Cat serum amyloid protein A and preparation method and application of monoclonal antibody of cat serum amyloid protein A

A serum starch and protein technology, applied in the field of bioengineering, can solve problems such as being unsuitable for rapid diagnosis, easy to delay treatment, cumbersome operation, etc., and achieve the effects of improving detection sensitivity, eliminating interference, and improving expression level.

Inactive Publication Date: 2019-03-26
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

[0003] At present, the diagnosis of early inflammation of feline infectious diseases on the market is mainly to detect and analyze feline blood...

Method used

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  • Cat serum amyloid protein A and preparation method and application of monoclonal antibody of cat serum amyloid protein A
  • Cat serum amyloid protein A and preparation method and application of monoclonal antibody of cat serum amyloid protein A
  • Cat serum amyloid protein A and preparation method and application of monoclonal antibody of cat serum amyloid protein A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0009] Example 1: Selection of Predominant Antigen Epitope of Feline Serum Amyloid A

[0010] Taking feline serum amyloid A as the target antigen, using the biological software DNAssist2.0 to analyze the hydrophilicity and antigenicity of the epitope sequence, and selecting 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

[0011] Example 2: Cat Serum Amyloid A Predominant Antigen Epitope Concatenation

[0012] In order to enhance the stimulation of the selected antigenic epitope on the immune system of mice and facilitate subsequent experiments, the two dominant antigenic epitope sequences of cat serum amyloid A, A and B, were connected by flexible fragments (four consecutive glycines). Repeat four more times 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

[0013] Embodiment 3: optimize the nucleotide sequence of coding recombinant protein

[0014] In order to improve 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. The specific sequence is shown in the sequence table Shown in SEQ ID No: 4, and the nucleotide sequences corresponding to the restriction sites BamHI and EcoRI were added in the upstream and downstream, respectively, and 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 bioengineering and relates to a recombinant protein. The recombinant protein contains two dominant epitopes of a cat serum amyloid protein A; and an amino acid sequence of the recombinant protein is converted into a corresponding nucleotide sequence by virtue of an escherichia coli preferred codon, the nucleotide sequence is chemically synthesized, and a recombinant expression vector is constructed, so that the yield of the recombinant protein in a prokaryotic expression system is increased. The invention further relates to preparation of the monoclonal antibody of the recombinant protein. A hybridoma cell strain is obtained through immunization, cell fusion and repeated screening, the monoclonal antibody is purified, europium ions (Eu3<+>) are respectively marked, and an optimal monoclonal antibody pairing combination is determined through an orthogonal experiment and can be applied to the early diagnosis of cat infectious inflammations.

Description

technical field [0001] The invention belongs to the technical field of bioengineering. Specifically, the present invention relates to a new recombinant protein, which encodes and synthesizes the nucleotide sequence of the recombinant protein through genetic engineering technology, constructs a plasmid vector containing the above-mentioned nucleotide sequence, and transforms the Escherichia coli strain with the above-mentioned plasmid vector, Express recombinant feline serum amyloid A; use the recombinant protein to prepare feline serum amyloid A monoclonal antibody, and apply it to the diagnosis of early inflammation of feline infectious diseases. Background technique [0002] Infectious diseases are caused by pathogens such as bacteria, viruses, fungi, and parasites. They are common and frequently-occurring diseases that seriously threaten the health of the body. Therefore, it is of great significance to find infectious markers that are beneficial to early diagnosis and tre...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/70C12N1/21C07K16/18C12R1/19
CPCC07K14/47C07K16/18C12N15/70
Inventor 刘清泉胡祥叶朱伟项美华武戌青王立童吴琼杉余铭恩
Owner 杭州贤至生物科技有限公司
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