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Recombinant strain capable of expressing fungal immunomodulatory protein from Flammulina velutipes (FIP-fve), construction method, protein expression and purification method, and protein application

A technology of immunomodulatory proteins and recombinant strains, applied in the direction of microorganism-based methods, peptide/protein components, recombinant DNA technology, etc., can solve the problems of low cell fragmentation rate, complicated purification process, and low expression rate of strains, and achieve fermentation The process is simple, the pollution rate is extremely low, and the effect of cost saving

Active Publication Date: 2013-02-13
INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem of low expression rate of existing Flammulina velutipes immunoregulatory protein engineering strains, mostly insoluble inclusion bodies, uncertain structure, low bacterial cell fragmentation rate, complex purification process and unsuitable for large-scale production and application. Multiple problems, but provide a kind of recombinant strain capable of expressing Flammulina velutipes immunomodulatory protein and its construction method, protein expression purification method and protein application

Method used

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  • Recombinant strain capable of expressing fungal immunomodulatory protein from Flammulina velutipes (FIP-fve), construction method, protein expression and purification method, and protein application
  • Recombinant strain capable of expressing fungal immunomodulatory protein from Flammulina velutipes (FIP-fve), construction method, protein expression and purification method, and protein application
  • Recombinant strain capable of expressing fungal immunomodulatory protein from Flammulina velutipes (FIP-fve), construction method, protein expression and purification method, and protein application

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specific Embodiment approach 1

[0027] Specific embodiment one: The recombinant bacterial strain capable of expressing Flammulina velutipes immunoregulatory protein of the present embodiment comprises the Escherichia coli expression plasmid pET30a (+) inserted with the cDNA encoding Flammulina velutipes immunomodulatory protein, so that the obtained recombinant expression plasmid is transformed into Transetta ( DE3) Escherichia coli can express Flammulina velutipes immunoregulatory protein.

[0028] This embodiment includes the following beneficial effects:

[0029] Compared with the few soluble products expressed in other Escherichia coli (the yield is 5 mg / L, KO JL et al., 1997) or mostly insoluble inclusion bodies, the denaturation and renaturation process is required ("Kong Xianghui et al., 2007") , The recombinant strain of this embodiment is selected as Transetta, a high-expression genetically engineered strain with chloramphenicol resistance gene and tRNA corresponding to 6 rare eukaryotic gene codons...

specific Embodiment approach 2

[0032] Specific embodiment two: the construction method of the recombinant bacterial strain capable of expressing Flammulina velutipes immunoregulatory protein of the present embodiment is carried out according to the following steps:

[0033] 1. Extract the total RNA of Flammulina velutipes with a kit;

[0034] 2. Using the total RNA of Flammulina velutipes extracted in step 1 as a template, carry out RT-PCR through a reverse transcription PCR kit to obtain cDNA capable of expressing Flammulina velutipes immunomodulatory protein;

[0035] 3. Insert the cDNA encoding the immunomodulatory protein of Flammulina velutipes obtained in step 2 into the expression plasmid pET30a(+) of Escherichia coli to obtain the recombinant vector pET30a(+)-FIP-fve ligation product;

[0036] 4. Transfer the recombinant vector pET30a(+)-FIP-fve connection product obtained in step 3 into Transetta (DE3) Escherichia coli to obtain a recombinant strain capable of expressing the immunomodulatory protei...

specific Embodiment approach 3

[0041] Specific embodiment 3: The difference between this embodiment and specific embodiment 2 is that the cDNA encoding the immunomodulatory protein of Flammulina velutipes described in step 3 is inserted into the expression plasmid pET30a (+) of E. coli. The cDNA encoding the immunoregulatory protein and the expression vector pET30a(+) were digested with BamHI and EcoRI. After digestion, the cDNA encoding the immunomodulatory protein of Flammulina velutipes and the expression vector pET30a(+) were purified and recovered, and then connected with ligase to obtain the recombinant vector pET30a(+)-FIP-fve ligation product. Others are the same as in the second embodiment.

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Abstract

The invention relates to recombinant strain capable of expressing fungal immunomodulatory protein from Flammulina velutipes (FIP-fve), a construction method, a protein expression and purification method, and protein application. Existing FIP-fve engineered strains have the disadvantages of low expression rate, expression product of insoluble inclusion body, uncertain structure and broken strain, complex purification process, and non-suitability for large-scale production. The invention adopts FIP-fve gene and Transetta (DE3) as host bacteria to construct recombinant strain; induces expression with isopropyl-beta-d-thiogalactoside (IPTG); treats by combining a lysozyme method and ultrasonic method; and desalts by adopting Ni-His.Band column chromatography and Sephadex G25 gel column chromatography, thereby finishing construction of recombinant strain, expression and purification. The invention is used for preparing drug for preventing or treating allergy. The purification process is simple and rapid, and suitable for large-scale fermentation production.

Description

technical field [0001] The invention relates to a recombinant bacterial strain and its construction method, protein expression and purification method and protein application. Background technique [0002] Flammulina velutipes immunomodulatory protein (fungal immunomodulatory protein from flammulina velutipes, FIP-fve) is a non-specific immune enhancer isolated from Flammulina velutipes. It prevents and treats allergies by regulating the content of cytokines in the body and maintaining balance, and has the advantages of less side effects and treating both symptoms and root causes. As a simple protein with small molecules without sugar groups, Flammulina velutipes immunomodulatory protein is easy to operate and apply, and it is a very promising clinically applicable medicinal protein. Therefore, it is necessary to develop it into an anti-allergic product for the benefit of mankind. [0003] However, due to the long production cycle of Flammulina velutipes, the natural FIP-fv...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P21/02A61K38/16A61P37/08C12R1/19
Inventor 孔祥辉张介驰张丕奇戴肖东韩增华马庆芳刘佳宁党阿丽
Owner INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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