CN101648992B - Small peptide B with antibacterial and antitumor functions and applications thereof - Google Patents

Abstract

The invention discloses small peptide B with antibacterial and antitumor activities. The peptide B has an amino acid sequence shown in SEQ ID NO.1, has a peptide sequence with antitumor and antibacterial activities at low concentrations and has no effect on normal growth of cells of humans at corresponding concentrations. The invention also discloses applications of the small peptide in preparing antitumor and anti-Gram negative bacteria drugs, in preparing genetically modified organisms and in preparing additives for feed, food and cosmetics.

Description

Have antibacterial and little peptide B and application thereof antitumous effect

Technical field

The present invention relates to little peptide and application thereof, relate in particular to a kind of little peptide B and application thereof, belong to biomedical sector with antibacterial and anticancer double activity.

Background technology

Cancer is a main cause of death in the whole world.2004, the cancer mortality number reached 7,400,000 (account for all death tolls 13%).In various cancers, lung cancer is one of common malignancy, and nearly many decades M ﹠ M all has the trend that obviously increases, and has become one of the highest cancer of mortality ratio in China.Chemotherapy and radiation is the main method of the big treatment of two outside excision cancer.Present employed most chemotherapeutics, also toxic to normal cell, particularly to the vigorous cell of those metabolism, as medullary cell and mucous membrane cell.So the problem that Internal Medicine-Oncology doctor and pharmacologist are faced is not just sought cytotoxic drug, but to seek under the situation of preserving host's important cells and function thereof can the selectively killing tumour cell medicine.If do not have the resistance phenomenon or lack medicine selectively, the chemotherapy of tumour is probably just as antituberculosis therapy effective (usually can remove infection fully).Be used for human most anticancer chemotherapies and do not reach this ideal state as yet.The tumor drug resistance problem reaches problems such as how overcoming chemotherapy resistance, remains the subject matter that the chemotherapy scholar pays close attention to.

Being extensive use of of antibacterials such as penicillin greatly reduces the mankind and is subjected to infectation of bacteria to cause dead threat.Also produced some new problems, bacterial drug resistance most importantly wherein, the resistance of pathogenic bacterium has day by day seriously threatened human beings'health.Corresponding resistance pathogenic strain has all appearred in all conventional microbiotic, and the microbiotic of seeking brand-new type is an effective way that solves the resistance problem.

In recent decades, the effect of peptide in human body caused the great attention of scientific circles.The experimental results shows that after protein was taken in, complete hydrolysis did not become amino acid, but most of form absorption with peptide finds that simultaneously the absorbed speed of polypeptide is than fast with the amino acid of forming.Polypeptide relates to the every field such as hormone, nerve, cell growth and reproduction of human body, can be widely used in medical treatment, health, health care, food,, aspect such as makeup.The principal feature of polypeptide drug is that consumption is little, and biological activity is strong, and disease such as cardiovascular and metabolism has significant curative effect and uses future widely to cancer, autoimmune disorder, hypomnesis, insane, hypertension and some.Along with the development of medical science and biochemical technology, polypeptide drug will become 21 century important diagnostic, monitoring, prevention and curative.Therefore, it is significant to carry out the research of polypeptide drugs.The research of polypeptide drug mainly comprises tumor protein p53, germ resistance bioactive peptide, polypeptide targeted drug, polypeptide vaccine, cytokine simulating peptide, diagnoses with 7 big classes such as polypeptide and other medicinal little peptides.

Summary of the invention

Based on the fundamental characteristics of peptide medicament and the needs in cancer and the infectation of bacteria clinical treatment, the purpose of this invention is to provide a kind of little peptide (the little peptide B of called after) and application thereof with antibacterial and anticancer double activity.

Little peptide of the present invention has the activity of antitumor cell growth and bacteria growth, with under the isoconcentration to human normal cell line growth unrestraint activity.Can be used for the treatment of cancer and infectation of bacteria and as the additive of industries such as animals and plants transgenosis source sequence and feed and food, cosmetic.

Little peptide B with antibacterial and anticancer double activity of the present invention is characterized in that: it is the aminoacid sequence shown in the SEQ ID NO.1; Information shown in it is:

(a) sequence signature

* length: 7 amino acid

* type: amino acid

* chain: strand

* topological framework: linearity

(b) molecule type: protein

(c) sequence description: SEQ ID NO.1

Ala?Ile?Ala?Cys?Cys?Arg?Arg。

Polypeptide amino acid number provided by the invention does not need any modification to connect less than 10, be linear polypeptide, and synthetic is convenient; Evidence has two kinds of functions of antibacterial and anticancer cell growth, simultaneously to the human normal cell line nontoxicity.

The application of little peptide B of the present invention in the preparation cancer therapy drug.

Functional experiment shows: little peptide B of the present invention can suppress the growth of lung cancer, cancer of the stomach, cervical cancer cell strain, points out it to have important development and application in the cancer clinical treatment and is worth.

In the application in the above-mentioned preparation cancer therapy drug, described cancer therapy drug is anti-lung-cancer medicament preferably.

Further functional experiment shows: little peptide B of the present invention is to lung cancer cell line and people's lung cancer taxol resistant strain H460 _TaxRGrowth have than the obvious suppression effect, with no influential under the isoconcentration, disclose little peptide B of the present invention and lay a good foundation and in the lung cancer clinical treatment, have the application prospect of Guan Kuo for the preparation treatment has medicine that the lung cancer of taxol resistance is correlated with to people's normal fibroblast growth.

The application of little peptide B of the present invention in the preparation antibacterials.

Functional experiment shows: little peptide B of the present invention can suppress the growth of various pathogens such as gram-positive microorganism, Gram-negative bacteria and mould, for the research and development of novel antibacterial medicine provide the foundation.

In the application of above-mentioned preparation antibacterials, the preferably anti-Gram negative bacteria drugs of described antibacterials.

The application of little peptide B of the present invention in the preparation genetically modified organism.

Experiment confirms: little peptide B of the present invention has antibacterial and anticancer cell growth effect; Therefore those of ordinary skills can be used as functional gene and are used to prepare transgenic animal and plant, creation new variety and expressive function albumen according to the anti-nucleotide sequence that forms that pushes away of the corresponding relation of amino acid and codon.

The application of little peptide B of the present invention in preparation feed, food or used for cosmetic additive.

In view of little peptide B of the present invention has antibacterial and anticancer cell growth effect; Therefore after those of ordinary skills can manually synthesize in a large number, in industry and agriculture production, use as the feed that fungistatic effect is arranged, food or used for cosmetic additive.

Description of drawings

Fig. 1 .2352 peptide (little peptide B) detects the restraining effect of H460 and the growth of NHFB cell.

Fig. 2 .2352 peptide (little peptide B) detects the restraining effect of intestinal bacteria growth.

Embodiment

Embodiment 1:

The design of polypeptide is with synthetic

1, extracts international main antibacterial peptide database (http://aps.unmc.edu/AP/main.php; Http:// penbase.immunaqua.com/; Http:// www.bbcm.univ.trieste.it/; The sequence of the antibacterial peptide that lands http://www.imtech.res.in/raghava/antibp/) is analyzed the corresponding relation of its primary structure and active relation, designs artificial short peptide sequence simultaneously.

2, utilize protein analysis database http://www.expasy.org/, the similarity of the secondary structure of the peptide sequence of analysis known activity and the peptide sequence of design is in conjunction with other the physical-chemical parameters prediction carrying out correction of implementation sequence.

3, it is synthetic or utilize automatic Peptide synthesizer to synthesize complete sequence that peptide sequence is delivered to biotech firm.HPLC detects purity and is greater than 96%, determines that by mass spectrometric detection polypeptide synthesizes quality simultaneously.Sample is preserved with lyophilised state.

4, the polypeptide after synthetic carries out antibacterial and anti-cancer function detects screening.

5, by Function detection, the final peptide sequence that obtains to have antibacterial and anticancer dual-use function, the test sequence number is No. 2352 peptides, the little peptide B of called after among the present invention, its aminoacid sequence are Ala Ile Ala Cys Cys Arg Arg.

Embodiment 2:

Utilize mtt assay to detect the external tumor-suppression activity of polypeptide

2.1 material and reagent

1) No. 2352 peptides (the little peptide B of called after among the present invention), Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is synthetic

2) H460 cell (people's nonsmall-cell lung cancer strain system), H460 _TaxRCell (human lung carcinoma cell H460 strain system, anti-taxol), NHFB cell (people's normal fibroblast strain system), Hela cell (human cervical carcinoma cell), SGC-7901 cell (human stomach cancer cell line system), Chinese Academy of Sciences's cell bank/Shanghai Inst. of Life Science, CAS cell resource center.

3) RPMI1640 substratum is U.S. GIBCO company product

4) DMEM substratum is U.S. HyClone company product

5) newborn calf serum is Hangzhou folium ilicis chinensis company product

6) dimethyl sulfoxide (DMSO) (DMSO), tetramethyl-azo azoles (MTT) and trypsinase are Sigma company product.

2.2 experiment

1) with diluent (aseptic PBS or sterilized water or corresponding cell culture medium) No. 2351 peptides are diluted to the initial measurement concentration of 1mM, filtration sterilization, after the packing-20 ℃ frozen.

2) cell cultures: cancer cells (contains 100U/mL penicillin and Streptomycin sulphate) in the RPMI RPMI-1640 that contains 10% calf serum cultivates, and goes down to posterity 1 time in the every 2-3 of cell days; Normal cell is cultivated in the DMEM substratum, goes down to posterity once in 4-5 days.When the cell growth is about 80% fusion states, adopt the method digestion isolated cell of trysinization, with corresponding medium preparation single cell suspension and adjust suitable cell concn, divide bottle to go down to posterity, placing 37 ℃, volume fraction is 5%CO: the humidifying environment cultivate, use the logarithmic phase cell during experiment.

3) cell adherent after, discard Central Plains, hole substratum, experimental group adds the 100 μ L substratum that contain gradient dilution (on the starting point concentration basis with 10 times of volume dilution) peptide respectively.Negative control group adds the same volume substratum that contains equal concentration dilution liquid, CO ₂Cultivate 48h in the constant incubator.

4) microscopically observation of cell growth conditions, every then hole add 20 μ l (5mg/mL) MTT solution.Cultivate 4h.

5) carefully exhaust liquid in the hole, every hole adds 150 μ l DMSO, places jog 10min on the horizontal shaking table of WD-9405B type, so that purple crystal fully is dissolved among the DMSO.

6) use the Model680 microplate reader to measure the light absorption value of every hole at the 570nm place, record data.

7) utilize sigmaplot10 and Excel software analysis processing data.

The result shows, No. 2352 peptides (the little peptide B of called after among the present invention) can suppress the growth of lung cancer, cancer of the stomach, cervical cancer cell strain under less than 1mM concentration, and it is less to suppress the effect concentration dependent.The survival rate of lung cancer cell line H460 all is about 70% when the lower concentration of 0.1mM and 0.1 μ M, is about 110% with isoconcentration servant normal fibroblast survival rate.The visible growth of cancer cells speed of microscopic examination reduces, and the generation of vesicle phenomenon appears in cell edges.The results are shown in accompanying drawing 1.

The selective killing ability to tumour cell that little peptide B sequence of the present invention has makes its clinical medicine that can be directly used in the preparation cancer therapy, and the clinical medicine that perhaps is developed as combined chemotherapy uses, and application promise in clinical practice is arranged.

Embodiment 3:

Utilize the liquid growth inhibition assay to detect the bacteriostatic activity of polypeptide

3.1 material, reagent

1) penbritin (Amp) is a solarbio company product, and disposable filter is U.S. PALL company product, and other medicine and reagent is homemade analytical pure.

2) select gram-positive microorganism for use: subtilis (Bacillus subtilis (Ehrenberg) Cohn), streptococcus aureus (Staphyloccocus aureus Rosenbach), micrococcus luteus (Micrococcus luteus), bacillus thuringiensis (Bacillus thuringiensis), Bacillus megatherium (Bacillus megaterium); Gram-negative bacteria: intestinal bacteria (Escherichia coli), vibrios (Vibrio), klebsiella pneumoniae (Klebsiellapneumoniae); Fungi: Fusarinm solani (Fusarium solani), Fusarium oxysporum (Fusarium oxysporum), melon anthrax bacteria (Colletetrichum lagenarium), apple anthrax bacteria (Gloeosporium album) verticillium dahliae (Verticillium dahliae) are tested.Microorganism National Key Laboratory of Shandong University bacterial classification chamber provides.

3) confining liquid: 0.4%BSA (w/v), 0.02% Glacial acetic acid, the degerming of preparation after-filtration.

4) PB liquid nutrient medium

Tryptones (Tryptone) 10g/L

Sodium-chlor (NaCl) 5g/L PH 7.4-7.6

3.2 experiment

1) in the employed hole of aseptic 96 hole enzyme plates, adds 200 μ L confining liquids, room temperature sealing 24h.

2), change to cultivate making bacterium be in logarithmic phase with the different bacterium of PB liquid nutrient medium incubated overnight.Adjust bacterium liquid OD with the PB substratum ₆₀₀Be about 0.002.

3) discard confining liquid, every hole adds 90 μ L OD ₆₀₀Be about 0.222 contain bacterium PB substratum, add the of the present invention little peptide B of the different concns gradient (starting point concentration 1mM) that 10 μ L sterilized waters prepare again.Negative control is that 90 μ L contain bacterium culture medium+10 μ L sterilized waters; Positive control is that to contain bacterium culture medium+10 μ L concentration be 0.25mg/mL penbritin solution (final concentration is 0.025mg/mL, about 62 μ M) to 90 μ l.

4) enzyme plate that will add bacterium liquid and medicine places 28 ℃ of shaking tables, 100rpm constant temperature culture.0h, 3h, 12h, 15h, 24h, 36h after cultivation respectively measure the 630nm light absorption value (OD in every hole with BIOTEK ELX800 microplate reader ₆₃₀).

5) utilize sigmaplot10 and Excel software analysis processing data.

The result shows that little peptide B of the present invention is not obvious to the gram-positive microorganism fungistatic effect, is subjected to concentration affects little to Gram-negative bacteria (is example with intestinal bacteria) fungistatic effect.Test shows little peptide B of the present invention when being low to moderate the concentration of 0.5 μ M, hatches with Gram-negative bacteria and shows bacteriostatic activity after 3 hours, at 15 hours maximum antibacterial vigor of performance.Concrete outcome is seen accompanying drawing 2.Little peptide B of the present invention is similar to the fungistatic effect of fungi and Gram-negative bacteria.Pointed out this little peptide to be expected to be used to prepare the relevant clinical novel antibacterial medicine of using.

Embodiment 4:

Small peptide sequence is used to produce the nucleotide sequence and the using method thereof of genetically modified organism

Because little peptide B of the present invention is a linear order, do not have any modification, according to the password sublist, this little peptide can obtain the corresponding nucleic acids sequence by the arbitrary combination of the triplet code base sequence of its amino acid correspondence, according to the degeneracy of codon, this sequence nucleotide sequence that can obtain can reach 7 relatively ⁴About.Then with this nucleotide sequence by carrier or be injected directly in animal, plant or the microbe that needs transgene, make genetic information carry out accurate translation and just can.Existing in this respect ripe, detailed method is looked concrete biological variety and is carried out with reference to operational guidance.

For example: with the transgenic microorganism is example, be summarized as follows, can obtain following nucleotide sequence by little peptide B of the present invention: 5 ' ttacatcatgctattgct 3 ', behind this nucleotide sequence synthetic, be connected into engineering carrier such as colibacillary expression vector pET30a with ordinary method, transform BL21 (DE3), after obtaining positive colony, incubated overnight, change and cultivate after 3-4 hour, use the IPTG abduction delivering, just can obtain to change over to this nucleic acid fragment, the transgenic microorganism new variety that have allogenic polypeptide to produce simultaneously.

Sequence table

＜110〉Shandong University

＜120〉have antibacterial and little peptide B and application thereof antitumous effect

<141>2009-9-10

<160>1

<210>1

<211>7

<212>PRT

＜221〉artificial sequence

<222>(1)…(7)

<400>1

Ala?Ile?Ala?Cys?Cys?Arg?Arg

5

Claims (1)

1. little peptide B with antibacterial and anticancer double activity, it is characterized in that: its aminoacid sequence is shown in SEQ ID NO.1; Information shown in it is:

(a) sequence signature

* length: 7 amino acid

* type: amino acid

* chain: strand

* topological framework: linearity

(b) molecule type: protein

(c) sequence description: SEQ ID NO.1

Ala?Ile?Ala?Cys?Cys?Arg?Arg。

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