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Gene of recombinant fungal immunomodulatory protein between ganodermas, protein coded thereby, and application thereof

An immunomodulatory protein, fungal technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of reports on the optimization of fungal immunomodulatory proteins that have not yet been found, and achieve a simple expression method, simple separation and purification process, and easy construction. Effect

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

[0004] So far, no reports have been found on the optimization of fungal immunomodulatory proteins using recombinant DNA technology

Method used

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  • Gene of recombinant fungal immunomodulatory protein between ganodermas, protein coded thereby, and application thereof
  • Gene of recombinant fungal immunomodulatory protein between ganodermas, protein coded thereby, and application thereof
  • Gene of recombinant fungal immunomodulatory protein between ganodermas, protein coded thereby, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, gene recombination of fungal immunomodulatory protein

[0025] 1. Fragment Synthesis

[0026] According to the preferred codons of Escherichia coli, under the premise of not changing its protein sequence, the red sesame fungal immunoregulatory protein gene (FIP-glu), Flammulina velutipes fungal immunoregulatory protein gene (FIP-fve) and variola volvacea fungal immunoregulatory protein gene ( FIP-vvo) recoded. Divide the FIP-glu, FIP-fve and FIP-vvo genes recoded according to the preferred codons of Escherichia coli into 50-60bp fragments according to their respective DNA sequences, and there are about 18bp nucleosides between each two adjacent fragments Acid overlaps (SEQ ID NO. 3 to SEQ ID NO. 28), and these fragments were synthesized.

[0027] 2. Primer-free PCR

[0028]The oligonucleotides shown in SEQ ID NO.3 to SEQ ID NO.28 were mixed in equal amounts, and the gene fragment was subjected to PCR reaction using KOD plus polymerase (Toyobo, Japan). ...

Embodiment 2

[0041] Example 2, Construction and Screening of Recombinant Fungal Immunomodulatory Protein Expression Vector

[0042] 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 cloned plasmid is extracted to obtain the recombinant fungal immunoregulatory protein expression vector. The obtained transformant is the recombinant fungal immunoregulatory protein gene recombination library. The recombinant fungal immunoregulatory protein gene transformants were screened. When the transformant colony grows to a diameter of 1 mm, the colony is blotted on a PVDF membrane. Then move the membrane to an LB plate containing 100 mg / mL car...

Embodiment 3

[0043] Example 3, Induced Expression and Purification of Recombinant Fungal Immunomodulatory Protein

[0044] 1. Induced expression of recombinant fungal immunomodulatory protein and Western blot detection

[0045] 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. 100 μL 2×SDS-PAGE Sample 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 kept in a 95°C water bath for 5 minutes. Then use two 15% SDS-PAGE gels to detect. One piece was used fo...

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Abstract

The invention discloses a gene of a recombinant fungal immunomodulatory protein (FIP) between ganodermas, a protein coded thereby, and application thereof, and belongs to the technical field of gene engineering. The nucleotide sequence is shown as SEQ ID NO.1, and the nucleotide sequence of the protein is shown as SEQ ID NO.2. Recombination of the gene of the fungal immunomodulatory protein is carried out, the recombinant gene is expressed in Escherichia coli, and a target protein is obtained; moreover, the target protein still has good immunomodulatory activity, and a new thought and exploitation way is provided for large-scale production of the FIP and use thereof as medicines 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 fungal 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 Japanese scholar Kino et al. isolated and extracted the first fungal immunomodulatory protein (LZ-8) from the fruiting body of Ganoderma lucidium in 1989, there have been LZ-8 (FIP-glu), FIP-gts, FIP Seven fungal immunomodulatory proteins including -fve, FIP-vvo, FIP-gja, FIP-gmi and FIP-gsi were obtained from G. tsugae, Flammulina velutipes, Volvariella volvacea, Zizhi (G.japoncium), Xiaospore Ganoderma (G.microsporum) and Chinese Zizhi (G.sinense) were isolated. And these sev...

Claims

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Application Information

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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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