CN106188254B - A kind of immune modulator and its application - Google Patents
A kind of immune modulator and its application Download PDFInfo
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- CN106188254B CN106188254B CN201610565579.5A CN201610565579A CN106188254B CN 106188254 B CN106188254 B CN 106188254B CN 201610565579 A CN201610565579 A CN 201610565579A CN 106188254 B CN106188254 B CN 106188254B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
The present invention provides a kind of new immune modulator with genetic engineering means.The present invention also provides and synthesized the gene of the above-mentioned albumen of coding.It is verified by experiments, albumen of the invention is low with the most fungal immunomodulatory protein homologys having found, is a kind of novel immunological regulation and antineoplastic biologic preparation.
Description
Technical field:
The present invention relates to genetic engineering fields, and in particular, to a kind of immune modulator and its gene.
Background technique:
Obtained from fungi immune modulator (Fungal immunomodulatory proteins, hereinafter referred to as
It is a kind of small protein with a variety of important physiological functions such as strengthen immunity, antiallergy, antitumor for FIPs).
The FIPs albumen that can be developed and used is obtained from the fungi for be completed gene order-checking by gene engineering method, both
The type that FIPs family can be enriched, more can provide new immunomodulator for the health care and treatment of human health.
Summary of the invention:
The purpose of the present invention is the FIPs albumen that can be developed and used is obtained from the fungi for be completed gene order-checking.
The present inventor is screened a kind of from fungi Stachybotrys by bioinformatics method
The albumen FIP-sch2 of chlorohalonata IBT 40285 is typical immune modulator.
The fungal immunomodulatory protein FIP-sch2,112 amino acid of overall length, theoretical molecular weight 12.555kDa,
Amino acid sequence is as shown in SEQ ID NO.1.
FIP-sch2 albumen is a kind of novel fungal immunomodulatory protein.The present inventor is by existing its amino acid sequence
Carry out BLAST in GenBank and compare discovery: FIP-sch2 albumen is exempted from small spore ganoderma lucidum, red ganoderma, Ganoderma Sinense and needle mushroom fungi
The sequence homology of epidemic disease regulatory protein GMI, FIP-gja, LZ-8 and FIP-fve are only respectively 59.5%, 52.3%, 54.1% and
53.6%.Illustrate that FIP-sch2 is a kind of new fungal immunomodulatory protein.
The present invention also provides and synthesized the gene of the above-mentioned fungal immunomodulatory protein FIP-sch2 of coding: drawn by a pair
Object FIP-sch2-F (EcoRI): 5'-CCC GAA TTC TCA GCT CCA ACC-3' and FIP-sch2-R (XhoI): 5'-
AAA AAA CTC GAG CTT CCA TTG G-3', has cloned this fungal immunomodulatory protein with the method for gene chemical synthesis
The gene of FIP-sch2, DNA complete sequence analysis the result shows that, fip-sch2 overall length 339bp, DNA sequence dna such as SEQ ID NO.2
It is shown.
The present invention also provides the recombinant expression carriers comprising albumen FIP-sch2 gene.Method is by fungi of the invention
Immune modulator FIP-sch2 gene is inserted between suitable restriction enzyme cleavage sites of the expression vector, and makes its nucleotide sequence
It is operable to be linked to the expression control sequence.Preferred expression vector is pGEX-6T-1.
As the most preferred embodiment of the invention, between the suitable restriction enzyme site of expression vector
It is to obtain recombination large intestine expression vector pGEX-fip- between EcoRI the and XhoI restriction enzyme site on pGEX-6T-1
sch2。
Above-mentioned recombinant expression carrier is converted into host cell E. coli.The host cell is preferably Escherichia coli
Rosetta obtains recombinant bacterial strain Rosetta (pGEX-fip-sch2).
The present invention also provides the recombinant bacterial strains comprising fungal immunomodulatory protein FIP-sch2 gene, preferably recombinant bacterium
Strain Rosetta (pGEX-fip-sch2).
The present invention also provides the methods for preparing fungal immunomodulatory protein FIP-sch2, comprising the following steps:
1) recombinant expression carrier of the gene containing FIP-sch2 is converted into host cell, obtains recombinant bacterial strain；
2) recombinant bacterial strain is cultivated, the expression of recombinant fungus immune modulator FIP-sch2 is induced；And
3) it recycles and purifies expressed fungal immunomodulatory protein FIP-sch2.
The present invention by it is following experiment confirm present invention discover that new albumen function.
1, anti tumor activity in vitro
Experimental result: FIP-sch2 has toxic effect to Lung Adenocarcinoma A 549 Cell, and semilethal measures IC50For 9.48 μ g/
ML (Fig. 2)；The fungal immunomodulatory protein comparative experiments of the separate sources of 8 μ g/ml the result shows that, poison of the FIP-sch2 to A549
Property effect it is suitable with Ganoderma lucidum immunoregulation protein LZ-8, be significantly higher than gold needle mushroom immunomodulatory protein FIP-fve.
Conclusion: FIP-sch2 has extremely strong high toxicity effect (embodiment 3) to tumour cell.
2, inducing apoptosis of tumour cell is analyzed
Experimental result: FIPs has apoptotic effect to A549 cell, at FIP-sch2, LZ-8 and FIP-fve of 8 μ g/ml
After managing A549 cell for 24 hours, apoptosis rate is respectively 50.19%, 38.82%, 10.88% (Fig. 3).
Conclusion: FIP-sch2 has apoptotic effect to tumour cell A549, and apoptosis-induced effect is similar to LZ-8, but shows
It writes and is higher than FIP-fve, be the anti-tumor drug (embodiment 4) of great potential.
3, inhibit tumor cell migration detection
Experimental result: the FIP-sch2 of 8 μ g/ml promotes wound healing situation to be slightly better than LZ-8, is significantly smaller than FIP-fve.
Conclusion: FIP-sch2 can inhibit tumour cell A549 to migrate, and FIP-sch2 acts on the inhibition of metastasis of A549 and omits
It is faint in LZ-8, be but significantly stronger than FIP-fve.
Advantages and beneficial effects of the present invention:
1, a considerable amount of immune modulator matter FIP-sch2 sterlings can be provided, clinic is able to satisfy completely and is answered with medicine
Use demand.
FIP-sch2 realizes high efficient expression in the escherichia expression system, and expression quantity is 31mg/L (embodiment 2)；Through
After column purification, it is pure (Fig. 1) that the content of protein reaches electrophoresis.And milligram grade usually can only be extracted from several kilograms of edible mushrooms at present
Albumen.
2, FIP-sch2 has anti-tumor activity, has treatment use potentiality.
Anti-tumor effect testing result shows that FIP-sch2 has toxic effect (embodiment 3) to A549, can induce
A549 apoptosis (embodiment 4) inhibits A549 migration (embodiment 5)；Its anti-tumor effect and Ganoderma lucidum immunoregulation protein LZ-8 phase
When, but it is significantly higher than gold needle mushroom immunomodulatory protein FIP-fve, great application potential.
3, the immune modulator of a new originated from fungus is provided with genetic engineering means for the first time.
It yet there are no report due to carrying out industrialization production fungal immunomodulatory protein FIP-sch2 product with genetic engineering means
Road.The present invention provides the immune modulator FIP-sch2 of a new originated from fungus for the first time.Moreover, skill according to the present invention
Art scheme, which can be realized, produces fungal immunomodulatory protein FIP-sch2 using genetic engineering means.
Detailed description of the invention:
FIG. 1 to FIG. 4 is to carry out analysis and function reality to the fungal immunomodulatory protein FIP-sch2 of present invention purifying preparation
It tests, in which:
Fig. 1 is 75 gel filtration of superdex and SDS-PAGE analysis: where Fig. 1 a is 75 gel filtration of superdex
Analysis, thus it is speculated that FIP-sch2 activated protein is dimer；Fig. 1 b is SDS-PAGE analysis.
Fig. 2 is that FIP-sch2 albumen inhibits tumour cell A549 proliferation results.
Fig. 3 is the protein induced tumour cell A549 apoptosis result of FIP-sch2.
Fig. 4 is that FIP-sch2 albumen inhibits tumour cell A549 migration results.
Specific embodiment
Method of the invention that following examples are for illustration only, does not limit the scope of the invention.
Test material
1, cell strain: human A549 cell lines are bought in BJ Union Hospital's cell resource center.
2, reagent consumptive material
CCK kit: Quan Shijin cytoactive detection kit；
TransDetctTMKit: Quan Shijin cell of Annexin V-FITC/PI Cell Apoptosis Detection
Apoptosis detection kit；
PCR primer synthesis and gene sequencing are completed by Shanghai Sheng Gong biotech firm；
Enzyme: restriction endonuclease EcoRI and XhoI are purchased from TaKaRa company, and ligase is purchased from Invitrogen company；
Prescission Protease is prepared by laboratory；
Instrument: centrifuge, oscillation shaking table, microscope, microplate reader, flow cytometer；
Biochemical reagents: ampicillin, penicillin (sodium salt), streptomycin sulphate, bromjophenol blue, Thiazolyl blue (MTT), tire ox blood
(FBS) clearly, dimethyl sulfoxide (DMSO) are Sigma product.
3, culture medium
10%FBS, 100U/ml penicillin, 100 μ g/ml streptomysins, 0.22 μm of filter membrane mistake is added in DMEM in high glucose culture solution
4 DEG C of preservations after filter sterilization.
Illustrate: not making the experimental methods of molecular biology illustrated in following embodiment, referring to " Molecular Cloning: A Laboratory
Guide " specific method listed in book of (third edition) J. Pehanorm Brooker one carries out, or according to kit and product description
It carries out.
Anti-tumor effect detection in the present embodiment, with Ganoderma lucidum immunoregulation protein (LZ-8) and needle mushroom immunological regulation
Albumen (FIP-fve) is positive control.
The screening of 1 fungal immunomodulatory protein FIP-sch2 encoding gene of embodiment, clone and expression vector establishment
Using gold needle mushroom immunomodulatory protein FIP-fve as bait, BLAST ratio is carried out in NCBI fungal gene group database
It is right, discovery and FIP-fve homology in 40285 genome of ascomycetous fungus Stachybotrys chlorohalonata IBT
For 53.6% FIP-sch2 encoding gene, full length gene 339bp (the additional terminator codon TAA of 336bp), DNA sequence
Column are as shown in SEQ ID NO.2.After codon optimization, gene is synthesized.
Design and synthesize primers F IP-sch2-F (EcoRI): 5'-CCC GAA TTC TCA GCT CCA ACC-3' and
FIP-sch2-R (XhoI): 5'-AAA AAA CTC GAG CTT CCA TTG G-3' synthesizes gene with the above primer amplification.
With EcoRI and XhoI digestion pcr amplification product, target gene fip-sch2 is obtained, target gene is connected to through identical digestion
Expression vector pGEX-6T-1, obtain the recombinant expression carrier pGEX- containing fungal immunomodulatory protein FIP-sch2 gene
Fip-sch2 converts Escherichia coli Rosetta, obtains recombinant bacterial strain Rosetta (pGEX-fip-sch2).
2 fungal immunomodulatory protein FIP-sch2 expression and purification of embodiment
Positive transformant Rosetta (pGEX-fip-sch2) bacterial strain is taken, the LB that 20mL contains 50 μ g/ml ammonia benzyls is inoculated in
In culture solution, 37 DEG C of 200rpm are activated overnight.Next day connects bacterium amount with 2%, is inoculated in the LB that 200mL contains 50 μ g/ml ammonia benzyls
In culture solution, 37 DEG C of 200rpm are cultivated to OD600For 0.8-1.0, after adding 0.1mM IPTG, 20 DEG C of 200rpm to induce 16h overnight,
Thalline were collected by centrifugation.The PBS of 0.2 volume dissolves thallus, and supernatant is collected by centrifugation in ultrasonic disruption.After GST column purification, 100U/ is used
The overnight digestion of 4 DEG C of Prescission Protease of ml, removes GST label.Cross Q column and molecular sieve purification FIP-sch2 egg
It is white.
Experimental result:
Theoretical molecular weight through above expression system expression FIP-sch2 albumen is 14.1KDa (including partial vector sequences
With restriction site translated amino acid)；FIP-sch2 is in E. coli, expression quantity 31mg/L；It is pure through column
After change, it is pure (Fig. 1) that the content of protein reaches electrophoresis.
Conclusion:
FIP-sch2 realizes high efficient expression in the escherichia expression system, therefore can quickly provide a considerable amount of
Protein sterling, to meet clinical and medical applications demand.
Embodiment 3CCK method measures fungal immunomodulatory protein FIP-sch2 anti tumor activity in vitro
Experimental method:
1) the secondary culture A549 cell in DMEM culture solution counts tumour cell, is diluted to final concentration of 2.5 × 105
Cell/ml.
2) 100 μ l tumor cell suspensions are added in every hole in 96 well culture plates, and after continuing culture for 24 hours, 100 μ l weight is added
Histone sample, so that the μ g/ml of final concentration of 0,1,2,4,8,16,32 and 64.8 μ g/mL of same procedure preparation are added simultaneously
Ganoderma lucidum and gold needle mushroom immunomodulatory protein LZ-8 and FIP-fve do positive control.5, each sample parallel.
3) after mixing, it is put in 5%CO2, cultivate 24 hours in 37 DEG C of incubators.
4) after 24 hours, CCK method detects cell activity (concrete operations are referring to specification).
Experimental result:
FIP-sch2 has toxic effect to Lung Adenocarcinoma A 549 Cell, and semilethal measures IC50For 9.48 μ g/mL (Fig. 2)；8μ
The fungal immunomodulatory protein comparative experiments of the separate sources of g/ml the result shows that, toxic effect and spirit of the FIP-sch2 to A549
Sesame immune modulator LZ-8 is suitable, is but significantly higher than gold needle mushroom immunomodulatory protein FIP-fve.
Conclusion:
FIP-sch2 has extremely strong high toxicity effect and with potential applications to tumour cell.
The analysis of 4 fungal immunomodulatory protein FIP-sch2 inducing apoptosis of tumour cell of embodiment
Experimental method:
1) in 5%CO2, DMEM culture solution culture A549 cell is used in 37 DEG C of incubators, cell count keeps its final concentration of
l x106Cell/ml.
2) 0.8ml tumor cell suspension is added in every hole in 6 well culture plates, after continuing culture for 24 hours, is separately added into
Recombinant fungus immune modulator FIP-sch2, LZ-8 and FIP-fve of the final concentration of 8 μ g/ml of 0.2ml, with normal growth
Tumour cell does negative control；In 5%CO2, cultivate 24 hours in 37 DEG C of incubators.
3) supernatant, pancreatin digestion, 3000rpm, room temperature centrifugation 5 minutes, collection tumour cell are removed.The inspection of apoptosis detection kit
Apoptosis is surveyed, concrete operations are referring to specification.
Experimental result:
FIPs has apoptotic effect to A549 cell, and FIP-sch2, LZ-8 and FIP-fve of 8 μ g/ml handle A549 cell
After for 24 hours, apoptosis rate is respectively 50.19%, 38.82%, 10.88% (Fig. 3).
Conclusion:
FIP-sch2 has apoptotic effect to tumour cell A549, and apoptosis-induced effect is similar to LZ-8, significantly high
It is the anti-tumor drug of great potential in FIP-fve.
5 fungal immunomodulatory protein FIP-sch2 of embodiment inhibits tumor cell migration detection
Experimental method:
4) in 5%CO2, DMEM culture solution culture A549 cell is used in 37 DEG C of incubators, cell count keeps its final concentration of
5x105Cell/ml.
5) above-mentioned tumor cell suspension is added in 6 well culture plates, in 5%CO2, culture 24 is small in 37 DEG C of incubators
When, plating cells cover with, and are crossed among culture plate cell monolayer with tip, PBS washes away suspension cell and fragment.
6) it is separately added into recombinant fungus immune modulator FIP-sch2, LZ-8 and FIP-fve of final concentration of 8 μ g/ml,
Negative control is done with the tumour cell of normal growth；In 5%CO2, cultivate 24 hours in 37 DEG C of incubators.Micro- sem observation cell
Growing state, and take pictures.
Experimental result:
After continuing culture for 24 hours, untreated cell (NC) marks wound and obviously heals, after the LZ-8 processing of 8 μ g/ml, wound
The phenomenon that heals is unobvious；The FIP-sch2 wound healing situation of 8 μ g/ml is more significant；The FIP-fve processing A549 of 8 μ g/ml is thin
Born of the same parents for 24 hours after, wound healing situation is extremely significant, very much like (Fig. 4) with negative control.
Conclusion:
FIP-sch2 can inhibit tumour cell A549 to migrate, and FIP-sch2 acts on the inhibition of metastasis of tumour cell A549
Slightly it is weaker than LZ-8, is but significantly stronger than FIP-fve, is the anti-tumor drug of great potential.
Claims (3)
1. amino acid sequence albumen as shown in SEQ IDNO.1 is preparing the application in anti-tumor agent.
2. application described in claim 1, the tumour is lung cancer.
It is by the gene as shown in SEQ ID NO.2 3. including the recombinant expression carrier of the gene as shown in SEQ ID NO.2
It is inserted between EcoRI the and XhoI restriction enzyme site on expression vector pGEX-6T-1, obtained recombinant expression carrier
pGEX-fip-sch2。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102241751A (en) * | 2011-04-20 | 2011-11-16 | 中国农业科学院饲料研究所 | Novel fungal immunomodulatory protein FIP-NHA with antineoplastic activity and gene thereof |
| CN103980354A (en) * | 2014-05-30 | 2014-08-13 | 中国农业科学院农产品加工研究所 | Immunomodulatory protein FIP-ppl of fungus and gene of protein |
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- 2016-07-18 CN CN201610565579.5A patent/CN106188254B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102241751A (en) * | 2011-04-20 | 2011-11-16 | 中国农业科学院饲料研究所 | Novel fungal immunomodulatory protein FIP-NHA with antineoplastic activity and gene thereof |
| CN103980354A (en) * | 2014-05-30 | 2014-08-13 | 中国农业科学院农产品加工研究所 | Immunomodulatory protein FIP-ppl of fungus and gene of protein |
Non-Patent Citations (1)
| Title |
|---|
| hypothetical protein S40285_08095 [Stachybotrys chlorohalonata IBT 40285];Jeremy Semeiks;《GenBank: KFA61840.1》;20140724;氨基酸序列 * |
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