Preparation and application of myoglobin monoclonal antibody

A myoglobin and single-chain antibody technology, which is applied in the field of eukaryotic expression vectors expressing myoglobin monoclonal antibodies and early diagnosis of acute myocardial infarction. Large discrepancies, poor detection accuracy, etc.

Active Publication Date: 2021-03-19
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

However, due to the uncertainty of ascites production in a single mouse and the large individual differences, the obtained anti-myoglobin Myo monoclonal antibody has large differences between batches, which makes the detection accuracy poor.

Method used

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  • Preparation and application of myoglobin monoclonal antibody
  • Preparation and application of myoglobin monoclonal antibody
  • Preparation and application of myoglobin monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0007] Example 1: Myoglobin (Myo) dominant antigen epitope selection

[0008] Taking myoglobin (Myo) as the target antigen, the hydrophilicity and antigenicity of its 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 results of sequence comparison showed that the selected two dominant epitope sequences of A and B have a broad spectrum and are the common epitopes of all myoglobin (Myo); and the A and B epitopes have no obvious identity with other protein sequences. Origin, only exists in the myoglobin (Myo) sequence.

Embodiment 2

[0009] Example 2: Concatenation of myoglobin (Myo) dominant antigenic epitopes

[0010] 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 myoglobin (Myo) were respectively repeated and then passed through the flexible fragment (four consecutive Glycine) connection to obtain the amino acid sequence of the recombinant protein.

Embodiment 3

[0011] Embodiment 3: optimize the nucleotide sequence of encoding recombinant Myo protein

[0012] 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 General Biosystems Anhui Co., Ltd. The synthesized target gene was cloned into the pMD25-T vector (Bao Bioengineering Dalian Co., Ltd.).

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Abstract

The invention belongs to the technical field of biology. The invention provides a recombinant protein. An amino acid sequence of the recombinant protein is formed by repeatedly connecting two dominantepitopes of myoglobin (Myo) in series, an escherichia coli preferred codon is adopted to convert the 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. Therefore, the expression quantity of the recombinant protein in escherichia coli is increased. The invention also relates to a phage library established by immunizing mice with the recombinant protein, a corresponding myoglobin single-chain antibody scfv sequence is obtained through selecting 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, and an optimal monoclonal antibody pairing combination is determined through an immunofluorescence orthogonal experiment.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention expresses a new recombinant protein, involves using the above-mentioned 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 Express myoglobin (Myo) monoclonal antibody, and apply to the early diagnosis of acute myocardial infarction (Acute Myocardiac infraction, AMI). Background technique [0002] Acute Myocardial Infarction (AMI) is one of the common diseases that seriously endanger human health. In recent years, the incidence of this disease in my country has shown an obvious upward trend. Myoglobin (Myo) is one of the earliest markers of myocardial injury when AMI occurs. The content of Myo in serum begins to increase within 2 hours of onset and reaches its peak at 6-9 hours. In addition,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/00C12N15/13C12N15/85
CPCC07K16/18C07K16/005C12N15/85C07K2317/56
Inventor 项美华李小平刘清泉余铭恩吴琼杉
Owner 杭州贤至生物科技有限公司
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