Genetic engineering expression and preparation methods of cecropin LL-37 and application thereof

A LL-37, genetic engineering technology, applied in the field of genetic engineering, can solve the problems of unmentioned yield, complicated cutting process, difficult recovery and purification, etc., and achieve the effects of low sterilization concentration MIC, good sterilization effect and cost reduction.

Inactive Publication Date: 2013-10-23
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, yeast fermentation takes 5 days and 5 nights, and the amount of small peptides secreted into the fermentation broth is small, so it is very difficult to recover and purify from a large amount of fermentation broth
In 2002, the Shanghai Bioengineering Research Center of the Chinese Academy of Sciences reported that Pichia pastoris was used to express the bactericidal peptide magainin, but the yield and recovery method of the target polypeptide were not mentioned in the report
The cutting process of other methods is complex, and cutting errors are common, so the yield of the target peptide is often low
2001 U.S. patent prepared buforin II by Escherichia coli using cyanogen bromide to cut off the fusion protein, the yield was not mentioned in the report
In 2010, Portugal reported the production of LL-37 fusion protein in Escherichia coli to avoid the use of cyanogen bromide and formic acid to cut off and purify. The downstream process is not complicated, but the yield is only 1mg / L
None of the above research has been transformed into industrial production

Method used

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  • Genetic engineering expression and preparation methods of cecropin LL-37 and application thereof
  • Genetic engineering expression and preparation methods of cecropin LL-37 and application thereof
  • Genetic engineering expression and preparation methods of cecropin LL-37 and application thereof

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Effect test

Embodiment 1

[0039] According to the amino acid sequence of LL-37, a DNA gene fragment is designed and synthesized, and the genetically engineered bacterium is a genetically engineered bacterium (E. Coli.). For the carrier of synthetic genetically engineered bacteria (LL-37), the present embodiment amplifies and connects the codon containing Met-LL-37 by conventional PCR method, and clones it into the Lac plasmid, and the enzyme cutting sites are BamH I and SalI, The fusion protein gene is formed, the expression plasmid is constructed, and the positive recombinant plasmid is confirmed by PCR. The positive recombinant plasmid was transfected into Escherichia coli E.Coli.JM109 to construct LL-37 genetically engineered bacteria. see figure 1 .

[0040] The single-stranded DNA structure SEQ ID in the expression vector of LL-37 genetically engineered bacteria:

[0041] -CTG-CTG-GGT-GAT-TTC-TTC-CGT-AAA-AGC-AAA-GAA-AAA-ATC-GGT-AAA-GAA-TTC-AAA-CGT-ATC-GTT-CAG-CGT-ATC-AAA - GAT-TTC-CTG-CGT-AAC-...

Embodiment 2

[0058] 1: Construction of expression plasmids

[0059] The genetically engineered bacterium is a genetically engineered bacterium (LL-37).

[0060] In order to synthesize the carrier of genetically engineered bacteria (LL-37), we amplified and joined the codons containing Met-LL-37 by conventional PCR method, and cloned it into the Lac (pUC18) plasmid, and the restriction sites were BamH I and Sal 1, form the fusion protein gene, construct the plasmid, and confirm the positive recombinant plasmid with the method of PCR. The positive recombinant plasmid was transfected into Escherichia coli E.Coli.JM19 to construct genetically engineered bacteria.

[0061] 2: fermentation

[0062] 250ml of seed culture solution (1000ml of seed culture solution contains 10g of peptone, 5g of yeast extract, 20ml of 0.02mol / L phosphate buffer, pH7.0) in a 1000ml Erlenmeyer flask, sterilized at 120°C for 20 minutes, cooled and then added 20% glucose solution 5ml. Add 1ml of the strain stored in...

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Abstract

The invention relates to genetic engineering expression and preparation methods of cecropin LL-37 and application thereof. The preparation method comprises the steps of synthesizing a DNA (Deoxyribonucleic Acid) sequence coding the cecropin LL-37, and connecting the DNA sequence to a suitable expression vector for expression and purification, wherein the DNA sequence coding the cecropin LL-37 adopts Escherichia coli preferred codons, and the LL-37 expressed in an Escherichia coli fusion and expression system accounts for about 20% of the total protein of a thallus; and cutting off the purified fusion protein by using cyanogen bromide so as to obtain the target polypeptide LL-37, and obtaining LL-37 powder with the purity higher than 90% through HPLC (High-Performance Liquid Chromatography). The LL-37 of low concentration can play a significant bactericidal effect in the aspect of human preparations for external use, serves as a drug for external use and is applied to the treatment of diseases, such as skin infection. The methods have the advantages that the cecropin LL-37 is expressed and prepared in the form of fusion protein, the process is simple, and the yield of fermentation liquor per cubic liter can reach 20 mg, so that the methods have significance in industrial batch production.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene engineering expression method of cecropin LL-37, a preparation method thereof and an application of cecropin LL-37 in skin infection. Background technique [0002] Since Austrian scientists first reported it in 1909, and German scientists were the first to apply the synthetic sulfa drug Prontosil in 1932, the research and development of antibacterial drugs has occupied an important position in the entire field of drug research and development, and the struggle between humans and pathogens once had the upper hand . However, with the widespread use of antibiotics, the abuse of antibiotics, the accompanying bacterial resistance, and the side effects of antibiotics have gradually become serious problems for human beings. The high frequency of multidrug-resistant bacteria (multiple drug-resistant, MDR), extremely drug-resistant bacteria (extensively dru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/70C12N1/21C07K14/00A61K38/16A61P31/04A61P31/10
Inventor 吴晓琰陆怡龚铁军
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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