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Nosiheptide resistance ribosomal RNA methyltransgerase, and preparation method and application thereof

A methyltransferase and ribosome technology, applied in the biological field, can solve problems such as antibiotic failure and bacterial drug resistance, and achieve the effect of reducing drug resistance

Inactive Publication Date: 2011-06-29
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The methylated 23S rRNA can no longer be bound by the antibiotic thiostrepton, so the antibiotic is ineffective and the bacteria develop drug resistance

Method used

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  • Nosiheptide resistance ribosomal RNA methyltransgerase, and preparation method and application thereof
  • Nosiheptide resistance ribosomal RNA methyltransgerase, and preparation method and application thereof
  • Nosiheptide resistance ribosomal RNA methyltransgerase, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Discovery of nosiheptide-resistant RNA methyltransferase

[0065] According to the amino acid sequence of the homologous protein thiostrepton drug-resistant RNA methyltransferase, a Blast comparison was performed on the website of the European Center for Biology, and the results showed that there was a highly conserved homology in Streptomyces actuosus protein, accession number AAB17875. Previous studies have shown that the fragment containing this protein can endow the bacteria Streptomyces lividans 1326 with resistance to nosiheptide antibiotics. The present invention named it Nosiheptide Resistance RNA Methyltranferase (NHR). The full-length NHR is a protein containing 274 amino acids with a molecular weight of about 29Kd. It is divided into two parts, N-terminal and C-terminal. domain. Among them, the N-terminal contains 4 α-helices and 4 β-sheets, and forms a sandwich structure responsible for recognizing and binding substrate RNA. The C-terminus cont...

Embodiment 2

[0066] Example 2: Cloning of nosiheptide-resistant RNA methyltransferase gene and construction of prokaryotic expression plasmid pET28A-NHR

[0067] According to the coding sequence of the nosiheptide drug-resistant RNA methyltransferase gene, primers were designed, and PfuDNA polymerase was used to amplify the nosiheptide drug-resistant RNA methyltransferase gene fragment, which was ligated into The pET28A plasmid forms the pET28A-NHR recombinant plasmid, which is transformed into Escherichia coli BL21, the positive clones are screened by enzymatic hydrolysis, and the nucleotide sequence analysis confirms that the gene sequence is the same as the design. It was confirmed that positive clones were obtained. figure 2 The construction procedure of the pET28A-NHR vector is shown.

[0068] Restriction endonucleases were purchased from Takara Company, Escherichia coli BL21, and plasmid pET28A was preserved in our laboratory.

Embodiment 3

[0069] Embodiment 3: the prokaryotic expression of NHR gene

[0070] Pick several positive clonal colonies, shake culture overnight at 30°C in LB medium, then dilute with new LB medium 1:50, shake culture at 30°C until OD600=0.5, add final concentration of 1mmol of IPTG, Expression was induced for 4 hours. After the induction ended, the bacteria were collected by centrifugation, washed twice with PBS (pH=7.4), and the expression product was analyzed by SDS-PAGE. After staining with Coomassie Brilliant Blue R-250, it could be seen that the supernatant after induction was at a molecular weight of 30Kd (considering that the protein There are 6 histidine tags at the end, so the molecular weight of the expressed product is slightly higher).

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Abstract

The invention belongs to the field of biotechnology, and relates to a novel antibiotic resistance methyltransgerase, in particular to gene cloning and protein preparation for a nosiheptide resistance ribosomal RNA methyltransgerase, and use of the nosiheptide resistance ribosomal RNA methyltransgerase as a novel antibiotic screening target. The invention provides a bacterial ribosomal RNA methyltransgerase, which comprises a 1-274 position amino acid sequence shown in a sequence table SEQ ID NO:2; and a nucleotide coding sequence comprises a 1-822 position nucleotide sequence shown in SEQ ID NO:1. By utilizing the characteristic that NHR protein methylates an in-vivo RNA target of a bacterium, the bacterial ribosomal RNA methyltransgerase is used as a medicine target for screening novel antibiotics and medicines for reducing nosiheptide resistance. The invention provides new thinking for research of reducing the nosiheptide resistance.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a novel antibiotic-resistant methyltransferase. More specifically, the present invention relates to gene cloning of nosiheptide-resistant ribosomal RNA methyltransferase, protein preparation, and its application as a screening target for new antibiotics. Background technique [0002] The discovery of antibiotics has brought good news to humans in the effective treatment of various bacterial infections. However, the widespread use and even misuse of antibiotics has resulted in a huge increase in antibiotic resistance, causing a large number of antibiotics to lose their effect. According to statistics, the number of people who die from various bacterial infections in the world every year is 8 times higher than that of ten years ago. Therefore, it is very important to discover new antibiotics, and the key point is to find new targets of antibiotics. [0003] Ribosome is the place where b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N1/21C12N15/70C12Q1/48C12Q1/68C12R1/465C12R1/19
Inventor 王哲阿拉斯太尔・马歇尔陈东戎沈妍
Owner FUDAN UNIV
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