Shuttle plasmid and method for efficiently expressing cecropin and lysozyme genes

A shuttle plasmid, high-efficiency expression technology, applied in genetic engineering, plant genetic improvement, chemical instruments and methods, etc., can solve the problems of low expression amount, high cost, and inability to large-scale production of bactericidal peptides

Active Publication Date: 2010-12-01
SHANGHAI E K M BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The object of the present invention is to provide a shuttle plasmid and a method for efficiently expressing cecropin and lysozyme genes. The shuttle plasmid and the method for efficiently expre...

Method used

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  • Shuttle plasmid and method for efficiently expressing cecropin and lysozyme genes
  • Shuttle plasmid and method for efficiently expressing cecropin and lysozyme genes
  • Shuttle plasmid and method for efficiently expressing cecropin and lysozyme genes

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Embodiment 1

[0040] The construction of Escherichia coli and Bacillus subtilis shuttle plasmids is as follows: figure 1 As shown, the Escherichia coli pUC18 plasmid and the Staphylococcus aureus pE194 plasmid were respectively digested with PstI, and the enzyme digestion system was 20uL: ddH 2 O 7uL, 10xH buffer 2uL, PstI 1uL, pUC18 or pE194 10uL, digestion overnight at 37°C. Enzyme digestion was completed by 1.0% agarose gel electrophoresis, and the corresponding fragments were recovered and purified. Then in 20uL system (ddH 2 O 5.5uL, T4 ligation buffer 2uL, T4 ligase 0.5uL, pUC18 and pE194 each 6uL) for T4 DNA ligase ligation at 16°C overnight, CaCl 2 Transform competent Escherichia coli DE3 by this method, after the transformation solution is absorbed by the solid medium, culture it upside down in a 37°C incubator overnight, transformants grown on the Amp+ plate are transferred and stored, and the plasmid is extracted to identify the size of the increased molecular weight and deter...

Embodiment 2

[0050] The specific construction method of the shuttle plasmid is as in Example 1. According to the human lysozyme gene sequence published by Agric Biol Chem, 1986, 50(3): 713-723, etc., the human lysozyme gene was chemically synthesized, and all codons were replaced with preferred codons suitable for expression in prokaryotes. Human lysozyme gene NheI and KpnI enzyme cutting sites are added to the two ends. Human lysozyme gene and shuttle plasmid were digested with NheI and KpnI. Gene 15uL (or shuttle plasmid 8uL), medium salt 2uL, NheI and KpnI each 0.8uL, make up to a total volume of 20uL with water. Digestion at 37°C for 4 hours, 1% agarose electrophoresis, recovery of gene and vector fragments and purification. The connection system is 20uL (ddH 2 (05.5uL, T4 ligation buffer 2uL, T4 ligase 0.5uL, human lysozyme gene 8uL, vector 4uL), T4 DNA ligase ligation overnight at 16°C on a PCR instrument, CaCl 2 Transform competent Escherichia coli DE3 by using the method. After...

Embodiment 3

[0055]The specific construction method of the shuttle plasmid is as in Example 1, and the expression, purification, and lysostaphin digestion of the fused shuttle plasmid are as in Example 2. A 15-amino acid peptide segment GlyTrpLysCysArgArgArgTrpGlnTrpArgTrpLys Lys Leu Gly with high-efficiency bactericidal function composed of the 17th to 32nd amino acids of bovine lactoferrin is fused in the human lysozyme gene. The 45 bp sequence of bovine lactoferrin is fused to the C-terminus of the human lysozyme gene DNA molecular sequence, and the 17th amino acid is mutated into Gly, and the 18th and 26th amino acids are mutated into Trp. In order to effectively cut the activated bovine lactoferrin active peptide after the fusion protein is expressed, its sequence is mutated and modified, and the cleavage recognition site sequence of lyso(staphylococcus) enzyme is added, and all amino acids use prokaryote preferred codons. The base sequence of the fusion protein constructed by human l...

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Abstract

The invention belongs to the field of bio-genetic engineering and provides a shuttle plasmid and a method for efficiently expressing cecropin and lysozyme genes. The invention constructs an escherichia coli-bacillus subtilis shuttle plasmid suitable for being expressed in prokaryotes (escherichia coli, bacillus subtilis and bacillus sphaericus). Cecropins expressed in the prokaryotes does not have a toxic action on hosts. The shuttle plasmid can successfully express the fusion protein of various cecropins such as human lysozyme, bovine lactoferrin, and the like. 6His is introduced into the expression plasmid constructed by the invention, which is convenient to purify, and the purified fusion protein identifies and correctly cut enzyme cutting sites among all fusion peptide segments through lysostaphin so as to release all functional peptides and enable the functional peptides to recover the activity; and moreover, enzyme separation and cutting are also saved, thus the method can be effectively, simply and conveniently used. The shuttle plasmid also has a certain common availability, and all suitable genes can be cloned and expressed.

Description

Technical field: [0001] The invention relates to the field of bioengineering, in particular to a shuttle plasmid and a method for efficiently expressing cecropin and lysozyme genes. Background technique: [0002] Lysozyme widely exists in various organs, tissues, serum or saliva of organisms, and is found in organisms such as animals, plants, and bacteriophages. So far, there are hundreds of related reports such as papaya lysozyme, mouse-derived lysozyme, dog-derived lysozyme, bird lysozyme, T4 lysozyme, P22 lysozyme, egg white lysozyme, etc. Among them, the content of lysozyme in egg white is particularly rich, accounting for 3.4% to 3.5% of the total protein of egg white, and the research articles are the most. Egg white lysozyme is an alkaline protein with a molecular weight of 14,000 and an optimum pH of 6 to 7. It has the characteristics of heat resistance and acid resistance. For example, at pH 3.0, lysozyme still has more than 85% of its activity when heated at 96°C...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/56C12N15/62C12N9/36C07K19/00C07K14/435A61K38/47A61P31/00A61K38/17
Inventor 张培德黄易明
Owner SHANGHAI E K M BIOTECH
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