Preparation method and application of recombinant protein and monoclonal antibody thereof

A recombinant protein and protein technology, which is applied in the field of early diagnosis of acute myocardial infarction and the preparation of cardiac fatty acid binding protein monoclonal antibody, can solve the problems of low expression level, poor specificity of monoclonal antibody, and distortion of detection results, etc., to improve the expression level , improve sensitivity, enhance the effect of stimulation

Active Publication Date: 2012-09-12
杭州贤至生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immunogen used in the preparation of conventional human heart-type fatty acid binding protein monoclonal antibody is a complete protein expressed by genetic engineering technology, but due to the base codon, it is difficult to express the protein in Escherichia coli, and the expression level is extremely low, resulting in Subsequent purification work is difficult to carry out, which seriously hinders the preparation of its monoclonal antibody
In addition, due to the homology of the amino acid sequence of the antigenic epitope, the monoclonal antibody prepared using the full-length sequence of the heart-type fatty acid binding protein as the immunogen has poor specificity and may recognize other proteins, resulting in distortion of the test results

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Selection of dominant epitopes of heart-type fatty acid binding protein

[0014] Taking human heart-type fatty acid binding protein as the target antigen, using the biological software DNAssist2.0 to analyze the hydrophilicity and antigenicity of the epitope sequence, and select the A dominant epitope (SEQ ID No: 2) and the B dominant antigenic epitope (SEQ ID No: 3). 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

[0015] Example 2: Concatenation of Cardiac Fatty Acid Binding Protein Predominant Epitopes

[0016] In order to enhance the stimulation of the selected antigenic epitopes to the immune system of mice to facilitate subsequent experiments, the two dominant antigenic epitope sequences of heart-type fatty acid binding protein A and B were respectively repeated and then passed through the flexible fragment (four consecutive glycines) Linked to obtain the amino acid sequence of the recombinant protein, the specific sequence of which is shown in SEQ ID No: 1 in the sequence table.

Embodiment 3

[0017] Embodiment 3: optimize the nucleotide sequence of coding recombinant protein

[0018] In order to improve the expression of the recombinant protein in E. 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 E. coli. The specific sequence is shown in the sequence table Shown in SEQ ID No: 4, after adding the nucleotide sequences corresponding to the enzyme cutting sites BamHI and EcoRI in its upstream and downstream, respectively, it was 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 pertains to the field of the biotechnology. The invention relates to a recombinant protein. The recombinant protein includes two dominant antigenic epitopes of humanized heart type fatty acid binding protein, the amino acid sequence of the recombinant protein is converted into corresponding nucleotide sequence by adopting codon preferred by escherichia coli, chemical synthesis is performed on the nucleotide sequence and recombinant expression vector is constructed, so that protein expression level of the recombinant protein in the escherichia coli is increased. The invention also relates to the preparation of monoclonal antibody of the recombinant protein. Immunization, cell fusion and several rounds of screening are carried out to obtain hybridoma cell strain, the monoclonal antibody is purified and labeled with horse radish peroxidase (HRP), the best matching combination of the monoclonal antibody is determined by ELISA orthogonal experiment, and an early diagnosis application of myocardial infarction is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention relates to a new recombinant protein, a nucleotide sequence encoding the recombinant protein, and a plasmid vector containing the above-mentioned nucleotide sequence, a bacterial strain transformed with the above-mentioned plasmid vector, and the use of the above-mentioned recombinant protein Preparation of cardiac fatty acid binding protein monoclonal antibody and its application in early diagnosis of acute myocardial infarction (AMI). Background technique [0002] Acute myocardial infarction refers to myocardial necrosis caused by acute and persistent ischemia and hypoxia (coronary insufficiency), and is a cardiovascular disease that seriously affects human health. In recent years, the incidence of this disease has been increasing year by year, and the trend of younger patients is becoming more and more serious. However, the treatment effect is not satisfactory....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N1/21C07K16/18C12R1/19
Inventor 吴琼杉汪俊余铭恩李会强冯俊涛李晓照
Owner 杭州贤至生物科技有限公司
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