Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of leech tyrosine sulfonate transferase gene and its application

A tyrosine sulfonic acid and transferase technology, applied in the field of biomedicine, can solve the problems of hindering the biosynthesis of hirudin in the analysis of natural hirudin, and the analysis work is stagnant, and achieves improved anticoagulant activity and strong substrate preference. sexual effect

Active Publication Date: 2022-03-15
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] With the development of genome sequencing technology, transcriptome sequencing technology, synthetic biology technology and protein characterization technology, a variety of leech source genome and transcriptome data have been published, but the related analysis work has been stagnant, which seriously hinders the development of hirudin sulphur. Analysis of Acidification Modification Pathway and Biosynthesis of Natural Hirudin

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
  • A kind of leech tyrosine sulfonate transferase gene and its application
  • A kind of leech tyrosine sulfonate transferase gene and its application
  • A kind of leech tyrosine sulfonate transferase gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Mining leech tyrosine sulfonate transferase gene based on the leech transcriptome database

[0031] 1. Mining of candidate leech tyrosine sulfonate transferase genes

[0032] Through the Blastx analysis of Japanese leech genome data, the candidate gene fragment of leech tyrosine sulfonyltransferase EN-133k-group2100.Contig1 was mined. ORF program (http: / / www.ncbi.nlm.nih.gov / orffinder) was used to analyze the open reading frame of the candidate gene, and Protein Blast was used to perform homologous comparison of the database. After comparison, the sequence SEQ ID NO. 3 was determined to be The nucleotide sequence of alternative leech tyrosine sulfonate transferase, SEQ ID NO. 1 is the amino acid sequence of alternative leech tyrosine sulfonate transferase.

[0033] 2. Construction of leech cDNA library

[0034] After the Japanese medical leech was fed with fresh duck blood for 2 h, the above leech was quick-frozen in liquid nitrogen. After grinding with liq...

Embodiment 2

[0037] Example 2 Preparation of leech tyrosine sulfonate transferase by using microbial cells

[0038] 1. Preparation of leech tyrosine sulfonate transferase by recombinant Escherichia coli

[0039] In view of the codon preference of microbial cells when expressing proteins, in order to obtain high-efficiency expression of the leech tyrosine sulfonate transferase gene, we artificially synthesized the codon-optimized leech tyrosine sulfonate transferase gene TPS , assembled by Gibbson, integrated into pET28a (+) by homologous recombination Nde I and not I site, construct the recombinant expression vector pET28a(+)- TPS ,in TPS The sequence is shown in SEQ. ID NO. 2. Transformation using the above-mentioned recombinant expression vector E . coli BL21 Obtaining Escherichia coli Recombinant Expression Strain of Leech Tyrosine Sulfonyltransferase E . coli BL21 pET28a(+)- TPS , for the subsequent preparation of leech tyrosine sulfonyltransferase ( image 3 in A...

Embodiment 3

[0044] Example 3 Establishment of leech tyrosine sulfonate transferase catalytic system

[0045] 1. Purification of leech tyrosine sulfonyltransferase

[0046] After equilibrating the Ni-NTAAgarose Fast Flow column with Buffer A (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 7.4), permeate the leech tyrosine sulfotransferase crude enzyme solution 1 and 2 respectively through 0.22 μm After filtration, the supernatant was passed through the Ni-NTA Agarose Fast Flow column at a rate of 3 mL / min. After binding for 10 min, Buffer B (20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, pH 7.4) was used to elute Enzyme active fractions were collected, concentrated, and freeze-dried.

[0047] 2. Preparation of natural hirudin

[0048] Using recombinant hirudin 1 (Sigma) or recombinant hirudin 2 (Sigma) as raw materials, the reaction solution includes 1-200 mMTris-HCl (pH5.0), 0.1-50 mM mM PAPS, 0.1-100 μM leech tyrosine sulfonate Acid transferase, 0.1-50 mM recombinant hirudin, 10-50°C,...

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 discloses a leech tyrosine sulfonate transferase gene and its application, and belongs to the field of biomedicine. The invention is based on the Japanese medical leech transcriptome database, and excavates and identifies leech tyrosine sulfonate by means of bioinformatics On the basis of the transferase cDNA sequence, hirudin tyrosine sulfonate transferase is synthesized through codon optimization, gene synthesis, and microbial cell expression, and the enzyme is used to catalyze the sulfonation modification of recombinant hirudin into natural hirudin. Its catalytic ability is greatly improved compared with acyl sulfonate transferase, human tyrosine sulfonate transferase and bovine source tyrosine sulfonate transferase. The leech tyrosine sulfonate transferase obtained in the present application has stronger substrate preference for recombinant hirudin than other tyrosine sulfonate transferases, and is suitable for large-scale preparation of natural hirudin. The synthesis provides a new way.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a leech tyrosine sulfonate transferase gene and its application, in particular to a method for excavating, expressing and sulfonating the recombinant hirudin of the leech tyrosine sulfonate transferase gene . Background technique [0002] According to the statistics of the World Health Organization, about 18 million people die from cardiovascular diseases every year, accounting for 31% of the global death toll. In order to curb and reduce the mortality rate of cardiovascular diseases, it is necessary to actively search for and develop drugs for the treatment of cardiovascular diseases. Natural hirudin (Sulfo-hirudin) is the strongest thrombin inhibitor discovered so far, and it is widely used clinically in the treatment of cardiovascular diseases. However, as a small molecular active peptide in leeches, the isolation and purification of natural hirudin is extremely difficult....

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 Patents(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/70C12N15/81C12N1/21C12N1/19C12P21/02C12R1/19C12R1/84
CPCC12N9/13C12N15/70C12N15/815C12P21/02C07K14/815C12Y208/0202C12N2800/22C12N2800/101C12N2800/102
Inventor 周正雄汪仁曾道平高萌徐晟李洁孙彬周佳宇
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com