Preparation of canine brain natriuretic peptide (BNP) monoclonal antibody

A technology of canine brain natriuretic peptide and single-chain antibody, which is applied in the direction of antibody mimics/scaffolds, anti-animal/human immunoglobulins, peptides, etc., and can solve the problems of large batch-to-batch differences, long monoclonal antibody cycles, and low purity, etc. problems, to achieve the effect of ensuring natural activity, shortening the preparation cycle, and improving immune efficiency

Active Publication Date: 2021-04-06
杭州贤至生物科技有限公司
View PDF8 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Design purpose: In order to solve the shortcomings of traditional mouse ascites preparation monoclonal antibody long cycle, low purity, and large batch-to-batch variation, the monoclonal antibody was prepared by designing and synthesizing canine BNP recombinant protein and establishing phage library and eukaryotic cell expression , not only saves time, but also improves the specific identification and detection of canine BNP

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation of canine brain natriuretic peptide (BNP) monoclonal antibody
  • Preparation of canine brain natriuretic peptide (BNP) monoclonal antibody
  • Preparation of canine brain natriuretic peptide (BNP) monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Example 1: Canine Brain Natriuretic Peptide (BNP) Predominant Epitope Selection

[0011] Taking canine brain natriuretic peptide (BNP) 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 canine brain natriuretic peptides (BNP); Homology exists only in the canine brain natriuretic peptide (BNP) sequence.

Embodiment 2

[0012] Example 2: Synthesis of polypeptides containing canine brain natriuretic peptide (BNP) dominant epitopes

[0013] In order to enhance the activation effect of the selected epitope on the mouse immune system and shorten the preparation time of monoclonal antibodies, two dominant epitope sequences of canine brain natriuretic peptide A and B were chemically synthesized and connected in series, and cysteine ​​was connected at the end (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) to obtain the polypeptide.

Embodiment 3

[0014] Embodiment 3: polypeptide coupling KLH protein

[0015] Dissolve 20 mg of SMCC in 2 ml of DMF (dimethylformamide), add 0.8 ml of KLH into a 25 ml round bottom flask, and add 1×PBS (pH 7.2) to make the final protein concentration 15 mg / ml. The dissolved SMCC solution was slowly added dropwise to the 120 mg KLH protein system, and the reaction was stirred at room temperature for 1 h. Dialyze with 1L 1×PBS (pH7.4) solution at 4°C for 6 hours to remove free SMCC. Pour the dialyzed KLH protein into a 50ml centrifuge tube to obtain a volume of 20ml. Take out 417ul KLH-SMCC solution and transfer it to a 5ml centrifuge tube. Dissolve 3.0 mg of canine BNP polypeptide in 0.6 ml of 1×PBS (pH 7.2) solution. The sulfhydryl group in the polypeptide was detected by Ellman reagent, and the OD value was 0.18. The polypeptide liquid was added dropwise to the KLH-SMCC tube, and mixed with a vertical mixer at room temperature for 4 hours. The OD value was 0.02 detected by Ellman reagen...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention belongs to the technical field of biology. The invention relates to a polypeptide containing two dominant epitopes of canine brain natriuretic peptide (BNP). The immune effect is enhanced by coupling the polypeptide with KLH protein immune mice. The invention also provides a recombinant protein. The recombinant protein comprises two dominant epitopes of canine BNP, and in order to improve the yield of the recombinant protein in a prokaryotic expression system, escherichia coli preferred codons are 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 protein expression vector is constructed. The invention further relates to a phage library established by coupling polypeptide with KLH protein immune mice, a corresponding canine BNP 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, monoclonal antibodies are expressed through transient HEK293F cells, the monoclonal antibodies are purified, and the optimal monoclonal antibody pairing combination is determined through an immunofluorescence orthogonal experiment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel canine brain natriuretic peptide (BNP) recombinant protein. It also involves immunizing mice with polypeptide-coupled KLH protein to establish a phage library, and screens to obtain a specific single-chain antibody scfv sequence, and also involves constructing the obtained scfv sequence into a eukaryotic expression vector to express anti-canine brain natriuretic peptide (BNP) recombinant protein monoclonal antibody, and applied to the early diagnosis of canine heart failure. Background technique [0002] Heart failure (heart failure), also known as cardiac insufficiency, is not an independent disease, and often occurs secondary to other diseases. Severe dysfunctional syndrome in the whole body. The incidence rate is high and the harm is great. It can be divided into two types: acute and chronic. Acute heart failure is often caused by a momentary overload of the...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/26C12N15/13C12N15/85C07K19/00C12N15/70C12R1/19
CPCC07K16/26C07K16/005C12N15/85C07K14/58C07K14/795C12N15/70C07K2317/622C07K2317/56C07K2317/14C12N2800/107C12N2800/208C07K2319/00
Inventor 武戌青曹丹琴陈安琪余铭恩吴琼杉
Owner 杭州贤至生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap