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Preparation of canine adenovirus II recombinant protein monoclonal antibody

An adenovirus and single-chain antibody technology, applied in the field of bioengineering, can solve the problems of large batch-to-batch variation, long monoclonal antibody cycle, and low purity

Active Publication Date: 2021-03-16
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

[0005] Design purpose: In order to solve the problems of long cycle time, low purity and large batch-to-batch variation in the preparation of monoclonal antibodies from traditional mouse ascites, the monoclonal antibodies were prepared by designing and synthesizing the canine type Ⅱ adenovirus protein and establishing phage library and eukaryotic cell expression. Cloning antibodies not only saves time, but also reduces batch-to-batch variability and improves detection accuracy

Method used

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  • Preparation of canine adenovirus II recombinant protein monoclonal antibody
  • Preparation of canine adenovirus II recombinant protein monoclonal antibody
  • Preparation of canine adenovirus II recombinant protein monoclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0008] Example 1: Selection of dominant antigenic epitopes of canine type Ⅱ adenovirus protein

[0009] Taking the canine type Ⅱ adenovirus protein as the target antigen, the hydrophilicity and antigenicity of the epitope sequence were analyzed by using the biological software DNAssist2.0, and the A-dominant epitope and the B-dominant epitope were selected. At the same time, the sequence comparison results showed that the selected two dominant antigenic epitopes A and B have high sequence specificity and no obvious homology with other protein sequences.

Embodiment 2

[0010] Example 2: Concatenation of dominant antigenic epitopes of canine type Ⅱ adenovirus protein

[0011] In order to enhance the stimulation of the selected antigenic epitope to the immune system of mice so as to facilitate subsequent experiments, the two dominant antigenic epitope sequences of A and B of the canine type II adenovirus protein were repeated respectively and passed through the flexible fragment (4 consecutive glycines). ) to obtain the amino acid sequence of the recombinant protein.

Embodiment 3

[0012] Embodiment 3: optimize the nucleotide sequence of coding recombinant protein

[0013] 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 The nucleotide sequences corresponding to the restriction sites BamHI and EcoRI were added respectively, which were synthesized by Hangzhou Xianzhi Biotechnology Co., Ltd. The synthesized target gene was cloned in the pMD19-T vector (Bao Bioengineering Dalian Co., Ltd.).

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Abstract

The invention belongs to the technical field of bioengineering. The invention relates to recombinant protein. The recombinant protein comprises two dominant epitopes of canine adenovirus II protein. In order to improve the yield of the recombinant protein in a prokaryotic expression system, preferred codons of escherichia coli are adopted to convert an amino acid sequence of the recombinant protein into a corresponding nucleotide sequence, the nucleotide sequence is chemically synthesized and a recombinant expression vector is constructed. The invention also relates to a method for establishing a phage library by immunizing a mouse with the recombinant protein. A corresponding canine adenovirus II protein 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 by transiently transferring HEK293F cells; the monoclonal antibody is purified and europium ions (Eu<3+>) are marked; and an optimal monoclonal antibody pairing combination is determined through orthogonal experiments and can be used for early diagnosis of canine infectious laryngotracheitis and pneumonia.

Description

technical field [0001] The invention belongs to the technical field of bioengineering. Specifically, the present invention relates to a new recombinant protein, involves using the above recombinant protein to immunize mice to establish a phage library, screens to obtain a specific single-chain antibody scfv sequence, and also involves constructing the obtained scfv sequence into a eukaryotic expression vector for expression Canine type Ⅱ adenovirus protein monoclonal antibody, and used in the early diagnosis of canine infectious laryngotracheitis and pneumonia symptoms. Background technique [0002] Canine adenovirus (Canineadenovirus CAV) is the most pathogenic virus in mammalian adenovirus. There are two serotypes. Type Ⅰ can cause canine infectious hepatitis, (an acute septic infectious disease characterized by hepatic lobular center necrosis, inclusion bodies and long bleeding time in liver parenchymal cells and cortical nuclei.) It can also cause fox encephalitis, so ...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/85C12N15/13
CPCC07K16/081C12N15/85C07K2317/622C07K2317/56
Inventor 洪淑凡曹丹琴余卫余铭恩吴琼杉
Owner 杭州贤至生物科技有限公司
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