

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Embodiment 1

[0043] Example 1 Antibody nucleic acid directional connection

[0044] 1. Prepare SPG-HUH fusion protein, connect with single-stranded DNA, and obtain protein-nucleic acid complex

[0045] ①Modify the gene sequence of Protein G (SPG) at a fixed point

[0046]Artificially synthesized SPG gene sequence, select a specific site on SPG, such as 12-position leucine, 18-position threonine, 20-position alanine, 23-position alanine, 26-position alanine, 28-position arginine Acid, 32-position glutamine, etc., using site-directed genetic modification technology, respectively mutate these sites into the amber codon TAG that can encode the unnatural amino acid L-p-benzoylphenylalanine (only one site needs to be modified , but different sites can be modified to obtain multiple different modified Protein G). see Figure 7 , the figure also shows three possible insertion sites on the Protein G protein (SPG).

[0047] ② According to the published sequence information, the gene sequence of ...

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Abstract

The invention discloses a precise antibody nucleic acid directional connection method. The precise antibody nucleic acid directional connection method comprises the following steps: (1) connecting HUH endonuclease with Protein G through a flexible Linker molecule to obtain a fusion protein; (2) synthesizing single-stranded DNA, wherein the single-stranded DNA contains a site which can be recognized by the HUH endonuclease; (3) mixing and reacting the fusion protein and the single-stranded DNA to obtain a protein nucleic acid compound; (4) mixing the protein nucleic acid compound with a target antibody, and performing reacting under the illumination catalysis condition to realize directional connection of a target antibody and the single- stranded DNA, wherein the step (1) and the step (2) are interchangeable. A codon of a non-natural amino acid specifically cross-linked by a light-induced site is inserted into a specific site of the Protein G gene. The method does not need any chemical or functional modification on the antibody and nucleic acid, and does not influence the antigen recognition capability of the antibody and the property and function of nucleic acid molecules. Only one nucleic acid molecule is connected to one antibody of the obtained antibody nucleic acid compound.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for directional connection of precision antibody nucleic acid. Background technique [0002] Immunoassay converts the information of target molecules into signals that can be interpreted quantitatively through specific immune recognition. It is widely used in the qualitative and quantitative detection of proteins, and is an important analytical method in the fields of clinical diagnosis, microbial detection, and food testing. However, traditional immunoassay methods are limited by the catalytic activity of enzyme molecules. For some trace analytes, such as rare receptors on the cell surface and trace disease markers in the circulatory system, it is difficult to achieve highly sensitive and accurate quantitative analysis. Immuno-PCR is a highly sensitive analytical method established by Professor Sano of the University of California in 1992. It combines the high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12Q1/686
CPCC07K14/00C12Q1/686C07K2319/30
Inventor 门冬曹姗姗周昆周娟张先恩
Owner 深圳伯生生物传感技术有限公司
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