CN102949707A

CN102949707A - Treatment on inflammation caused by respiratory syncytial virus by developing flammulina velutipes immune adjustment proteins and lucid ganoderma immune adjustment proteins and reduction of virus dosage

Info

Publication number: CN102949707A
Application number: CN2011102497388A
Authority: CN
Inventors: 柯俊良; 张语琦
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-08-23
Filing date: 2011-08-23
Publication date: 2013-03-06

Abstract

The invention discloses HEp-2 cell strains infected with respiratory syncytial virus (RSV) treated by flammulina velutipes immune adjustment proteins, which can effectively inhibit the infection of the RSV after FIP-fve is treated. Phosphorylation of IkBalpha and NF-kB is sensed in a western blotting method; the expression amount of the two phosphorylation proteins are both inhibited after the treatment on FIP-fve is found so as to further observe the distribution condition of NF-kB in cells by a conjugate focal plane microscope; and the result shows that Nf-kB nuclear introduction caused by RSV can be obviously reduced by the FIP-fve, and IL-4 expression amount of inflammatory factors related to the NF-kB is obviously reduced; and the flammulina velutipes proteins FIP-fve has a function of reducing the RSV infection so as to reduce curative effect generated by inflammation substances related to an NF-kB path; and finally, BALA/C mice are used as an animal mode, the mice which orally take the flammulina velutipes proteins can inhibit asthma caused by titrating the RSV from the nasal cavity.

Description

Exploitation gold needle mushroom immunomodulatory protein and Ganoderma lucidum immunoregulation protein treatment respiratory tract merge the viral inflammation that causes and reduce viral dosage

Technical field

The present invention relates to biotechnology and agriculture chemistry.

Background technology

One, respiratory tract merges the introduction of tumor virus (Respiratory Syncytial Virus:RSV)

It is the main cause that causes the acute viral respiratory tract diseases such as the serious viral bronchitis of baby and child and viral pneumonia that respiratory tract merges tumor virus (Respiratory Syncytial Virus:RSV).It is a kind of RNA viruses, and mumps virus, german measles virus belongs to paramyxovirus (Paramyxovirus) together, in multiple different tissues, cultivate and all can produce special Cell fusion (syncytial cytopathology), can cause behind the intrusion respiratory tract that tracheal epithelial cell is downright bad, mucus is secreted, inflammatory cells infiltrates, the Submucosa edema.Respiratory tract merges tumor virus and misses potter the respiratory tract epidermis cell that infection has cilium, can cause the infiltration of bronchiolar obstruction and inflammation and Submucosa cell at respiratory tract, occur that expiratory duration prolongs, the symptoms such as pulmonary atelectasis (atelectasis) and pneumonia.In the patient of old people and immunological incompetence, respiratory tract merges tumor virus can cause serious respiratory tract disease equally.From sanitarian viewpoint, the health organization of countries in the world and the health unit of government all classify make respiratory tract fusion tumor virus vaccine as one of important process of current control viral infection as.

It is paramyxovirus section (Paramyxoviridae) that respiratory tract merges tumor virus, and pneumonitis virus belongs to (Pneumovirus), has outer embrane (envelope), its genome is sub-thread (-) RNA, formed by 15,222 nucleic acid, comprise 10 sections mRNA.These mRNA can translate out 11 kinds of protein that have been identified existence, comprise 4 kinds of nucleocapsid protein (nucleocapsid protein), respectively called after nucleocapsid N protein, phosphoprotein P, large polymerase subunit transcription elongation factor M2-1; 3 kinds of overcoat glucoprotein matter that penetrate film are respectively fusion F protein, attachment G protein and small hydrophobic SH protein; 2 kinds of nonstructural proteins NS1, NS2; 1 kind of stroma protein M protein and the protein such as M 2-2 that are speculated as at present negative regulatory factor.At present function is indefinite NS1, NS2 and a SH protein etc.Merge the result of the test of tumor virus vaccine according to past scientists development respiratory tract, the immunoreation that we can summarize dead malicious vaccine (inactivated vaccine) initiation is a series of reactions of Th 2 T-helper cell, meeting is so that the state of an illness of lower respiratory tract (referring to the positions such as bronchus, lungs) is more serious, because cytohormone IL-4, IL-10 etc. that Th 2 T-helper cell produce, can cause more serious cellular infiltration phenomenon, increase the weight of and accelerate the state of an illness., there is no at present specific drug and mostly be supportive treatment for suffering from this sick peak period (Lee et al., 2007) with the per 2 years spring and autumn of north Taiwan area Epidemiological study 2001/01-2005/12.And if with glucocorticoids treatment and can't improve its symptom (Patel et al., 2008).And utilize us in current research is carried out, to find the reaction that FIP-fve may can induce the establishment viral infection and alleviate the Th 1 T-helper cell of the respiratory tract disease state of an illness, be the effective ways that meet our desired development clinical medicine.

Two, the function of Mycophyta immune modulator

At some edible mushrooms, as: the purified protein of separating in Ganoderma, Volvariella volvacea (Bull.Ex Franch.) Singer． and the JINZHENGU, they have similar amino acid sequence and immunoloregulation function, we are with the protein with this kind, called after Mycophyta immune modulator matter (Fungal immunomodulatory proteins, FIPs) (Hsu et al., 1997; Ko et al., 1995; Lin et al., 1997).Especially become the outstanding figure with Ganoderma at Chinese medicine, to keeping health, greatest effect has been arranged.Past research finds that it has antiallergic inflammation (Lee et al., 2001), the liver protecting function (Shieh et al., 2001), antitumor and enhancing immunologic function, but mostly be confined to thick extract (Cuella et al., 1996) research, or micromolecular compound research (Lai et al., 2001; Zhang et al., 2002).Until Japanese Mingzhi's pharmacy in 1989 purification from Ganoderma gets an immunoloregulation function albumen, LZ8 (Kino et al., 1989), experimental result finds that LZ-8 can significantly suppress systemic anaphylaxis, treatment hepatitis and prevent diabetes, our laboratory recent findings Ganoderma lucidum immunoregulation protein suppresses lung carcinoma cell terminal enzymatic activity (Liao et al., 2006; Liao et al., 2007) and cause lung carcinoma cell old and feeble (Liao et al., 2008) can develop treatment pulmonary carcinoma and assist Yisheng Bio tech Development Co., Ltd. to apply for a patent.LZ8 has the mitogenic ability, and can suppress the CFW mouse by the caused anaphylaxis of bovine serum albumin BSA.This plan host once protein structure from the heavy chain district of another edible mushrooms-JINZHENGU (FIP-fve) discovery and LZ8 and immunoglobulin has the similarity of certain degree, and secondary structure mostly is b-Sheet, and when measuring it for human lymphocytes multiplication capacity, (Ko et al. when FIP-fve concentration is 100 ug/ml, 1995), its multiplication capacity reaches the strongest.And injection FIP-fve protein when its dosage is 8 mg/kgs of body weight, has the caused systemic anaphylaxis reaction of the BSA of inhibition in mouse body.Lumbar injection FIP-fve protein in advance in mouse toes swelling experiment when dosage is 5 mg/kgs of body weight, can reduce compound 48/80 caused inflammatory response.Learn that by above experimental result this protein has immunoregulatory activity, to turn be for promoting the Transcription of IFN-γ and TNF-α gene to immunoregulatory machine after further study.

What wherein true Mushrooms immunoloregulation function albumen research was saturating clear is Ganoderma, this immunoloregulation function protein can promote performance (the Haak-Frendscho et al. of ICAM-1 (intercellular adhesion molecule-1), 1993), and obviously increase IFN-γ, therefore Ganoderma increases the CD2 performance, cause that rosette forms, so Ganoderma can the adhesion molecule shows its property of medicine on the immunocompetent cell by regulating.And in the Ganoderma bacterial strain, identified G.applanatum., G.formosanum., G.fornicatum., G.lucidium, several Ganodermas such as G.tropicum and G.tsugae according to permitting to separate difference in the auspicious auspicious thesis for the doctorate.Its immunoregulatory activity difference is quite large between every kind of strain, the FIP-fve of at present G.lucidium and G.tsugae and this researcher discovery has been proved has immunoloregulation function, and this research team is simultaneously in the new immunoloregulation function albumen ability of exploitation in this year in addition.

In the Mushrooms plant, in their sporophore or mycelium, some are arranged in the Mushrooms plant, some close glucoprotein matter (lectins) are arranged in their sporophore or the mycelium, can promote lymphocytic propagation; Opposite, some then can suppress lymphocytic propagation.Comprise the Agaricus bisporus (Koyama et al., 2002) of Tricholoma matsutake (lto et lmai) Singer section, molecular weight is 64kDa, is made of 4 sub-cell bodies, understands anticancer U937 propagation even causes apoptosis (Apoptosis).These close glucoprotein matter all have the ability of coagulation cell.Close glucoprotein matter in the Mushrooms, how not identical the carbohydrate of institute's combination is on its protein, Hericium erinaceus (Bull. Ex Fr.) Pers. Hericium erinaceum (Kawagishi et al., 1994) carbohydrate on is to belong to sialic acid combination, Laetiporus sulfurens (Yoshikawa et al., 2001) be the N-acetyllactosamine combination, may promote the combination of cell surface receptor, and close glucoprotein is verified in the immunoloregulation function aspect, close glucoprotein matter not of the same race is with isoantibody or Cell binding ability are not quite alike, can be combined with Ig A (Irazoqui et al., 1997) such as: Agaricus bisporus; L-selectin impels the neutrophil coagulation; C.albicans is purified that manoprotein also can reduce IL-8 and its IL-8receptor binding ability, slows down the inflammatory response (Kemeny et al., 1998) that is caused by IL-8.Whether therefore this research is main intends inquiring into this type of coagulation albumen can be through the immunoloregulation function immunologic function of slowing down inflammatory response, the injury that alleviates RSV.

Three, gold needle mushroom immunomodulatory protein and Ganoderma lucidum immunoregulation protein

By JINZHENGU (Flammulina velutipes) sporophore via cation exchange resin CM-52 purification after resulting albumen: FIP-fve (Fungal immunomodulatory protein), be the protein with immunoloregulation function.Learnt by experiment before: FIP-fve can make the lymph corpuscle (HPBMCs) of the peripheral blood of normal human subject and the spleen cell of Balb/c mouse bring out the generation of IFN-γ, and cell is had the ability of proliferation.And be to belong to the cytohormone that the Th1 cell brings out by the IFN-γ that FIP-fve brings out, can suppress the cytohormone that the Th2 cell produces.Recombinant Ganoderma lucidum immunoregulation protein is the albumen that obtains 13kDa by the Ganoderma lucidum immunoregulation protein gene being inserted bread microzyme, being further purified.Therefore have in immune system and reduce by caused anaphylaxiss such as Th2cytokine regulation and control IgE, mast cell, comprise chronic asthma, respiratory inflammation etc.Therefore this piece of writing research makes p38 and ERK (extracellular signal-regulated protein kinase) phosphorylation the most obvious with FIP-fve (100 μ g/ml) stimulation HPBMCs, but can not activate JNK (the c-Jun NH2-terminal kinase), and anticipate the inhibitor of p38MAPkinase: SB203580 (10 μ M) can suppress the phosphorylation of p38.Prove that simultaneously processing HPBMCs with FIP-fve (100 μ g/ml) can obviously produce IFN-γ in 48 hours, and induce and also can be subject to SB203580, LY294002 (inhibitor of PI3-kinase) and U0126 (inhibitor of MEK1/2) suppresses.Except the FIP-fve albumen by JINZHENGU sporophore purification had the ability of bringing out IFN-γ, the FIP-fve fusion rotein of restructuring (recombinant FIP-fve) also had the FIP-fve activity of half nearly.This description of test the mechanism of FIP-fve regulation and control IFN-γ be the activated channel that can see through p38 MAP kinase pathway and ERK, rather than via activation JNK approach.The IFN-γ of a large amount that FIP-fve brings out but can be by the generation that suppresses the cytohormone that the TH2 cell produces, and expectation can be applied on the prophylactic treatment of genetic immunization in the future.

Summary of the invention

The object of the invention is to develop the medicinal application that gold needle mushroom immunomodulatory protein merges virus activity as preparation treatment respiratory tract.

The invention provides a kind of oral true Mushrooms immune modulator, comprise: gold needle mushroom immunomodulatory protein or Ganoderma lucidum immunoregulation protein, can suppress the respiratory tract overreaction that RSV causes.

The phosphorylation that gold needle mushroom immunomodulatory protein can suppress the NF-κ B that RSV induces with enter nucleus.

Oral gold needle mushroom immunomodulatory protein can suppress inflammatory response associated hormone and the viral performance amount of reduction that RSV induces.

Find that through experiment JINZHENGU protein FIP-fve has the reduction rsv infection, and then reduce the curative effect of the inflammation material generation of NF-κ B path coherence.

Description of drawings

Figure 1A is laboratory animal RSV (Respiratory Syncytial Virus) sensitization and fills with food FIP-fve experiment flow; Figure 1B is for presenting result behind the purification with 12%SDS PAGE running gel figure.

Fig. 2 is for counting experimental observation JINZHENGU and Ganoderma lucidum immunoregulation protein for the impact of the cell plaque that RSV was caused with plaque.

Fig. 3 is with the impact of immune Fluorescent Staining Observation FIP-fve for rsv infection power.

Fig. 4 is with the impact of ferment immunosorbent adsorption test assessment FIP-fve for rsv infection power.

Fig. 5 is with RT-PCR detecting RSV and IL-4mRNA performance amount.

Fig. 6 is with ELISA detecting IL-6 secretory volume.

Fig. 7 for west ink dot method assessment FIP-fve for NF-κ B that RSV was caused and the impact of I κ B α phosphorylation.

Fig. 8 for immune Fluorescent Staining Observation FIP-fve for rsv infection after NF-κ B enter the impact of nucleus situation.

Fig. 9 is after stimulating via RSV, intracellular IKK complex can be with I κ B phosphorylation, and the I κ B that is phosphorylated can degrade, NF-κ B is released, on the other hand, NF-κ B is also simultaneously by IKK complex phosphorylation and enter in the nucleus, NF-κ B enter behind the nucleus can bond to many inflammation material related genes (for example: transcribing on position IL-4), the gene performance will be activated, and so that infected cell produces inflammatory response.

Figure 10 is for observing JINZHENGU and Ganoderma lucidum immunoregulation protein for infecting the over-reactive impact of RSV Respiratory Tract of Mice.

Figure 11 is for observing JINZHENGU and Ganoderma lucidum immunoregulation protein for the impact of infecting RSV mouse lung IL-6 content.

The primary clustering symbol description

AHR respiratory tract overreaction is measured

Methacholine (mg/ml) methacholine

Penh pulmonary function pointer

The Control controlled quentity controlled variable

The RSV respiratory tract merges virus

The FIP-fve/RSV immune modulator

FIP-gts/RSV Ganoderma albumen

The specific embodiment

It is the modal virus of infant lower respiratory infection that respiratory tract merges virus (Respiratory syncytial virus, RSV), because its infection mechanism is not yet clear and definite at present, therefore can't develop effective vaccine or therapeutic modality.

FIP-fve be one from JINZHENGU (Flammulina velutipes) purification immune modulator out, molecular size range is 13kDa, be sorted in Mycophyta immune modulator (FIPs), this type of albumen has regulation and control IFN-γ to be increased, and reduces the effect of irritated inflammation.

The present invention processes in the HEp-2 cell strain that infects RSV virus with JINZHENGU protein FIP-fve, and detects in the following manner respectively viral performance amount:

1. plaque counting test (plaque assay)

2. immunofluorescence staining (Immunofluorescence)

3. ferment immunosorbent adsorption test (ELISA)

4. Polymerase Chain Reaction (RT-PCR)

5.BUXCO?SYSTEM-AHR

6. laboratory animal: the female Mus of large BALB/c of seven weeks

Provided by juridical person country animal center, and raise in middle mountain medical university Experimental Animal Center.

See also Figure 1A, be laboratory animal RSV (Respiratory Syncytial Virus) sensitization and fill with food FIP-fve experiment flow, Figure 1B is for presenting result behind the purification with 12%SDS PAGE running gel figure, and the JINZHENGU protein molecular weight is positioned at 13kDa.M represents marker, and C represents thick extract, and fve represents the gold needle mushroom immunomodulatory protein FIP-fve that is purified into.

See also Fig. 2, for counting experimental observation JINZHENGU and Ganoderma lucidum immunoregulation protein with plaque for the impact of the cell plaque that RSV was caused.In 12 hole culture dishs, plant 3 * 10 ⁵HEp-2 cell, the albumen of pre-treatment variable concentrations infected RSV 1 hour again, and covers it with 0.75% methylcellulose after 2 hour respectively every other day, observed the situation of plaque test after 6 days with violet staining.

See also Fig. 3, for the impact of immune Fluorescent Staining Observation FIP-fve for rsv infection power.Pre-treatment 200 μ g/ml JINZHENGU protein are after 2 hours in Human Lung Cancer epithelial cell A549, infect the RSV 48 hours of various dose, and with specificity fluorescent antibody detecting RSV, blue-fluorescence partly are nucleus again, and green fluorescence is RSV.

See also Fig. 4, for assessing FIP-fve for the impact of rsv infection power with the ferment immunosorbent adsorption test.Respectively pre-treatment 0,50,100 in Human Lung Cancer epithelial cell A549,200 μ g/ml JINZHENGU protein infect the RSV of various dose after 2 hours again, after 72 hours with the acetone fixed cell, and use specificity RSV antibodies, at last with wavelength 450nm detecting light absorption value.

See also Fig. 5, for detecting RSV and IL-4mRNA performance amount with RT-PCR.Pre-treatment 0,50,100 μ g/ml JINZHENGU protein infect RSV (moi=0.1) after 2 hours more respectively in human laryngeal cancer cell HEp-2, are purified into mRNA after 48 hours and detect its RSV and IL-4 gene performance amount in the mode of RT-PCR.

See also Fig. 6, for detecting the IL-6 secretory volume with ELISA.Pre-treatment 0,100,400 μ g/ml JINZHENGU protein infect RSV (moi=3) after 2 hours more respectively in human laryngeal cancer cell HEp-2, collect culture fluid after 24 hours and detect its IL-6 concentration in the mode of ELISA.

See also Fig. 7, for west ink dot method assessment FIP-fve for NF-κ B that RSV was caused and the impact of I κ B α phosphorylation.In A549 pulmonary carcinoma epithelial cell, infect RSV, and give 200 μ g/ml JINZHENGU protein, after 4 hours and 8 hours, isolate total protein in the cell respectively, and observe the albumen performance amount of phosphorylation NF-κ B (p-NF-κ B) and phosphorylation I κ B α (p-I κ B α) with west ink dot method.

See also Fig. 8, for immune Fluorescent Staining Observation FIP-fve for rsv infection after NF-κ B enter the impact of nucleus situation.Pre-treatment 200 μ g/ml JINZHENGU protein are after 2 hours in Human Lung Cancer epithelial cell A549, infect the RSV 24 hours of various dose, and with specificity fluorescent antibody detecting RSV, blue-fluorescence partly are nucleus again, and red fluorescence is NF-κ B.

See also Fig. 9, after stimulating via RSV, intracellular IKK complex can be with I κ B phosphorylation, and the I κ B that is phosphorylated can degrade, and NF-κ B is released, on the other hand, NF-κ B is also simultaneously by IKK complex phosphorylation and enter in the nucleus, NF-κ B enter behind the nucleus can bond (for example: transcribing on position IL-4), the gene performance will be activated, and so that infected cell generation inflammatory response to many inflammation material related genes.And the result presentation of this research goes out FIP-fve and may have the reduction rsv infection, and then reduces the potentiality that the inflammation material produces.

See also Figure 10, for observing JINZHENGU and Ganoderma lucidum immunoregulation protein for infecting the over-reactive impact of RSV Respiratory Tract of Mice.Give in advance per 12 hours of the JINZHENGU protein of every oral 200ug of mice or 50 μ g Ganoderma albumen once, give 10 with the nasal cavity titration mode after 48 hours ⁸The RSV virus of PFU, and continue to give oral protein, after the sensitization the 6th day with mice be exposed to variable concentrations be respectively 0 (saline), 5,10,20 and the vaporific methacholine of 40mg/ml in, and carry out the Respiratory Tract of Mice overreaction and measure (AHR).

See also Figure 11, for observing JINZHENGU and Ganoderma lucidum immunoregulation protein for the impact of infecting RSV mouse lung IL-6 content.Give in advance per 12 hours of the JINZHENGU protein of every oral 200 μ g of mice or 50 μ g Ganoderma albumen once, give the RSV virus of 108PFU after 48 hours with the nasal cavity titration mode, and continue to give oral protein, collect the mouse lung irrigating solution on the 6th day after the sensitization and detect IL-6 concentration with ELISA.

Finding by above experiment can establishment RSV Viral infection after processing FIP-fve.On the other hand, past studies have pointed out, RSV can irritation cell in phosphorylation and the degraded of I κ B α, and impel NF-κ B to enter in the nucleus and be attached in the transcription factor bond district of gene, make cell produce inflammatory response.In the present invention, phosphorylation with west ink dot method detecting I κ B α and NF-kB protein, after finding to process FIP-fve, the performance amount of both phosphorylated proteins has repressed situation, further with the distribution situation of confocal microscopic examination NF-κ B in cell, the result shows that FIP-fve can obviously reduce the NF-κ B that is caused by RSV and advance nuclear, and the relevant inflammatory factor IL-4 performance amount of NF-κ B also obviously descends.

Comprehensive above experimental result finds that JINZHENGU protein FIP-fve has the reduction rsv infection, and then reduces the curative effect of the inflammation material generation of NF-κ B path coherence.At last, carry out zootype with the BALA/C mice, find the mice of oral JINZHENGU protein, can suppress the asthma phenomenon that is caused after the virus with nasal cavity titration RSV.

Claims

1. oral true Mushrooms immune modulator comprises: gold needle mushroom immunomodulatory protein or Ganoderma lucidum immunoregulation protein, can suppress the respiratory tract overreaction that RSV causes.

The gold needle mushroom immunomodulatory protein phosphorylation that can suppress the NF-κ B that RSV induces with enter nucleus.

3. oral gold needle mushroom immunomodulatory protein can suppress inflammatory response associated hormone and the viral performance amount of reduction that RSV induces.