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Preparation of canine relaxin recombinant protein and preparation of monoclonal antibodies thereof

A technology of recombinant protein and relaxin, applied in the direction of relaxin, anti-hormone immunoglobulin, recombinant DNA technology, etc., can solve the problems of hindering the preparation of monoclonal antibodies, poor specificity of monoclonal antibodies, low expression level, etc., to achieve guaranteed detection The effect of broad spectrum, improved detection sensitivity, and improved expression level

Inactive Publication Date: 2018-11-16
杭州艾宠科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method usually requires the preparation of a monoclonal antibody that can recognize canine relaxin. The immunogen used in the preparation of conventional canine relaxin monoclonal antibody is a complete protein expressed by genetic engineering technology. Difficult to express and extremely low expression in medium, making it difficult to carry out subsequent purification work, 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 canine relaxin as the immunogen has poor specificity and has high homology with proteins of other species, resulting in distortion of the detection results

Method used

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  • Preparation of canine relaxin recombinant protein and preparation of monoclonal antibodies thereof
  • Preparation of canine relaxin recombinant protein and preparation of monoclonal antibodies thereof
  • Preparation of canine relaxin recombinant protein and preparation of monoclonal antibodies thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1: Selection of dominant antigenic epitopes of canine relaxin protein

[0022] Taking canine relaxin protein as the target antigen, using the biological software DNAssist2.0 to analyze the hydrophilicity and antigenicity of its antigenic epitope sequence, select A dominant antigenic epitope (SEQ ID No: 2) and B dominant antigenic epitope (SEQ ID No: 2) ID No: 3). At the same time, the sequence comparison results showed that the selected A and B two dominant antigenic epitope sequences had a broad spectrum and were common epitopes of all canine relaxin proteins; and the A and B epitopes had no obvious homology with other protein sequences, Only present in the canine relaxin protein sequence.

Embodiment 2

[0023] Example 2: Concatenation of canine relaxin dominant epitopes

[0024] In order to enhance the activation effect of the selected epitope on the immune system of mice and shorten the preparation time of monoclonal antibodies, the two dominant epitope sequences of canine relaxin nucleoprotein A and B were respectively linked by flexible fragments (four consecutive glycines) Repeat again 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

[0025] Example 3: Optimizing the Nucleotide Sequence Encoding Canine Relaxin Recombinant Protein

[0026] On the premise that the amino acid sequence of canine relaxin recombinant protein remains unchanged, in order to increase the expression of canine relaxin recombinant protein in Escherichia coli, the amino acid sequence encoding canine relaxin recombinant protein was converted into the corresponding nuclear Nucleotide sequence, the specific sequence is shown in the sequence table SEQ ID No: 4, and after adding the nucleotide sequences corresponding to the restriction sites BamHI and EcoRI in the upstream and downstream of it, it was synthesized by Hangzhou Xianzhi Biotechnology Co., Ltd. The synthesized target gene was connected to the pMD20-T vector (Bao Bioengineering Dalian Co., Ltd.).

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Abstract

Belonging to the biotechnology field, the invention provides a canine relaxin recombinant protein, which mainly contains two dominant antigen epitopes of canine relaxin. In order to increase the yieldof the recombinant protein in a prokaryotic expression system, an escherichia coli preferred codon is employed to convert the recombinant protein amino acid sequence into a corresponding nucleotide sequence, the nucleotide sequence is synthesized chemically and a recombinant expression vector is constructed. The invention also relates to preparation of monoclonal antibodies of the recombinant protein. After antigen immunization, cell fusion and multiple rounds of screening, monoclonal cell strains can be obtained, the monoclonal antibodies are purified and labeled with colloidal gold particles respectively, and orthogonal experiment is carried out to determine the optimal monoclonal antibody pairing combination for canine pregnancy diagnosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a canine relaxin recombinant protein, a nucleotide sequence encoding the recombinant protein, 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 recombination Protein preparation of canine relaxin nucleoprotein monoclonal antibody, and application in early diagnosis of canine pregnancy. Background technique [0002] In modern life, many people will keep pets, especially pet dogs. In order to give them better care, it is especially important to be able to make a correct diagnosis early in the dog's pregnancy. The pregnancy period of a dog is generally 9 weeks, but it will not appear until the last few weeks, so it is difficult to detect in the early stage of pregnancy; at the same time, the reproductive mechanism of dogs is different from that of other animals. I...

Claims

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Application Information

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IPC IPC(8): C07K14/64C07K16/26C12N15/16C12N15/70C12N1/21G01N33/74C12R1/19
CPCC07K14/64C07K16/26C12N2800/22G01N33/74G01N2333/64G01N2800/36
Inventor 胡祥叶吴琼杉曹丹琴王立童衣铭杨鹏英项美华刘清泉
Owner 杭州艾宠科技有限公司