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Preparation method and application of ganoderma atrum fungal immunomodulatory protein

A technology of immunoregulatory protein and black ganoderma, which is applied in the preparation and application of black ganoderma fungus immunomodulatory protein, and can solve the problems of low content and high cost

Active Publication Date: 2014-04-23
秦岭健康医药产业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the practice of clinical and medical applications, there are certain requirements for the dosage of proteins. Therefore, the primary problem in applying such proteins to the medical field is how to carry out large-scale mass production? Because the content of fungal immunomodulatory proteins in natural mushrooms (or mushrooms) is very low (20-80mg / Kg) (Lin Jingwei, Sun Fei, Zhang Ren, etc. Research progress of fungal immunomodulatory proteins. Chinese Journal of Immunology. 2005 , 21(6):477-480), coupled with the interference of fungal polysaccharides and other components in the protein extraction process, the cost of obtaining protein is relatively high

Method used

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  • Preparation method and application of ganoderma atrum fungal immunomodulatory protein
  • Preparation method and application of ganoderma atrum fungal immunomodulatory protein
  • Preparation method and application of ganoderma atrum fungal immunomodulatory protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Construction of the prokaryotic expression vector of the fungal immunomodulatory protein gene FIP-gas of Ganoderma lucidum:

[0047] 1) According to the sequence of the black Ganoderma fungus immunoregulatory protein gene FIP-gas, 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 Construction of prokaryotic expression vectors.

[0048] 2) Using the black ganoderma fungus immunomodulatory protein gene FIP-gas as a template, after PCR amplification, the FIP-gas gene was cloned into an intermediate vector (such as pMD18-T simple vector), and the sequencing results were consistent with the reported black ganoderma fungal immunity The sequence comparison of regulatory protein gene FIP-gas shows that the consistency is 100%, indicating that there is no mismatch in the amplification.

[0049] 3) Cut off the FIP-gas fragment from the pMD18-T simple ve...

Embodiment 2

[0053] Induced Expression and Purification of the Immunomodulatory Protein FIP-gas from Ganoderma lucidum

[0054] 1. Induced expression and Western blot detection of recombinant fungal immunomodulatory protein FIP-gas in Ganoderma lucidum

[0055] 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.

[0056] 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.

[0057] 3) Take out 1 mL every 0, 0.5, 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) glycerine, 2% (W...

Embodiment 3

[0064] Cell agglutination assay of recombinant fungal immunoregulatory protein FIP-gas from Ganoderma lucidum on human breast cancer cell line MDA-MB-231.

[0065] 1. Culturing of Cancer Cells

[0066] Breast cancer cell line MDA-MB-231 used DMEM medium containing 10% fetal bovine serum in 5% CO 2 , Cultured under saturated humidity conditions at 37°C, and cultured in a monolayer of tumor cells. After the cells covered the bottle, they were digested with 0.25% trypsin and 0.04% EDTA in PBS liquid, and prepared into a single cell suspension in 0.5% fetal bovine serum albumin (BSA) DMEM solution for later use. Human breast cancer cells MDA-MB- 231 Density adjusted to 5-7×10 3 / 100 μL.

[0067] 2. Cell Agglutination Experiment

[0068] 1) Add the breast cancer cells whose cell concentration was adjusted to 5-7×103 / 100 μL in Example 3.1 into a 96-well plate, and store in CO 2 Incubate overnight at 37°C in an incubator.

[0069] 2) Dialyze the recombinant protein FIP-gas with...

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Abstract

The invention relates to a preparation method and an application of a ganoderma atrum fungal immunomodulatory protein. A gene of the ganoderma atrum fungal immunomodulatory protein has the nucleotide sequence indicated by SEQ ID NO.1. When the recombined fugal immunomodulatory protein is expressed on colibacillus to obtain a high-expression protein, and the ganoderma atrum fungal immunomodulatory protein has a good agglutination effect. By adopting the preparation method and the application, a novel direction is provided for producing a great amount of recombined immunomodulatory proteins by utilizing the gene engineering method and developing a novel anti-tumor preparation by utilizing the protein, and the preparation method can be widely applied to the fields of the healthcare food and biological medicine.

Description

technical field [0001] The invention relates to a method in the technical fields of molecular biology and genetic engineering, in particular to a preparation method and application of a black ganoderma fungus immunomodulatory protein. Background technique [0002] Fungal immunomodulatory proteins (fugal immunomodulatory proteins, FIPs) are another class of active substances isolated from Ganoderma lucidum after polysaccharides and triterpenoids. In the FIPs family, seven proteins have been reported, named LZ-8 (FIP-glu), FIP-gts, FIP-fve, FIP-vvo, FIP-gja (AY987805), FIP-gmi and FIP -gsi, derived from Ganoderma lucidum, G.tsugae, Flammulina velutipes, Volvariela volvacea, G.japoncium, G.microsporum and Zizhi (G. sinensis) [Li QZ, Wang XF, Zhou XW. Recent status and prospects of the fungal immunomodulatory protein family. Critical Reviews in Biotechnology. 2011, 31(4): 365-375]. In this protein family, there are only four fungal immunomodulatory proteins isolated directly f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/375C12N15/31C12N15/70C12N15/66A61K38/16A61P35/00
CPCA61K38/00C07K14/375C12N15/70
Inventor 周选围孔应予丛蔚然
Owner 秦岭健康医药产业科技有限公司
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