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Method for preparing intragenus recombinant fungal immunomodulatory proteins and application of intragenus recombinant fungal immunomodulatory proteins

A technology of immunomodulatory proteins and fungi, which is applied in the fields of genetic engineering and molecular biology, and can solve problems that have not been found in literature reports

Active Publication Date: 2013-08-14
秦岭健康医药产业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the analysis to existing document, do not find to have the document report identical with the subject of the present invention so far

Method used

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  • Method for preparing intragenus recombinant fungal immunomodulatory proteins and application of intragenus recombinant fungal immunomodulatory proteins
  • Method for preparing intragenus recombinant fungal immunomodulatory proteins and application of intragenus recombinant fungal immunomodulatory proteins
  • Method for preparing intragenus recombinant fungal immunomodulatory proteins and application of intragenus recombinant fungal immunomodulatory proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Construction of prokaryotic expression vector of recombinant fungal immunomodulatory protein gene reFIP-SN15 within the genus:

[0045] (1) According to the sequence of the recombinant fungal immunomodulatory protein gene reFIP-SN15 within the genus, design primers that can amplify the full length of the gene, and add restriction endonuclease sites Bam H I and Hind III to the upstream and downstream primers, respectively , to construct prokaryotic expression vectors.

[0046] (2) Using the recombined fungal immunomodulatory protein gene reFIP-SN15 within the genus as a template, after PCR amplification, the reFIP-SN15 gene was cloned into an intermediate vector (such as pMD18-T simple vector), and the sequencing results were consistent with those reported within the genus. The sequence comparison of the recombined fungal immunoregulatory protein gene reFIP-SN15 shows that the identity is 100%, indicating that there is no mismatch in the amplification.

[0047] (3) Cut ...

Embodiment 2

[0051] Induced expression and purification of recombinant fungal immunomodulatory protein reFIP-SN15 in Ganoderma lucidum:

[0052] 1. Induced expression and Western blot detection of recombinant fungal immunomodulatory protein reFIP-SN15 in Ganoderma lucidum

[0053] (1) Inoculate the monoclonal obtained in Example 1 in LB liquid medium containing 50 mg / mL kanamycin, and cultivate overnight at 37°C with shaking.

[0054] (2) The next day, inoculate a larger volume of culture medium with 2% inoculum. Continue culturing at 37°C until OD 600 When it reaches 0.5-0.7, add IPTG to make the final concentration of IPTG reach 1mM.

[0055] (3) Take out 1 mL every 0, 1, 2, 3, 4, and 5 hours and store it at -20°C for later use. Centrifuge the obtained bacterial liquid sample at 5,000 rpm for 20 min to collect the bacterial cells, remove the supernatant, and wash the bacterial cells with 100 μL 2×SDS-PAGE Sample buffer (100 mM pH6.8 Tris-Cl, 4% (W / V) SDS, 0.2% (W / V) / V) BPB, 20% (W / V...

Embodiment 3

[0062] Q-tof MS Analysis of Recombinant Fungal Immunomodulatory Protein reFIP-SN15 in Ganoderma lucidum

[0063] 1. Recombinant fungal immunomodulatory protein reFIP-SN15 pre-treatment in Ganoderma lucidum

[0064] (1) Cut off the target band of SDS-PAGE gel with a clean blade, transfer to a 1.5mL centrifuge tube, and use a 200μL pipette tip to crush the film into 1-2mm 2 glue blocks of size;

[0065] (2) Wash the gel twice with ultrapure water, add 500 μL decolorization solution (50% acetonitrile, 25mM NH 4 HCO 3 ), shake at room temperature for 15-20min, centrifuge and discard the supernatant. Repeat this step 1-2 times until the glue block is colorless;

[0066] (3) Add 500 μL of 50% acetonitrile, shake at room temperature for 10-15 minutes, and centrifuge to discard the supernatant;

[0067] (4) Dry the sample in a concentrated drying apparatus (50-55°C for 5 minutes or room temperature for 10 minutes);

[0068] (5) Add 100 μL of 10mM DTT (25mM NH 4 HCO 3 dissolved...

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Abstract

The invention discloses a method for preparing intragenus recombinant fungal immunomodulatory proteins and application of the intragenus recombinant fungal immunomodulatory proteins. The intragenus recombinant fungal immunomodulatory proteins have a nucleotide sequence shown in SEQ ID NO. 1 in a sequence table. According to the application, genes of the intragenus recombinant fungal immunomodulatory proteins are expressed in Escherichia coli so as to obtain high-expression target proteins, and the condition that the target proteins have good anti-tumor activity to breast and lung cancers is proved, so that novel application directions and exploration ways are provided for the large-scale production of the recombinant fungal immunomodulatory proteins and the application of the recombinant fungal immunomodulatory proteins in development of novel anti-tumor drugs.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to the expression and application of a novel fungal immunoregulatory protein gene nucleotide sequence. Background technique [0002] Fungal immunomodulatory proteins (fugal immunomodulatory proteins, FIPs) are isolated from Ganoderma lucidum, following polysaccharides and triterpenoids, another class of active substances. Since 1989, Kino et al first isolated this protein from the fruiting bodies of Ganoderma lucidum, and named it Ling Zhi-8 (LZ-8 or FIP-glu). So far, from red Ganoderma lucidum (G.lucidum), pine and fir Ganoderma (G.tsugae), Flammulina velutipes (Flammulina velutipes), Straw mushroom (Volvariella volvacea), Purple Ganoderma (G.japoncium), Microspore Ganoderma (G. .microsporum) and Zizhi (G.sinensis) cloned seven kinds of FIPs, respectively named as LZ-8 (FIP-glu), FIP-gts, FIP-fve, FIP-vvo, FIP-gja (...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/31A61K38/16A61P35/00
Inventor 周选围丛蔚然
Owner 秦岭健康医药产业科技有限公司
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