Antigen peptide of DNA methyltransferase 1 and polyclonal antibody thereof

A polyclonal antibody, methyltransferase technology, applied in the direction of transferase, genetic engineering, plant genetic improvement, etc., can solve the problems of low specificity and complex analysis process.

Pending Publication Date: 2022-04-15
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
View PDF8 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above detection methods all use fluorescent dyes for the quantitative analysis of DNA methyltransferase 1, but have the disadvantages of low specificity and compl

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
  • Antigen peptide of DNA methyltransferase 1 and polyclonal antibody thereof
  • Antigen peptide of DNA methyltransferase 1 and polyclonal antibody thereof
  • Antigen peptide of DNA methyltransferase 1 and polyclonal antibody thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 Amplification of pig DNMT1 gene and construction of eukaryotic expression vector

[0031] Primers were designed according to the predicted sequence of the porcine DNMT1 gene (XM_021082029.1) in GenBank, the RNA was reverse-transcribed into cDNA, and the cDNA was used as a template for PCR amplification. The size of the fragment band was consistent with the expected size.

[0032] The pCMV-Flag14 plasmid and DNMT1 gene were double digested and ligated using BamHI and HindIII respectively, and then verified by sequencing that the pFlag14-DNMT1 (cloned) recombinant plasmid had been successfully constructed, wherein the amino acid sequence of the predicted sequence of the porcine DNMT1 gene As shown in SEQ ID NO.3, its nucleotide sequence is shown in SEQ ID NO.4. figure 1 It is the porcine DNMT1 gene identification result (5102bp) that utilizes RT-PCR method to amplify from alveolar macrophage in the example of the present invention; Wherein, M: DNA Maker; Swim...

Embodiment 2

[0033] Example 2 Preparation of porcine DNMT1 polyclonal antibody

[0034] As shown in SEQ ID NO.1, the polypeptide fragment with the amino acid sequence of SSPVKRPRKEPVDED was selected as the antigenic peptide of the polyclonal antibody, coupled to the KLH carrier protein for purification, and then the antigenic peptide was diluted with physiological saline, and the ratio was 1:1 Mix and emulsify with Freund's adjuvant to immunize New Zealand white rabbits. Before the formal immunization, venous blood collection is required as a negative control. The immunization method is subcutaneous injection or intramuscular injection. The total amount of antigen is 500ug. Complete adjuvant booster immunization once, 7 days after immunization six times to obtain polyantiserum by venous or cardiac blood collection. The titer of the polyclonal antibody determined by the indirect ELISA method is greater than 1:50k, which can ensure that the polyclonal antibody can be used for Western Blot de...

Embodiment 3

[0035] The specificity analysis of embodiment 3 polyclonal antibody

[0036]In order to verify whether the obtained DNMT1 protein synthetic peptide polyclonal antibody can be used in subsequent Western Blot experiments, the obtained DNMT1 protein synthetic peptide polyclonal antibody was used to detect the overexpression of pFlag-DNMT1 (cloned) plasmid in HEK 293T cells, by using Mouse anti-Flag antibody and rabbit anti-DNMT1 polyclonal antibody were used as primary antibodies for detection, figure 2 It is the protein result chart expressed by the recombinant plasmid pFlag14-DNMT1 (cloned), such as image 3 As shown, both antibodies were able to display the overexpressed DNMT1 protein band, and the expected size was consistent.

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 particularly relates to an antigen peptide of DNA methyltransferase 1 and a polyclonal antibody of the antigen peptide, and belongs to the technical field of molecular cloning. According to a predicted pig DNMT1 sequence published by NCBI (National Center of Biotechnology Information), a pig DNMT1 gene is obtained through RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification, a pFlag14-DNMT1 (Cloned) recombinant plasmid is constructed by utilizing an eukaryotic expression system, analysis is started from the pig DNMT1 protein sequence, and the pig DNMT1 protein synthetic peptide polyclonal antibody meeting the subsequent detection requirement can be constructed. The polyclonal antibody disclosed by the invention is high in detection sensitivity and strong in specificity, and is suitable for rapid detection and related research of DNMT1 proteins from human, pigs, monkeys, cattle and dogs.

Description

technical field [0001] The invention belongs to the technical field of molecular cloning, and in particular relates to an antigenic peptide of DNA methyltransferase 1 and a polyclonal antibody thereof, a preparation method and application thereof. Background technique [0002] DNA methyltransferase 1 (DNA methyltransferase 1, DNMT1) is a key gene for DNA methylation in mammalian genome epigenetic modification, and its encoded protein is a large molecular weight and complex functional enzyme with multiple regulatory functions , participate in multiple biological processes such as stem cell growth, cell proliferation, organ development, aging and tumorigenesis during body development. Once the Dnmt activity in the organism changes, it will cause abnormal methylation level of the genome, which will increase the local methylation level of the 5'-CG-3' nucleotide island and reduce the overall methylation level of the genome, resulting in abnormalities in the genome. Stability, s...

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
IPC IPC(8): C12N9/10C12N15/54C07K16/40G01N33/573
Inventor 邱亚峰马志永朱琳魏建超刘珂李宗杰李蓓蓓邵东华
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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