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Medusa cystatin gene, and expression and use thereof

A technology of cysteine ​​protease and inhibitor, applied in the field of new genes

Inactive Publication Date: 2003-10-29
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] At present, there are no research reports on jellyfish cysteine ​​protease inhibitors at home and abroad. The unique jellyfish resources in my country have been studied, and new jellyfish cysteine ​​protease inhibitors with application prospects have been found. Gene recombination, expression, Site-directed mutagenesis, structure and function research are of great significance

Method used

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  • Medusa cystatin gene, and expression and use thereof
  • Medusa cystatin gene, and expression and use thereof
  • Medusa cystatin gene, and expression and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Construction of the jellyfish tentacles cDNA library

[0052] The extraction of total RNA from jellyfish tentacles refers to TRIZOL from Gibcol BRL  LS reagent instructions; cDNA synthesis according to Clontech company SMART TM cDNALibrary Construction Kit manual operation.

[0053] Using TRIZOL  The tentacle total RNA extracted by LS reagent was detected by 1% formaldehyde denaturing gel electrophoresis as follows: figure 1 The clear 28S and 18S rRNA bands indicated that the integrity of the total RNA was good. Adopt SMART TM The cDNA synthesized by the cDNA Library Construction Kit was electrophoresed on a 1% agarose gel, and the result showed a uniform distribution, such as figure 2 As shown, the size ranged from 200bp to 5kb, mainly concentrated in the region below 2kb, indicating that the integrity of the cDNA was good. The cDNA was inserted into the plasmid vector pcDNA3 to construct a cDNA library, and the number of library clones was 5.1×10 ...

Embodiment 2

[0054] Example 2 Cloning, sequencing and analysis of jellyfish tentacles cDNA library

[0055] Select the clones of the jellyfish tentacles cDNA library, and extract the plasmid DNA according to the method of Vitagene 96-easyplasmid Mini-prep Kit. to T 7 The universal primers are sequencing primers, and the 5' ends of 2153 random cDNA sequences were sequenced using the automatic sequencer ABI Prism 3700 sequencer (Perkin-Elmer). The obtained sequences were clustered, carrier sequences removed, and Blast X analyzed. The results showed that the jellyfish tentacles cDNA library contained a variety of genes, among which the abundance of cysteine ​​protease inhibitor genes was the highest, with 68 cDNA sequences named as Cystatin J, the jellyfish cysteine ​​protease inhibitor gene encodes a protein of 131 amino acids, as shown in the sequence table, which includes a signal peptide of 18 amino acid residues and a mature protein of 113 amino acid residues, and the mature protein has...

Embodiment 3

[0056] Example 3 Construction of Recombinant Jellyfish Cysteine ​​Protease Inhibitor Expression Plasmid

[0057] According to the sequence at both ends of the mature protein encoded by the Cystatin J gene and the multi-restriction site of the prokaryotic fusion expression vector pETTrx, a pair of primers were synthesized with the following sequence: upstream primer, 5'GCT GAA TTC GGT ACC CTACTT CCT GGC GGA ATA AGA CG 3' single underlined part is Kpn I restriction sequence, double underlined part is enterokinase restriction sequence downstream primer, 5'CGT GCG GCC GC AGC TCG GAG GCA TTTTGI 3' The single underline part is the Not I restriction sequence, and the double underline is the stop codon

[0058] PCR amplification and gene cloning were carried out according to conventional methods. The PCR product is about 450bp such as Image 6 As shown, encoding 113 amino acid residues. The target gene was cloned into the prokaryotic fusion expression vector pETTrx to constr...

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Abstract

A cystatin gene of meduse, its expression, and its use in suppressing the hydrolysis of protease are disclosed. The protein coded by said gene is a precursor protein composed of 131 amino acids, including the signal peptide containing 18 amino acid and the mature protein containing 113 amino acid. The purified recombinant protein has obvious suppression action on papain.

Description

technical field [0001] The invention relates to a new gene, especially a jellyfish cysteine ​​protease inhibitor gene; the invention also relates to the expression of the gene and the inhibitory effect of the encoded protein on papain. Background technique [0002] Protease inhibitors are a kind of water molecular protein that has inhibitory activity on proteolytic enzymes, and are ubiquitous in plants, animals and microorganisms. In vertebrates, protease inhibitors are an integral part of the immune system and act by combining their active centers with proteases to form stable complexes. There are few studies on protease inhibitors in invertebrates. It is worth noting that there is no immune system in the body, so the role of protease inhibitors cannot be ignored. Protease inhibitors are classified according to the type of protease they act on, and are mainly divided into: serine protease inhibitors that inhibit trypsin, chymotrypsin, etc.; cysteine ​​protease inhibitors t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/57A61P25/00A61P31/00A61P35/00A61P43/00C07H21/04C12N15/12C12N15/63
Inventor 徐安龙杨彦臻彭立胜于萃玲卫剑文杨文利
Owner SUN YAT SEN UNIV
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