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

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...

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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

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