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Novel fungal immunomodulatory protein FIP-NHA with antineoplastic activity and gene thereof

A technology of FIP-NHA and immunoregulatory protein, applied in the field of genetic engineering, can solve the problems of high consumption, time-consuming, and difficulty of natural FIPs

Active Publication Date: 2013-06-26
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To develop and apply such medicines, a considerable amount of pure protein must be obtained. However, mushrooms contain a large amount of polysaccharides and other complex components. It is difficult to extract natural FIPs from mushrooms and the amount is small. Only milligrams of proteins can be extracted from mushrooms per kilogram. Therefore, mushrooms Extraction of protein is costly, time-consuming, laborious, high consumption, and small amount

Method used

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  • Novel fungal immunomodulatory protein FIP-NHA with antineoplastic activity and gene thereof
  • Novel fungal immunomodulatory protein FIP-NHA with antineoplastic activity and gene thereof
  • Novel fungal immunomodulatory protein FIP-NHA with antineoplastic activity and gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Screening and cloning of the fungal immunoregulatory protein FIP-NHA enzyme coding gene fip-nha

[0052] Fungal genes were screened from the NCBI Fungal Genome Database based on the published conserved sequences of fungal immunomodulatory proteins. A pair of primers FIP-nha-F (EcoRI 5'- GAA TTC GCT ACT ACC AAC G-3') and FIP-nha-R (XhoI 5'- CTC GAG TTA CTT CCA TTG GGC GAC-3'), the fungal immunomodulatory protein FIP-NHA gene derived from the fungus Nectria haematococca was cloned by gene synthesis. The results of DNA sequence analysis showed that the full length of FIP-NHA was 342bp, and its cDNA sequence As shown in SEQ ID NO.2.

Embodiment 2

[0053] Example 2 Preparation of recombinant fungal immunoregulatory protein FIP-NHA.

[0054] The expression vector pGEX-4T-1 was subjected to double enzyme digestion (EcoRI and XhoI), and at the same time, the synthetic gene encoding the fungal immunoregulatory protein FIP-NHA was double enzyme digested (EcoRI and XhoI) to cut out the mature fungal immunoregulatory protein FIP -The gene fragment of NHA was connected with the expression vector pGEX-4T-1 to obtain the recombinant plasmid pGEX-FIP-nha containing the fungal immunoregulatory protein FIP-NHA gene of the fungus Nectriahaematococca and transform it into Escherichia coli Rosetta to obtain the recombinant strain ROS / GST-FIP- NHA.

[0055] Take the Ros / GST-FIP-NHA strain containing the recombinant plasmid, inoculate it in 200 mL of LB culture solution containing 0.1 mM IPTG and 50 μg / ml ampicillin, induce culture at 25°C and 250 rpm for 6 hours, and collect the bacteria by centrifugation. It was then sonicated and the ...

Embodiment 3

[0056] Example 3 Determination of Hemagglutination Activity of Recombinant Fungal Immunomodulatory Protein

[0057] (1) 2 mL of healthy human cells (type A, B, AB or O) and rabbit blood cells were centrifuged at 2000 rpm for 5 minutes at room temperature.

[0058] (2) Carefully suck out the plasma supernatant, collect the red blood cells at the bottom of the centrifuge tube, carefully wash with pH 7.2 10mM PBS to fully resuspend the blood cells, centrifuge at 2000rpm for 5 minutes at room temperature, and wash 5 times in total.

[0059] (3) Finally, the blood cells were suspended in 1.5% (V / V) PBS.

[0060] (4) Prepare 0.2% gelatin PBS solution.

[0061] (5) Accurately weigh 1 mg of the recombinant Ganoderma lucidum immunomodulatory protein, dissolve it in 1 mL of PBS, and adjust the sample concentration so that the final working concentration is 1, 2, 4, 8 and 16 μg / mL.

[0062] (6) The hemagglutination reaction solution is mixed with 25 μl blood cell suspension, 25 μL reco...

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Abstract

The invention relates to the field of genetic engineering, in particular to a novel fungal immunomodulatory protein FIP-NHA with antineoplastic activity and a gene thereof. The fungal immunomodulatory protein FIP-NHA according to the invention has an amino acid sequence as shown in SEQ ID NO. 1, and the gene fip-nha for coding the fungal immunomodulatory protein has a nucleotide sequence as shownin SEQ ID NO. 2. The fungal immunomodulatory protein FIP-NHA provided by the invention has the effects on promoting splenic lymphocyte division, increasing synthesis of interleukin 2, and obviously inhibiting proliferation of three tumor cells including hepatoma cell HepG2, gastric cancer cell MGC823 and leukemia cell HL60, therefore, the fungal immunomodulatory protein FIP-NHA as a novel antineoplastic biological agent can be widely used in the fields of health products and biological medicines.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a novel fungal immunoregulatory protein FIP-NHA with antitumor activity and its gene. Background technique [0002] Fungal immunomodulatory proteins (FIPs) are a class of small molecular proteins extracted from the fruiting bodies of some higher basidiomycetes (mushrooms) in recent years. Immunity and other biological activities, so FIPs have important health care, medicine, and pharmaceutical value. It is similar to the traditional immunomodulatory protein plant lectin, but has its own obvious characteristics and advantages, which are manifested in the following aspects: 1) The molecular weight of lectins is generally large, 20-100kD, while the molecular weight of FIPs is small, 13kD; 2) Monosaccharides and disaccharides do not weaken the hemagglutination and immunomodulatory activities of FIPs, while typical lectins are inhibited by monosaccharides or disaccharides; 3) α-hel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/63C12N15/70C12N1/21A61K38/16A61P35/00A61P35/02C12R1/01C12R1/645
Inventor 姚斌赵珩李淑英罗会颖孟昆杨培龙王亚茹黄火清石鹏君袁铁铮柏映国
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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