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Isovalerylspiramycin I component high-content high-yield genetic engineering bacteria

By introducing the acyB2 regulatory gene into the isovalerylspiramycin-producing bacteria to activate ist gene expression, a strain with high content and high yield of isovalerylspiramycin I was constructed, which solved the problem of difficult control of the extraction and purification process of multi-component antibiotics. It simplifies quality control and testing, is suitable for the preparation of injections, and improves clinical treatment effects.

Active Publication Date: 2010-12-15
SHENYANG TONGLIAN GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fermentation unit of this strain is very low, which is not conducive to large-scale production

Method used

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  • Isovalerylspiramycin I component high-content high-yield genetic engineering bacteria
  • Isovalerylspiramycin I component high-content high-yield genetic engineering bacteria
  • Isovalerylspiramycin I component high-content high-yield genetic engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] "Example 1" Constructing the recombinant plasmid pSET52-ia containing ist-acyB2 double genes

[0030] From the recombinant plasmid pSW4 originally constructed in our laboratory [Shang Guangdong et al., Bioengineering 1999, 15(2): 171], digested with SmaI-BamHI to obtain a 1820bp fragment containing the complete ist gene and part of the acyB2 gene, and obtained from China The total DNA of Streptomyces thermotolarences CGMCC4.1501 provided by the General Microbiology Center of the Culture Collection of Microorganisms was used as a template, and primers p1 were designed according to the sequence of acyB2 published by NCBI: 5'-CGCTCAGGGACGCAAGACC-3' and

[0031] P2: 5'-CC GGAATTCGCCCCGTGACCTCACCGTC-3'(EcoRI), a 1013bp fragment of the cyB2 partial gene was obtained by PCR reaction, the PCR product was digested with BamHI-EcoRI to obtain a 934bp fragment, and the above SmaI-BamHI (1820bp), BamHI-EcoRI (934bp) fragments were ligated into The SmaI-EcoRI restriction site of pB...

Embodiment 2

[0032] 《Example 2》Recombinant plasmid pSET52-ia transforms isovalerylspiramycin I producing bacteria

[0033] Isovalerylspiramycin I-producing strain WSJ-2 was constructed in our laboratory and published in [Chunyan Ma et al. Current Microbiology 2010, DOI 10.1007 / s00284-010-9664-8]. Bacteria in slant medium [2.0% soybean cake powder, 1.0% glucose, 3.0% starch, CaCO 3 0.5%, NaCl 0.4%, agar 2.0%] and cultured at 28°C for 7-10 days, prepared protoplasts according to the method described in the literature [Genetic manipulation of Streptomyces such as D.A.Hopwood, A Laboratory Manual, Norwich; John Innes Foundation UK, 1985], Specifically, inoculate strains into sucrose-containing R2YE medium [10.3% sucrose, 1.0% glucose, 0.4% yeast extract, 0.2% tryptone, 0.4% peptone, 0.4% casamino acid, K 2 SO 4 0.025%, CaCl 2 0.216%, KH 2 PO 4 0.005%, MgCl 2 6H2O1.012%, add NaOH (1mol / L) 0.5ml, Tris-HCl (0.25mol / LpH7.2) 10ml, trace element solution [ZnCl 2 0.04%, FeCl 3 6H2O 0.02%...

Embodiment 3

[0034] "Example 3" WSJ-IA strain fermentation titer detection

[0035] WSJ-IA strains were cultured in the above-mentioned slant medium at 28°C for 7-10 days, and inoculated into the seed medium by excavating pieces: soybean cake powder 1.5%, starch 3.0%, NaCl 0.4%, CaCO 3 0.5%, fish peptone 0.3%, KH 2 PO 4 0.05%. Cultivate at 28°C for 48 hours, transfer to fermentation medium formula: glucose 0.5%, starch 6.0%, yeast powder 0.5%, fish meal 2.0%, NH 4 NO 3 0.6%, NaCl 1.0%, CaCO 3 0.5%, KH 2 PO 4 0.05%, MgSO 4 0.1%. Fermentation at 28°C for 96 hours. The fermented liquid was centrifuged to take the supernatant, which was diluted with phosphate buffer solution of pH 7.8-8.0 containing 3% NaCl. Bacillus [CMCC (B) 63501] was used as the test bacteria, and the medium [peptone 0.6%, beef extract powder 0.15%, yeast extract extract 0.6%, glucose 0.2%, agar 1.5-2.0%], according to the Chinese Pharmacopoeia 2010 version II Part Appendix XI A described standard curve met...

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Abstract

The invention relates to an antibiotic single-component high-content high-yield strain constructed and obtained by using regulator gene technology, in particular an iIsovalerylspiramycin I component high-content high-yield genetic engineering bacterial strain obtained by transferring ist-acyB2-containing digenic recombinant plasmids pSET52-ia into iIsovalerylspiramycin I producing bacteria. Thus, better conditions are created for the industrial production and preparation of injections.

Description

Technical field: [0001] The invention relates to the construction and acquisition of antibiotic single component high-content and high-yield strains by means of regulatory gene technology. Background technique: [0002] Bitespiramycin (formerly known as Shengjimycin) [patent number: ZL97104440.6] is a spiramycin derivative developed by genetic engineering technology. Bitspiramycin has strong activity against Gram-positive bacteria, especially against Streptococcus pneumoniae, Mycoplasma pneumoniae, and Chlamydia, and against erythromycin and β-lactam antibiotic-resistant bacteria, influenza bacillus, and Legionella , Clostridium perfringens has antibacterial activity; there is no complete cross-resistance with similar drugs. It has high lipophilicity, rapid oral absorption, strong tissue permeability, wide distribution, long maintenance time in vivo, and good post-antibiotic effect. At present, the Phase III clinical trial of genetically engineered Bitespiramycin has been ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/63C12R1/465
CPCC12R1/465C12P19/62C07K14/36C12N1/20C12N9/1029Y02A50/30C12N15/03C12N15/63C12N15/76C12N1/205C12R2001/465
Inventor 王以光武临专姜洋郝玉有杨生武林灵周红霞戴剑漉赫卫清马春燕
Owner SHENYANG TONGLIAN GRP CO LTD
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