Preparation method of clostridium difficile recombinant protein monoclonal antibody and application thereof

A single-chain antibody and heavy chain technology, applied in anti-bacterial immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of distortion of test results, poor specificity of monoclonal antibodies, large batch-to-batch differences, etc. question

Active Publication Date: 2021-04-16
杭州贤至生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, in the traditional preparation of monoclonal antibodies, natural antigens are generally used as immunogens, and some sequence epitopes of natural antigens are conservative and have high homology with other species, resulting in

Method used

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  • Preparation method of clostridium difficile recombinant protein monoclonal antibody and application thereof
  • Preparation method of clostridium difficile recombinant protein monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: GDH dominant antigen epitope selection

[0012] Clostridium difficile GDH was used as the target antigen, and the hydrophilicity and antigenicity of its epitope sequence were analyzed by biological software DNAssist2.0, and the A dominant antigen epitope and the B dominant antigen epitope were selected. At the same time, the sequence comparison results showed that the selected two dominant antigenic epitopes, A and B, had high sequence specificity and broad spectrum, and were the common epitopes of GDH, and had no obvious homology with other protein sequences.

Embodiment 2

[0013] Example 2: Concatenation of GDH dominant antigenic epitopes

[0014] In order to enhance the stimulation of the selected epitope on the immune system of mice and facilitate subsequent experiments, the two dominant epitope sequences of A and B of GDH were respectively repeated and then connected by a flexible fragment (four consecutive glycines) to obtain Amino acid sequence of the recombinant protein.

Embodiment 3

[0015] Embodiment 3: optimize the nucleotide sequence of encoding recombinant GDH protein

[0016] In order to increase the expression of recombinant protein in Escherichia coli, under the premise that the amino acid sequence of the recombinant protein remains unchanged, the amino acid sequence encoding the recombinant protein is converted into the corresponding nucleotide sequence according to the preferred codons of Escherichia coli, and the upstream and downstream After adding the nucleotide sequences corresponding to the restriction sites BamHI and EcoRI respectively, they were synthesized by Hangzhou Xianzhi Biotechnology Co., Ltd. The synthesized target gene was cloned into the pMD19-T vector (Bao Bioengineering Dalian Co., Ltd.).

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Abstract

The invention belongs to the technical field of biology. The invention relates to a recombinant protein which is formed by repeatedly connecting two sections of dominant epitopes of clostridium difficile membrane protein-glutamate dehydrogenase (GDH) in series, and in order to improve the expression quantity of the recombinant protein in Escherichia coli, Escherichia coli preferred codons are adopted to convert the amino acid sequence of the recombinant protein into a corresponding nucleotide sequence, and the nucleotide sequence is chemically synthesized and a recombinant expression vector is constructed. The invention also relates to a phage library established by immunizing a mouse with the recombinant protein, a corresponding GDH single-chain antibody scfv sequence is obtained through panning and screening, the obtained scfv sequence is constructed into a complete mouse IgG1 antibody sequence expression vector, a monoclonal antibody is expressed through transient HEK293F cells, the monoclonal antibody is purified, horse radish peroxidase (HRP) is marked respectively, and an optimal monoclonal antibody pairing combination is determined through an ELISA orthogonal experiment.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention expresses a new recombinant protein. The present invention involves using the above recombinant protein to immunize mice to establish a phage library, and obtains a specific single-chain antibody scfv sequence through screening, and also involves constructing the obtained scfv sequence into a eukaryotic The expression vector expresses the GDH monoclonal antibody and is applied to the early diagnosis of Clostridium difficile infection (CDI). Background technique [0002] Clostridium difficile (Clostridium difficile) is extremely sensitive to oxygen, and it is difficult to isolate and culture. Therefore, it is named Clostridium difficile. Clostridium difficile is a Gram-positive clostridium toxin-producing bacillus that grows anaerobically, and the human intestine is a relatively anaerobic environment, so it is a normal flora in the human intestine. When antibiotics...

Claims

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Application Information

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IPC IPC(8): C07K16/40C07K16/12C12N15/13C12N15/85G01N33/573G01N33/569G01N33/535
CPCY02A50/30
Inventor 武妮妮武戌青刘清泉余卫余铭恩
Owner 杭州贤至生物科技有限公司
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