Leech tyrosine sulfonate transferase gene and application thereof

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: 2021-12-14
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • 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

Method used

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  • Leech tyrosine sulfonate transferase gene and application thereof
  • Leech tyrosine sulfonate transferase gene and application thereof
  • Leech tyrosine sulfonate transferase gene and application thereof

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,...

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Abstract

The invention discloses a leech tyrosine sulphonate transferase gene and application thereof, and belongs to the field of biomedicine. The leech tyrosine sulphonate transferase gene is characterized in that on the basis of a hirudo nipponia transcriptome database, a leech tyrosine sulphonate transferase cDNA sequence is excavated and identified through a bioinformatics means, and on this basis, the leech tyrosine sulfotransferase is synthesized through codon optimization, gene synthesis and microbial cell expression, and the recombinant hirudin is catalyzed by the leech tyrosine sulfotransferase to be subjected to sulfonation modification to obtain the natural hirudin. The catalytic ability of the transferase is greatly improved compared with that of acyl sulfonate transferase, human-derived tyrosine sulfonate transferase and bovine-derived tyrosine sulfonate transferase. Compared with other tyrosine sulfonic acid transferase, the leech tyrosine sulfonic acid transferase obtained in the invention has stronger substrate preference for recombinant hirudin, is suitable for large-scale preparation of natural hirudin, and provides a new way for synthesis of natural hirudin.

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

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Application Information

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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
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