CN104558121B - A kind of Mercuric chloride antibacterial peptide and its encoding gene and application - Google Patents
A kind of Mercuric chloride antibacterial peptide and its encoding gene and application Download PDFInfo
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Abstract
The present invention provides a kind of Mercuric chloride antibacterial peptide and its encoding gene and application, the sequence of the Mercuric chloride antibacterial peptide such as SEQ ID NO：Shown in 2, encoding gene SEQ ID NO：Shown in 1；The Mercuric chloride antibacterial peptide and its encoding gene can be used for preparing the therapeutic agent of cause pathogeny imcrobe infection disease.Mercuric chloride antibacterial peptide of the present invention has the characteristics that simple structure, artificial synthesized convenience, antibacterial pedigree is wide, antibacterial activity is strong.
Description
Technical field
The invention belongs to gene engineering technology field more particularly to a kind of Mercuric chloride antibacterial peptide and its encoding gene with answer
With.
Background technique
In recent years, with the generation of conventional antibiotic drug resistance, people start the research of growing interest antibacterial peptide.Antibacterial peptide
It is a kind of small peptide with antibacterial activity, is widely present in living nature, is the important defensive substance of congenital immunity, it is anti-with wide spectrum
Bacterium, antiviral, antimycotic, anti parasitic and the bioactivity such as antitumor, and it is not likely to produce drug resistance, mechanism of action has antibiosis
The incomparable superiority of element can kill the bacterium for having generated drug resistance, and energy selective killing tumour cell is without destroying
Normal cell.The skin of amphibian animal forms the three sets of defence system for resisting cause pathogeny imcrobe infection during natural evolution
System, and antibacterial skin peptide is the important component of its innate immune system.The amphibian animal biology ancient as one kind, they
The acquired immune system day after tomorrow it is extremely fragile compared with mammal, they develop in long-term evolution course forms one
Cover the very effective system of defense for resisting microbiological attack, wherein antibacterial frog skin peptide be its innate defence system it is main at
Point, have the characteristics that it is efficient, wide spectrum, be not likely to produce drug resistance.
It has been investigated that antibacterial peptide to Escherichia coli, salmonella, golden yellow streptococcus, Escherichia coli, streptococcus fecalis,
More than 40 categories such as Pseudomonas aeruginosa proteus, Streptococcus viridans, beta hemolysis streptococcus have inhibitory effect, part antibacterial peptide
There are killing or killing effect to fungi.From the antibacterial peptide magainin of Africa xenopus and from Chinese Bombina maxima
Antibacterial peptide maximin is in micro-molar concentration level just to the lethal effect of tumour cell showed different.From Japanese horse
The anti-polyphemusins and tachyplesin of family name's crab hemolymph just show one to AIDS virus in 5 μ g/mL of concentration
Fixed inhibiting effect.Antibacterial maximin 3 from Chinese Bombina maxima skin fights AIDS when concentration is 1.5 μ g/mL
Virus selects coefficient for 7.6.This is the AIDS virus resisting antibacterial peptide in the amphibian animal source reported for the first time.In addition,
The antibacterial peptide dermaseptin that Ye Pao frog skin obtains from South America also shows strong inhibiting effect to hcrpesviruses.It derives from
There is the melittin melitin of honeybee strong red blood cell to dissolve activity；From the antibacterial peptide of amphibian animal, such as China is big
The antibacterial peptide maximin H of the web bell toad and antibacterial peptide bombinH of Bombina orientalis also shows strong red blood cell dissolution activity.
MaximinH can lead to the dissolution of rabbit erythrocyte 100% in 50 μ g/m L.From amphibian animal Pseudisparadoxa
Obtained antibacterial peptide pseudin has adjustment effect to the Apoptosis of control tadpole Tail Morphology of Adult variation.Pseudin is in structure
A kind of upper one section of region with apoptosis factor DEFY shows very high similitude.
The cDNA clone of Mercuric chloride antibacterial peptide gene, the research of sequence analysis are carried out using Mercuric chloride resource, if can be from
The bioactive substance with important value is obtained in Mercuric chloride skin, will be widened the exploitation of Mercuric chloride, be utilized new way, tool
There is important realistic meaning, while arousing people to amphibian animal skin bioreactivity peptide, especially China's characteristic species is special
The concern of molecular diversity, and pay attention to their meanings in biology, biomedical basic research and natural drug exploitation.
Summary of the invention
The main purpose of the present invention is to provide a kind of Mercuric chloride antibacterial peptides.
The encoding gene one of of the invention for being designed to provide above-mentioned Mercuric chloride antibacterial peptide.
Purpose one of of the invention provides above-mentioned Mercuric chloride antibacterial peptide as preparing controlling for cause pathogeny imcrobe infection disease
Treat the application in terms of drug.
Another object of the present invention is to provide the encoding genes of Mercuric chloride antibacterial peptide in treatment cause pathogeny imcrobe infection disease
The application of sick aspect.
The invention is realized in this way a kind of Mercuric chloride antibacterial peptide, sequence such as SEQ ID NO：Shown in 2.Wherein, this hair
Mercuric chloride antibacterial peptide (Frog1- albumen) in bright is a kind of single chain polypeptide of Chinese amphibian animal Mercuric chloride gene coding, point
Son amount 1367.673Da, isoelectric point 6.04.
Invention further provides the encoding gene of above-mentioned Mercuric chloride antibacterial peptide, sequence such as SEQ ID NO：Shown in 1.
The full amino acid sequence of the encoding gene such as SEQ ID NO：Shown in 3, wherein the Mercuric chloride antibacterial peptide is the encoding gene
177th~213 nucleotide.
Invention further provides above-mentioned Mercuric chloride antibacterial peptides as the treatment for preparing cause pathogeny imcrobe infection disease
Application of the encoding gene of application and Mercuric chloride antibacterial peptide in terms of drug in terms for the treatment of cause pathogeny imcrobe infection disease.
The present invention overcomes the deficiencies of the prior art and provide a kind of Mercuric chloride antibacterial peptide and its encoding gene and application, passes through
Mercuric chloride antibacterial is screened to Mercuric chloride antibacterial peptide Total RNAs extraction, RNA concentration mensuration, RNA reverse transcription, design of primers and PCR amplification
Peptide, the amplification Mercuric chloride antibacterial peptide gene 3 ' end RACE and the design of primers at 5 ' ends etc., obtain Mercuric chloride antibacterial peptide gene and extremely compile
The Mercuric chloride antibacterial peptide of code.Wherein, Mercuric chloride antibacterial peptide gene amplimer is：
CaSPI-F：5 '-ATGTTCACCATGAAGAAATCCCT-3 ',
CaSPI-R：5'-ATTCTACAGGCCGAGGCGGCCGACATG-3'.
Obtained positive monoclonal carries out gene nucleotide series measurement, Mercuric chloride antibacterial peptide encoding gene such as SEQ ID NO：
Shown in 1, Mercuric chloride antibacterial peptide such as SEQ ID NO：Shown in 2, Mercuric chloride antibacterial peptide is a kind of single chain polypeptide, molecular weight
1367.673Da, isoelectric point 6.04.The full amino acid sequence of Mercuric chloride antibacterial peptide encoding gene such as SEQ ID NO：Shown in 3,
In, the Mercuric chloride antibacterial peptide is the 177th~213 nucleotide of the encoding gene.
Mercuric chloride antibacterial peptide gene prepares the application of Mercuric chloride antibacterial peptide as genetic engineering.
Compared with the prior art the shortcomings that and deficiency, the invention has the advantages that：The present invention is by Mercuric chloride antibacterial
DNA encoding peptide derives its mature antibacterial peptide amino acid primary sequences using bioinformatics method, and artificial synthesized Mercuric chloride is anti-
Bacterium peptide has significant antibacterial action；Mercuric chloride antibacterial peptide of the present invention simple, artificial synthesized convenience, antibacterial pedigree with structure
Extensively, the strong feature of antibacterial activity.
Detailed description of the invention
Fig. 1 is that 3 ' RACE pcr amplification products identify electrophoretogram in the embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
The clone of 1 Mercuric chloride antibacterial peptide gene of embodiment
1, Mercuric chloride skin mucus Total RNAs extraction
Centrifuge tube, pipette tips, gun case used etc. is extracted to handle through 0.1%DEPC water soaked overnight and high pressure sterilization, it is used
Mortar, glass homogenizer, tweezers and scissors are through 180 DEG C of high-temperature process 2.5h.
Concrete operation step is：
It takes Mercuric chloride skin mucus sample about 100mg that liquid nitrogen grinding is added into powder, pours into glass homogenizer
↓ the further ground sample of 1ml Trizol Reagent is added
Homogenised sample is transferred to 1.5mlEP pipe (DEPC pipe) 15~30 DEG C of placement 5min
↓ 200 μ l chloroforms are added, 3min is acutely vibrated, 4 DEG C, 12000 × g is centrifuged 15min
Supernatant is drawn to new centrifuge tube and addition and the isometric isopropanol of supernatant, is stored at room temperature 10min
↓ 4 DEG C, 12000 × g is centrifuged 10min
It abandons supernatant and the ethyl alcohol cleaning precipitating of 1ml 75% is added
↓ 4 DEG C, 7500 × g is centrifuged 10min
Supernatant is abandoned, drying is placed at room temperature for, adds 20~50 μ l of DEPC water to dissolve, -70 DEG C save backup.
2, RNA concentration mensuration：
The above-mentioned total serum IgE for being dissolved in DEPC water of 1 μ l is taken to be placed in Nanodrop 2000c measurement RNA concentration.It is dense according to surveyed RNA
Added RNA template consumption is 1 μ g when degree adjustment reverse transcription.
3, RNA reverse transcription：
According to following preparation cDNA synthetic reaction system and carry out reverse transcription reaction.
Reaction condition：
42℃1h
↓
70℃15min
Reaction solution is stored in -20 DEG C after reaction and is used for PCR amplification.
4, design of primers and PCR amplification condition：
According to the allied species such as batrachia announced in GenBanK code area conserved sequence and amino terminal sequencing result,
Utilize 5.0 software design upstream and downstream primer of Premier.
KFB-F：TGTAGGTGCCAAGGGTAG
KFB-R：TGAACAGGTCCACAGAGC
Primer is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Using antibacterial peptide cDNA as template, PCR amplification, PCR amplification system such as following table are carried out：
5, agarose gel electrophoresis：
According to isolation of DNA fragments size, 1% Ago-Gel is prepared with 1 × TAE, heating and melting is cooled in micro-wave oven
EB to final concentration of 0.5 μ g/ml is added at 50 DEG C or so, pours into gel after mixing well in the plastic plate of miniature electrophoresis tank,
After cooled and solidified, gel is put into electrophoresis tank.The sample of appropriate amount is taken to mix with 6 × electrophoresis sample-loading buffer, when loading makes
With a certain size DNA Marker.Electrophoretic buffer is 1 × TAE, and voltage is selected as 120V, moves to bromophenol blue indicating strip solidifying
Stop electrophoresis when 2/3 position of glue, is observed under ultraviolet transilluminator.
6, DNA gel purifies：
Target fragment is recycled using the raw work SanPrep pillar DNA plastic recovery kit in Shanghai, specific step is as follows：
(1) agarose gel electrophoresis detects and is tapped and recovered purpose band rapidly to 1.5ml centrifuge tube.
(2) 3 times of blob of viscose weight of Buffer B2,50 DEG C of water-bath 5~10min colloidal sols are added.
(3) sol solutions are moved into adsorption column, 8000 × g is centrifuged 30s, outwells liquid in collecting pipe.
(4) 500 μ l Wash Solution, 9000 × g centrifugation 30s are added, outwell liquid in collecting pipe.
(5) it is primary that step (4) are repeated.
(6) suction attached column is centrifuged 1min in 9000 × g.
(7) adsorption column is put into a clean 1.5ml centrifuge tube, 15~4l μ Elution is added in adsorbed film center
Buffer after being stored at room temperature 1min, is centrifuged 1min.It is spare in -20 DEG C to save DNA solution in pipe.
7, connection reaction：
Coupled reaction system and 16 DEG C of reaction 4h are established in 0.5ml centrifuge tube by following conditions, connection liquid is for converting.
10 μ l coupled reaction systems are as follows：
8, the competence preparation of bacillus coli DH 5 alpha：
(1) the E.coli BL21 bacterium solution saved in -80 DEG C of glycerol is taken with the direct libation at an ancient wedding ceremony of sterile platinum filament, is drawn in LB planar surface
Line, 37 DEG C of inversion overnight incubations；
(2) it from picking single colonie on LB plate, is inoculated in 5ml LB liquid medium, 37 DEG C, 200rmp shake culture
Overnight；
(3) bacterium solution for taking 100 μ l to be activated overnight accesses in 5ml LB fresh culture, and 37 DEG C of oscillation 2.5h of 200rpm are left
The right side, until the visible pico- muddiness of naked eyes.
(4) culture solution is transferred to 1.5ml centrifuge tube, ice bath 10min, 4 DEG C of 4000rpm are centrifuged 10min.
(5) supernatant is abandoned, thallus is collected, adds the 1.0ml 0.1M CaCl2 of pre-cooling, light outstanding, ice bath 10min, 4 DEG C of 4000rpm
It is centrifuged 10min.
(6) supernatant is abandoned, 100 μ l 0.1M CaCl2 (pre-cooling) are added, it is light outstanding, it can be used after ice bath at least 4h, if thinking to protect for a long time
Deposit, add sterile glycerol saved in -70 DEG C to final concentration 15%.
9, connection product converts：
(1) full dose (10 μ l) is added into 100 μ l competent cells, and 30min is placed in ice.
After (2) 42 DEG C of heating 45s, then 1min is placed in ice.
(3) 890 μ l LB liquid mediums, 37 DEG C of 200rpm shaken cultivation 60min are added.
(4) 4000rpm is centrifuged 1min, abandons supernatant, stays bacterium solution, and piping and druming suspends.
(5) it is spread evenly across in the LB solid medium tablets containing X-Gal, IPTG, Amp and cultivates.
1h or so is placed in (6) 37 DEG C of incubator fronts, after bacterium solution is cultured base absorption completely, 37 DEG C of inversion overnight incubations
To the visible single colonie of formation naked eyes.
(7) several single colonies selected at random be inoculated in 5ml contain in the LB liquid medium of corresponding antibiotic 37 DEG C,
4~6h of 200rmp shaking table culture is identified for bacterium solution PCR.
10, bacterium solution PCR is identified：
Using the bacterium solution of culture as template, PCR amplification, reaction system such as following table are carried out：
Amplification
Product is identified through 1% agarose gel electrophoresis.It will identify that correct bacterium solution is sent to Shanghai Sani biotech firm and carry out DNA survey
Sequence.
11, RACE expands the design of primers at the end of antibacterial peptide gene 3 ' and 5 ' ends in Mercuric chloride skin mucus：
The specific inner primer of 3 '-RACE and 5 '-RACE is used for according to the part antibacterial peptide gene sequence design of acquisition：
5 ' inner primers：CaSPI-F(1)：GGGTCTTGATAGATGTCATAGCG
3 ' inner primers：CaSPI-R(1)：CTTCAGCGTTGACTTCTCCA
Primer is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.In addition, primer：
3'race outer primer(1)：5′-taccgtcgttccactagtgattt-3′
5'race outer primer(1)：5′-catggctacatgctgacagccta-3′
3'race inner primer(2)：5′-cgcggatcctccactagtgatttcactatagg-3′
5'race inner primer(2)：5′-cgcggatccacagcctactgatgatcagtcgatg-3'
It is respectively from 3 '-Full RACE Core Set Ver.2.0 and 5 '-Full RACE Kit of TaKaRa.
12,3'RACE：
It can be highly sensitive by known cDNA sequence using 3 '-Full RACE Core Set Ver.2.0 of TaKaRa
The full length sequence of degree, the with high specificity 3 ' ends of amplification cDNA.Specific experiment operating method is as follows：
(1) reverse transcription reaction.Reaction system and reaction condition are as follows：
Reaction condition：
42℃1h
↓
70℃15min
After reaction for carrying out next step experiment.
(2) sleeve type PCR reacts
Outer PCR reaction
The experimental implementation carried out when Outer PCR reacts using TaKaRa LA Taq is as follows：
1) PCR after reaction, takes the PCR reaction solution of 5~10 μ l to carry out agarose gel electrophoresis, and confirmation PCR amplification produces
Object.And carry out Inner PCR reaction.
2) Inner PCR reacts.Reaction system and reaction condition are as follows：
(3) agarose gel electrophoresis
After reaction, the PCR reaction solution of 5~10 μ l is taken to carry out agarose gel electrophoresis, confirmation 3 ' RACEPCR amplification produces
Object.
(4) sequencing analysis
The purpose product of PCR amplification is cloned into after pMD-18T carrier and carries out DNA sequencing (specific experiment operating procedure again
It is identical as abovementioned steps 6~10).
13,5'RACE：
Utilize 5 '-Full RACE Kit amplification, 5 ' end DNA sequence dna, including step in detail below：
(1) phosphatizing treatment.
Phosphoric acid is carried out to 5 ' phosphate groups exposed in Total RNA using Alkaline Phosphatase (CIAP)
Reaction.
1) dephosphorylation reactant liquor is prepared by following component.
2) 50 DEG C of reaction 1h.
3) the 3M CH3COONa (pH5.2) of 20 μ l, the RNase Free dH of 130 μ l are added into above-mentioned reaction solution2O
Afterwards, it mixes well.
4) phenol/chloroform/isoamyl alcohol (25 of 200 μ l is added：24：1) 13000 × g room temperature centrifugation after, mixing well
Upper strata aqueous phase is transferred in new Microtube by 5min.
5) chloroform of 200 μ l is added, 13000 × g room temperature centrifugation 5min, upper strata aqueous phase is transferred to new after mixing well
In Microtube.
6) it is uniformly mixed after the NA Carrier of 2 μ l is added.
7) isopropanol of 200 μ l, after mixing well, cooled on ice 10min is added.
8) 13000 × g, 4 DEG C of centrifugation 20min abandon supernatant.
9) 70% cold ethyl alcohol (the RNase Free dH of 500 μ l is added2O is prepared) rinsing, 4 DEG C of 13000 × g centrifugations
5min abandons drying after supernatant.
10) the RNase Free dH of 7 μ l is added2O dissolution precipitating, obtains CIAP-treated RNA.
(2) " removing cap " reacts.
5 ' the cap sequences for removing mRNA using Tobacco Acid Pyrophosphatase (TAP) retain a phosphorus
Acid groups.
1) " removing cap " reaction solution is prepared by following component.
2) it reacts 1 hour for 37 DEG C.
3) this reaction solution is CIAP/TAP-treated RNA.5 μ l are taken to react for 5 ' RACE Adaptor connections, it is remaining
5 μ l be stored in -80 DEG C.
The connection of (3) 5 ' RACE Adaptor.
1) following solutions are prepared first
2) 65 DEG C of heat preservations are placed 2 minutes on ice after five minutes, and following reagent is then added
3) 16 DEG C of reaction 1h.
4) the 3M CH of 20 μ l is added into above-mentioned reaction solution3COONa (pH5.2), the RNaseFree dH of 140 μ l2After O,
It mixes well.
5) phenol/chloroform/isoamyl alcohol (25 of 200 μ l is added：24：1) 13000 × g room temperature centrifugation after, mixing well
Upper strata aqueous phase is transferred in new Microtube by 5min.
6) chloroform of 200 μ l is added, 13000 × g room temperature centrifugation 5min, upper strata aqueous phase is transferred to new after mixing well
In Microtube.
7) it is uniformly mixed after the NA Carrier of 2 μ l is added.
8) isopropanol of 200 μ l, after mixing well, cooled on ice 10min is added.
9) 13000 × g, 4 DEG C of centrifugation 20min abandon supernatant.70% cold ethyl alcohol (the RNase FreedH of 500 μ l is added2O matches
System) it rinses, 13000 × g, 4 DEG C of centrifugation 5min, abandon drying after supernatant.
10) the RNase Free dH of 6 μ l is added2O dissolution precipitating, obtains Ligated RNA.
(4) reverse transcription reaction.
1) inverse transcription reaction liquid is prepared by following component.
2) reverse transcription reaction condition is as follows：
30℃10min
↓
42℃1h
↓
70℃15min
3) next step experiment can be carried out after reaction.
(5) Outer PCR reacts
(6) Inner PCR reacts
(7) agarose gel electrophoresis
After reaction, the PCR reaction solution of 5~10 μ l is taken to carry out agarose gel electrophoresis, as shown in Figure 1, in Fig. 1, M is
DL2000 nucleic acid molecular weight standard, l and 2 are amplified production.3 ' RACE pcr amplification products can be confirmed from Fig. 1.
(8) sequencing analysis
The purpose product of PCR amplification is cloned into after pMD-18T carrier and carries out DNA sequencing (specific experiment operating procedure again
It is identical as abovementioned steps 6~10).
14, Mercuric chloride antibacterial peptide gene sequencing and result：
It extracts Plasmid DNA and measures nucleotide sequence with dideoxy, the use of instrument is Applied biosystems373A
Full-automatic nucleotide sequencing instrument.The result of genetic testing such as SEQ ID NO:Shown in 1, derived using bioinformatics method
The Mercuric chloride antibacterial peptide of artificial synthesized and antibacterial experiment verifying (such as embodiment 2) encodes albumen such as SEQ ID NO:Shown in 2.Tiger
The sequence of line frog antibacterial peptide gene nucleotide is shown as：Sequence length is 331 bases, sequence type：Nucleic acid, chain number：It is single-stranded,
Topology：Straight-chain, sequence type：CDNA, source：Mercuric chloride skin mucus.Encoding Mercuric chloride antibacterial peptide mature sequence is
177~213 nucleotide of Mercuric chloride antibacterial peptide gene, nucleotide sequence such as SEQ ID NO:Shown in 3, the Mercuric chloride antibacterial peptide
Gene prepares the application of Mercuric chloride antibacterial peptide as genetic engineering.
The detection of 2 Mercuric chloride antibacterial peptide (CaSPI) antibacterial activity of embodiment and minimal inhibitory concentration determine
Test strain：Escherichia coli Escherichia coli, staphylococcus aureus Staphylococcus
Aureus, bacillus subtilis Bacillus dysenteriae, the above strain have laboratory preservation.The brave line of antibacterial tests
Frog antibacterial peptide sample presses R V C A S F P L P I C Y sequent synthesis, 2mg/mL concentration is pressed after sample synthesis, with 0.1M phosphorus
Sour hydrogen sodium salt buffer solution (PH 6.0) is configured to solution, and it is spare to set -20 DEG C of refrigerators.
It takes 100 μ L of the bacterium solution being incubated overnight to be spread evenly across LB solid state rheology primary surface, is put in media surface cross
Enter the sterilized filter paper of 0.5cm diameter, the Mercuric chloride antibacterial peptide sample of 20 μ L synthesis is separately added on each filter paper, uses
Equivalent is compareed with 0.1M dibastic sodium phosphate salt buffer solution (PH 6.0).Then, 37 DEG C of inversions are cultivated 20 hours, have been seen whether
Inhibition zone is formed.Have inhibition zone formation shows there is antibacterial activity, the measurement of further progress minimal inhibitory concentration.
When measuring minimal inhibitory concentration, by the antibacterial detection method of standard, using LB liquid medium as culture medium.It chooses
In LB liquid medium, 37 DEG C are cultivated 20 hours strain after taking appropriate activation.Take a certain amount of LB liquid medium, then plus
Enter a certain amount of sample for being dissolved in 0.1M dibastic sodium phosphate salt buffer solution (PH 6.0) filtered through the aperture 0.22cm film, according to
Doubling dilution is diluted to a series of concentration gradients, and 37 DEG C are cultivated 20 hours, and light absorption is measured at 600nm wavelength.Minimum suppression
Bacteria concentration is the minimum sample concentration of invisible bacterial growth.Sample protein concentration is calculated using following formula：
Protein concentration (mg/mL)=(A215nm-A225nm) (0.144.
Antibacterial Establishing is as follows, is added in 1.9mL culture medium and is dissolved in 0.1M phosphorus through what the aperture 0.22cm film filtered
The sample of sour hydrogen sodium salt buffer solution (PH 6.0), 0.1mL take 1mL that the 2nd pipe is added, successively multiple proportions as the first pipe after mixing
Dilution is sucked out 1mL from the 8th pipe and discards, the 9th piping control tube, as shown in the table：
Bacterium solution (105cfu/mL) 0.05mL corrected will be added in each pipe, 37 DEG C of culture 18h are placed after mixing, in
Light absorption is measured at 600nm wavelength.Minimal inhibitory concentration is the minimum sample concentration of invisible bacterial growth.Sample protein concentration
It is calculated using following formula：
Protein concentration (mg/mL)=(A215nm-A225nm) (0.144.
Antibacterial testing result shows that the Mercuric chloride antibacterial peptide CaSPI of synthesis all has antibacterial activity to test strain,
CaSPI is weaker than bacillus subtilis to the inhibition of Escherichia coli, is better than staphylococcus aureus.
The minimal inhibitory concentration result of Mercuric chloride antibacterial peptide CaSPI is as shown in the table：
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (2)
1. a kind of Mercuric chloride antibacterial peptide, which is characterized in that its sequence such as SEQ ID NO：Shown in 2.
2. Mercuric chloride antibacterial peptide described in claim 1 is in terms of as the therapeutic agent for preparing cause pathogeny imcrobe infection disease
Using.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1803834A (en) * | 2006-01-12 | 2006-07-19 | 南京农业大学 | Antibacterial peptide of brown-spotted torrential frog, its gene and application in drug preparation |
| WO2007133033A1 (en) * | 2006-05-16 | 2007-11-22 | Promeditech, Inc. | Novel analogues of antimicrobial and anticancer peptide synthesized and produced from gaegurin 5 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1803834A (en) * | 2006-01-12 | 2006-07-19 | 南京农业大学 | Antibacterial peptide of brown-spotted torrential frog, its gene and application in drug preparation |
| WO2007133033A1 (en) * | 2006-05-16 | 2007-11-22 | Promeditech, Inc. | Novel analogues of antimicrobial and anticancer peptide synthesized and produced from gaegurin 5 |
Non-Patent Citations (2)
| Title |
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| Tigerinins: Novel Antimicrobial Peptides from the Indian Frog Rana tigerina;Korrapati Purna Sai,et al.;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20010126;第276卷(第4期);第2701-2707页 * |
| 虎纹蛙皮肤组织cDNA文库的构建及抗菌肽基因tigerinin-HRs的克隆;王辉等;《华北农学报》;20100131;第25卷(第1期);61-67 * |
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