Recombinant fungal immunomodulatory protein gene in Ganoderma lucidum, protein coded whereby and application thereof

A technology of immunomodulatory proteins and fungi, applied in the fields of application, genetic engineering, plant gene improvement, etc., to achieve the effects of easy construction, simple expression method, and simple separation and purification process

Inactive Publication Date: 2011-09-28
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of gene recombination technology to recombine the immunomodulatory protein of Ganoderma lucidum fungus and obtain effective protein

Method used

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  • Recombinant fungal immunomodulatory protein gene in Ganoderma lucidum, protein coded whereby and application thereof
  • Recombinant fungal immunomodulatory protein gene in Ganoderma lucidum, protein coded whereby and application thereof
  • Recombinant fungal immunomodulatory protein gene in Ganoderma lucidum, protein coded whereby and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1, Gene Recombination of Fungal Immunomodulatory Proteins in Ganoderma

[0025] 1. Fragment Synthesis

[0026] According to the preferred codons of Escherichia coli, the fungal immunoregulatory protein gene (FIP-glu) and the fungal immunoregulatory protein gene (FIP-gsi) of Chizhi were recoded without changing their protein sequences. The FIP-glu and FIP-gsi genes recoded according to the E. coli preferred codons were divided into 50-60bp length fragments according to their DNA sequence, and there was about 18bp nucleotide overlap between every two adjacent fragments (SEQ ID NO.3 to SEQ ID NO.20), and these fragments were synthesized.

[0027] 2. Primer-free PCR

[0028] The oligonucleotides shown in SEQ ID NO.3 to SEQ ID NO.20 were mixed in equal amounts. The mixed gene fragments were subjected to PCR reaction using KOD plus polymerase (Toyobo, Japan). There are no primers in the reaction system. The reaction program was as follows: after pre-denaturation ...

Embodiment 2

[0039] Example 2, Construction and Screening of Recombinant Ganoderma Fungus Immunomodulatory Protein Gene Expression Vector

[0040] The product II obtained in Example 1.3 was digested overnight with restriction endonucleases BamH I and Hind III, and ligated with the expression vector pQE-30 digested with the same restriction endonucleases. The constructed expression vector was transformed into Escherichia coli M15 host cells, and transformants were screened with LB plates containing 100 mg / mL carbenicillin sodium and 50 mg / mL kanamycin. The cloning plasmid is extracted to obtain the expression vector of the recombinant ganoderma fungus immunoregulatory protein. The obtained transformant is the recombination library of the immunoregulatory protein gene of the fungus of the genus Ganoderma lucidum. Screen the transformants of the recombinant Ganoderma lucidum fungal immunoregulatory protein gene. When the transformant colony grows to a diameter of 1 mm, the colony is blotted...

Embodiment 3

[0041] Example 3. Induced Expression and Purification of Recombinant Fungal Immunomodulatory Proteins in the Genus Ganoderma

[0042] 1. Induced expression and Western blot detection of recombinant fungal immunomodulatory proteins in Ganoderma lucidum

[0043] The single clone obtained in Example 2 was inoculated into LB liquid medium containing 100 mg / mL carbenicillin sodium and 50 mg / mL kanamycin, and cultured overnight at 37° C. with shaking. The next day, inoculate a larger volume of culture medium with 2% inoculum. Continue culturing at 37°C, and when OD600 reaches 0.5-0.7, add IPTG to make the final concentration of IPTG reach 1 mM. After culturing for 6 hours, the obtained bacterial liquid samples were centrifuged at 5,000 rpm for 20 minutes to collect bacterial cells. The supernatant was removed, and the cells were washed with 100 μL 2×SDS-PAGESample buffer (100 mM pH 6.8 Tris-Cl, 4% (W / V) SDS, 0.2% (W / V) BPB, 20% (W / V) glycerine, 2 % (W / V) β-ME) was suspended and k...

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Abstract

The invention, belonging to the filed of genetic engineering technology, relates to a recombinant fungal immunomodulatory protein (FIP) gene in Ganoderma lucidum, a protein coded whereby and an application thereof. The nucleotide sequence of the recombinant FIP gene in Ganoderma lucidum is shown in SEQ ID No.1, and the protein coded by the recombinant FIP gene is shown in SEQ ID No.2. According to the invention, the FIP gene in Ganoderma lucidum is recombined, an object protein is obtained by expressing the recombinant gene in escherichia coli, and the immunomodulating activity of the recombinant gene is proved to be good. The invention provides a new idea and approach for producing FIP in large scale and the application of using FIP as medicine or other health products.

Description

technical field [0001] The invention relates to a gene in the technical field of genetic engineering and its application, in particular to a gene of a recombinant ganoderma fungus immunoregulatory protein, the protein encoded by the gene and its application. Background technique [0002] Fungal immunomodulatory proteins are a class of small molecular proteins that are extracted from the fruiting bodies of higher fungi and are similar in structure and immune function to plant lectins and immunoglobulins. Since the Japanese scholar Kino et al. isolated and extracted the first fungal immunomodulatory protein (LZ-8) from the fruiting bodies of Ganoderma lucidium in 1989, it has been isolated from Ganoderma lucidum (G.tsugae) and Flammulina velutipes respectively. ), Volvariella volvacea, G.japoncium, G.microsporum and G.sinense were isolated from seven FIPs, namely LZ-8(FIP-glu), FIP-gts, FIP-fve, FIP-vvo, FIP-gja, FIP-gmi and FIP-gsi form a new protein family - FIPs. Lin Zhon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/375C12N15/31A61P37/02
Inventor 周选围王雪飞李奇璋苏恺琪丛蔚然
Owner SHANGHAI JIAO TONG UNIV
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