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Immunomodulatory protein FIP-ppl of fungus and gene of protein

An immunoregulatory protein, fip-ppl technology, applied in the field of genetic engineering, to achieve the effect of improving the body's immunity

Active Publication Date: 2014-08-13
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there are fewer FIPs that can actually be applied

Method used

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  • Immunomodulatory protein FIP-ppl of fungus and gene of protein
  • Immunomodulatory protein FIP-ppl of fungus and gene of protein
  • Immunomodulatory protein FIP-ppl of fungus and gene of protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Screening and Cloning of the Fungal Immunomodulatory Protein FIP-ppl Encoding Gene fip-ppl

[0050] Using the fungal immunomodulatory protein FIP-fve of Flammulina velutipes as bait, BLAST comparison was performed in the NCBI fungal genome database, and FIP-ppl with 52% homology to FIP-fve was found in the genome of the basidiomycete Postia placenta Mad-698-R Coding gene, codon optimization, synthetic gene; design and synthesis of primers FIP-P-GST-F (EcoRI: 5'-GAA TTC ATT ACT TAT GTT CTT CAG G-3') and FIP-P-22b-R ( XhoI: 5'-CTC GAG TTA CTG GGA CTG GG-3'), use the above primers to amplify the synthetic gene, connect it to the cloning vector T-vector, and perform sequencing verification; obtain the full-length 384bp of the FIP-ppl coding gene, and its cDNA sequence As shown in SEQ ID NO.2.

Embodiment 2

[0051] Example 2 Preparation of recombinant fungal immunoregulatory protein FIP-ppl.

[0052] Digest the above cloning vector with restriction enzymes EcoRI and XhoI to obtain the target gene fip-ppl, connect the target gene to the expression vector pGEX-4T-1 after the same digestion, and obtain the recombinant expression of the fungal immunomodulatory protein FIP-ppl gene The vector pGEX-fip-ppl was transformed into Escherichia coli Rosetta to obtain the recombinant strain ROS / GST-FIP-ppl.

[0053] Take the ROS / GST-FIP-ppl strain containing the recombinant plasmid, inoculate it in 200mL LB culture solution containing 50μg / ml ampicillin, and cultivate it to OD at 37°C and 200rpm 600 0.8-1.0, add 0.1mM IPTG, induce culture at 25°C and 200rpm for 6h, and collect the bacteria by centrifugation. 0.2 volume of PBS was used to dissolve the cells, ultrasonically disrupted, and the supernatant was collected by centrifugation. After GST column purification, use 100 U / ml thrombin at 3...

Embodiment 3

[0058] Example 3 Recombinant Fungal Immunomodulatory Protein FIP-ppl Determination of Mouse Spleen Lymphocyte Proliferation Activity

[0059] experimental method:

[0060] The mitogenic effect of recombinant fungal immunomodulatory protein on mouse spleen lymphocytes was determined by MTT assay. The antibiotics used, recombinant proteins FIP-ppl, ConA, LPS and MTT were all filtered with 0.22 μm filter membrane and stored at -20°C.

[0061] 1) Take BalB / C mice grown in a non-sensitized environment for 4-6 weeks, kill the mice by neck dislocation, soak in 75% alcohol for 5 minutes, and take the spleen under aseptic conditions in an ultra-clean workbench.

[0062] 2) Spleen lymphocytes were separated by grinding method (4-6 layers of sterilized gauze or 200-mesh cell sieve).

[0063] 3) Suspend splenocytes with 3ml RPMI1640 culture medium (without FBS and double antibody), and centrifuge at 2000rpm for 2 minutes.

[0064] 4) Carefully aspirate the supernatant and repeat step (...

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Abstract

The invention provides a novel immunomodulatory protein FIP-ppl derived from a fungus by applying a genetic engineering means for the first time. According to the invention, a gene which codes the protein is provided and synthesized. The protein FIP-ppl has immunomodulatory and anti-tumor effect. Experiments verify that the protein has a catalytic splitting effect on splenic lymphocyte and can add synthesis of interleukin 2; the protein has remarkable suppress proliferation and apoptosis-inducing effects of the protein on hepatoma carcinoma cell HepG2 and gastric carcinoma cell MCG823 are further verified. The protein FIP-ppl provided by the invention is lowly homological with almost found immunomodulatory proteins of fungi and is a novel immunomodulatory and anti-tumor biological preparation.

Description

Technical field: [0001] The invention relates to the field of genetic engineering, in particular to a fungal immunoregulatory protein FIP-ppl with immunoregulatory and antitumor activities and its gene. Background technique: [0002] Fungal immunomodulatory proteins (FIPs) are a class of small molecules that have been extracted from the fruiting bodies of some higher basidiomycetes (edible fungi) in recent years, and are similar in structure and function to plant lectins and immunoglobulins. protein. FIPs have many important physiological functions such as regulating immunity, anti-tumor, anti-virus, anti-fungal, lowering cholesterol and inhibiting the body's autoimmunity. [0003] FIPs have unique physiological functions, and are extremely stable and safe to eat. They can regulate immunity, inhibit tumorigenesis, and treat allergies, etc. Therefore, FIPs can be used not only as functional components of health care products to enhance body health, but also as natural medic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/375C12N15/31C12N15/70C12N1/21A61K38/16A61P37/02A61P35/00
CPCA61K38/00C07K14/375
Inventor 李淑英唐选明丁洋聂莹
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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