Tylosin gene engineering strain and application thereof

A technology of genetically engineered bacteria and tylosin, which is applied in the field of genetic engineering and bio-fermentation industry, can solve the problem that tylosin products do not meet the standards of Veterinary Pharmacopoeia, the content of component A of tylosin is low, and the content of component C is not Easy transformation and other problems, to achieve the effect of reducing the difficulty of separation and purification, reducing production costs, and long breeding cycle

Inactive Publication Date: 2012-09-26
宁夏泰瑞制药股份有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

Although there is already large-scale production of tylosin in China, the ability of the strain to produce toxins is low, the fermentation level of the strain is 11000-12000u/ml, and the content of tylosin A component is 80%-85%
Strain selection is still at the traditional breeding level, and the yi...

Method used

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  • Tylosin gene engineering strain and application thereof
  • Tylosin gene engineering strain and application thereof
  • Tylosin gene engineering strain and application thereof

Examples

Experimental program
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Effect test

Embodiment Construction

[0031] Construction of Genetic Engineering Bacteria TL-209

[0032] Design primers to PCR clone TylF gene and VHb gene respectively

[0033] PT1: 5'-GTGGCACCTTCCCCGGACCACGCC-3'

[0034] PT2: 5'-TCAGCCGCTGTGCCGCCAATAGG-3'

[0035] PV3: 5'-CGGGATCCATATGCTGGACCAGCAAACCAT-3'

[0036] PV4: 5'-CGGGATCCTTTATTCAACCGCTTGAT-3'

[0037] The PCR product was recovered from agarose gel, and then double-digested with the expression vector pFC119. After recovery, TylF-VHb and pFC119 were ligated with T4 DNA ligase at 16°C for 2 hours to construct a recombinant expression vector pFC120 (such as figure 2 ). Then pour into Escherichia coli ET12567 competent cells, activate and culture in LB medium for 1 hour, spread on the ampicillin-resistant plate, pick the transformant, extract the plasmid for verification, and then transfer the recombinant expression vector pFC120 Introduce Streptomyces flexneri.

[0038] Specific steps are as follows:

[0039] (1) Pick ET12567 / pUZ8002 monoclonal and...

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Abstract

The invention relates to a tylosin gene engineering strain. The construction method for the tylosin gene engineering strain comprises the following steps: cloning tylosin through polymerase chain reaction (PCR) to synthesize crucial rate-limiting enzyme gene TylF and Vitreoscilla hemoglobin VHb gene, constructing a recombinant plasmid by using an E. coli-streptomyces shuttle vector, importing the recombinant plasmid into Escherichia coli ET12567/pUZ8002, performing conjugal transfer culture on the Escherichia coli ET12567/pUZ8002 and streptomyces fradiae spores, converting streptomyces, screening a converter through apramycin resistance, further confirming the converter by using PCR, and finally obtaining the gene engineering strain. The tylosin gene engineering strain can be directly used in the industrial production of tylosin; and compared with the tylosin gene engineering strain used in the conventional industrial production, the tylosin gene engineering strain has the advantages that the yield of the tylosin can be obviously improved to be 17,000-18,000u/ml and the content of a component A in the tylosin is effectively improved to be 94-95 percent.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and biological fermentation industry, and in particular relates to a tylosin genetically engineered bacterium and its application. Background technique [0002] Tylosin is a macrolide antibiotic produced by the secondary metabolism of Streptomyces fradiae, Streptomyces romosus and Streptomyces hygroscopicus, among which Streptomyces fradiae is the main producer of tylosin. Tylosin is mainly composed of four components: tylosin A, desmycosin (desmycosin) B, macrocin (macrocin) C and relomycin (relomycin) D, among which Tylosin Mycocin A is the main component and has the highest biological activity. [0003] The main structure of Tylosin A is a cyclic lactone formed by 16 atoms, that is: Tylactone (Tylactone), and then connected to three deoxyaglycones in sequence, and the connection sequence of the three deoxyaglycones For: Mycaminose (Mycaminose), 5′-deoxy-D-allose (Mycinose), Mycar...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/76C12P19/62C12R1/54
Inventor 王义任勇刘建成王文超徐淑芬
Owner 宁夏泰瑞制药股份有限公司
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