CN103172730B - Bufo melanostictus schneider cystatin as well gene and application thereof - Google Patents
Bufo melanostictus schneider cystatin as well gene and application thereof Download PDFInfo
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- CN103172730B CN103172730B CN201310085704.9A CN201310085704A CN103172730B CN 103172730 B CN103172730 B CN 103172730B CN 201310085704 A CN201310085704 A CN 201310085704A CN 103172730 B CN103172730 B CN 103172730B
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Images
Abstract
The invention discloses a bufo melanostictus schneider cystatin and a gene of the bufo melanostictus schneider cystatin. The amino acid sequence of the bufo melanostictus schneider cystatin is shown as SEQ ID NO: 2, and the nucleotide sequence of the gene for coding the bufo melanostictus schneider cystatin is shown as SEQ ID NO: 1. The bufo melanostictus schneider cystatin obtained by means of prokaryotic expression has strong cathepsin-B inhibiting activity, is convenient to produce in vitro and can be used as a cathepsin-B inhibitor.
Description
Technical field
The invention provides a kind of Bufo melanostictus cystatin gene and application thereof, belong to field of biomedicine technology.
Background technology
Bufo melanostictus, Anura, Bufonidae, Bufo, is one of main source of traditional Chinese medicine material " dry toad ", generally in summer, Qiu Erji, catches; Taste is pungent, cool, poisonous, returns liver, spleen, lung channel, has the effect of subduing swelling and detoxicating, pain relieving, diuresis, is usually used in the disease treatments such as chronic tracheitis, carbuncle furuncle furunculosis, swelling and pain in the throat, oedema and dysuria; Also the proved recipe that is useful on cancer (especially mammary cancer) etc. among the people, but at present less about the Study on Molecular Mechanism of its effect.
In the process of tumor development, cathepsin B plays an important role, cathepsin B can directly degrade or plasminogen activation activator and matrix metalloproteinase etc. and many extracellular matrix components of indirectly degrading, thereby participates in the Invasion and Metastasis process of tumour.In addition it can also promote tumor vascular hyperplasia, strengthens the motor capacity of tumour cell, and cathepsin B transcribes, the height of expression level and the prognosis of tumour are proportionate.Think at present relevant with antitumor action to the inhibition of the L-Cysteine HCL Anhydrouss such as cathepsin B.
Bufo melanostictus cystatin amino acid complete sequence of the present invention and encoding gene thereof have been carried out to comparison search to Protein Data Bank and gene database respectively, all found no any identical sequence information.
Summary of the invention
The object of the present invention is to provide a kind of Bufo melanostictus cystatin, the aminoacid sequence of this Bufo melanostictus cystatin is as shown in SEQ ID NO:2, and it is a kind of single chain polypeptide, molecular weight 10882.84 dalton, iso-electric point 7.57.
Another object of the present invention is to provide a kind of gene of the described Bufo melanostictus cystatin of encoding, and its nucleotide sequence is as shown in SEQ ID NO:1.
The cloning process of Bufo melanostictus cystatin gene is as follows:
The total RNA extraction of Bufo melanostictus skin, reverse transcription and design primer and utilize PCR method screening Bufo melanostictus cystatin gene, wherein forward amplimer length is 23 Nucleotide, its sequence is 5 ’ – ATGGATCAAAAAGGAAATGTTCC-3 ', and oppositely amplimer sequence is 5 '-CTAAAAATAACAGATGGGGTCTTC-3 '; The positive monoclonal that obtains carries out gene nucleotide series mensuration, and gene sequencing result show the to encode gene of Bufo melanostictus cystatin is comprised of 309 Nucleotide, from 5 ' end to 3 ' terminal sequence, is:
atggatcaaa aaggaaatgt tcctggcgga ctgggtgcag aaaagcctgc taccccagaa 60
atacaggcgc tgtgtgacat ggtaaaatgc gcttttgagc aaaaatccgg gactaatgca 120
gcagagttta aggccatctg ttacgcaagt caagttgttg ctggaacaat gtactatgtg 180
aaggtggata ttggaggagg tcagtgctgt catctgaaaa tacttcaacc tctgcctact 240
ggagggaaag tgagtctgag cgactaccag tgcgggaaga agaaggaaga ccccatctgt 300
tatttttag 309
The ripe cystatin of coding Bufo melanostictus is 306 Nucleotide of the 1st –, and its aminoacid sequence is as shown in SEQ ID NO:2.
Prokaryotic expression, the purification process of Bufo melanostictus cystatin are as follows:
Build prokaryotic expression carrier, be transformed into BL21 competent cell, be incubated in the LB substratum containing penbritin, after IPTG abduction delivering, centrifugal collection thalline, ultrasonication centrifuging and taking supernatant, carries out affinity chromatography and obtains fusion Bufo melanostictus cystatin; After digesting with enteropeptidase after ultrafiltration, purifying obtains restructuring Bufo melanostictus cystatin, and it is active that this cystatin has strong inhibiting cathepsin B.
Another object of the present invention is that Bufo melanostictus cystatin is applied in and is prepared in cathepsin B inhibitors.
In the present invention, Bufo melanostictus used derives from Xishuangbanna Prefecture, Yunnan Province, from the market of farm produce, buys.
beneficial effect of the present invention is:
By Bufo melanostictus cystatin encoding gene its amino acid structure of deriving, and carry out prokaryotic expression, Bufo melanostictus cystatin after purifying has the activity of strong inhibiting cathepsin B, and this Bufo melanostictus cystatin has the advantages that produced in vitro is convenient, inhibiting cathepsin B activity is powerful.
Bufo melanostictus cystatin provided by the invention can suppress cathepsin B's activity of 75% in 2uM concentration, 8uM concentration Bufo melanostictus cystatin can suppress cathepsin B's activity of 90%.
Accompanying drawing explanation
Fig. 1 is that Bufo melanostictus cystatin of the present invention suppresses active result schematic diagram to cathepsin B.
Embodiment
Below by embodiment, the present invention is described in further detail, but content of the present invention is not limited to this, method all operations according to a conventional method if no special instructions in the present embodiment, the conventional reagent of agents useful for same employing if no special instructions or the according to a conventional method reagent of configuration.
embodiment 1: the clone of Bufo melanostictus cystatin gene
One, the total RNA of Bufo melanostictus skin extracts
A, live body Bufo melanostictus water is cleaned up, put into liquid nitrogen quick-frozen 4 hours, get skin histology, weigh, get 300mg skin histology, add total RNA Extraction buffer (the Trizol solution of 10ml, American I nvitrogen company product), homogenate 10 minutes in 20ml glass homogenizer;
B, add equal-volume phenol/chloroformic solution, vibration mixes, and room temperature is placed 10 minutes, and 4 ℃, centrifugal 10 minutes of 12000rpm, draws upper strata aqueous phase liquid;
C, in supernatant liquor, add the Virahol of supernatant liquor 1/2 volume, room temperature is placed 10 minutes, and 7500g is centrifugal 10 minutes at 4 ℃, and precipitation is washed once with 75% ethanol, dries, and manages end throw out and is the total RNA of Bufo melanostictus skin.
Two, Bufo melanostictus skin cDNA library is synthetic
Adopt the CreatorTM SMART TM cDNA Library Construction Kit of CLONTECH company Construction of Plasmid cDNA Library test kit, the operation of reference reagent box specification sheets method is as follows:
A, cDNA the first chain synthetic (mRNA reverse transcription), concrete steps are as follows:
1, at the aseptic centrifuge tube of 0.5ml, add the total RNA of 1 μ l Bufo melanostictus skin, 1 μ l SMART IV oligonucleotide, 1 μ l CDS III/3 ' PCR primer, add 2 μ l deionized waters and make cumulative volume reach 5 μ l;
2, the reagent mixing in centrifuge tube is also of short duration centrifugal, and 72 ℃ are incubated 2 minutes;
3, centrifuge tube is hatched on ice 2 minutes;
4, in centrifuge tube, add following reagent 2.0 μ l 5 * the first chain bufferings, 1.0 μ l 20mM dithiothreitol (DTT), 1.0 μ l 10mM dNTP mixtures, 1.0 μ l PowerScript ThermoScript II;
5, mix reagent of short duration centrifugal in centrifuge tube, 42 ℃ of insulations 1 hour;
6, centrifuge tube is placed in and ends the synthetic of the first chain on ice;
7, from centrifuge tube, get cDNA first chain of 2 μ l synthesizeds standby.
B, adopt long end polymerase chain reaction (LD-PCR) method second chain that increases, particular content is as follows:
1,95 ℃ of preheating PCR instrument;
2,2 μ l cDNA the first chains (mRNA reverse transcription), 80 μ l deionized waters, 10 μ l 10 * Advantage 2 PCR bufferings, 2 μ l 50 * dNTP mixtures, 2 μ l 5 ' PCR primers, 2 μ l CDS III/3 ' PCR primers and 2 μ l Escherichia coli polymerase centrifuge tubes are reacted;
3, in PCR instrument, by following program, increase:
1. 95 ℃ of 20 second
2. 22 circulations:
95 ℃ of 5 second
68 ℃ 6 minutes;
4; After loop ends, cDNA two strands synthetic in centrifuge tube is carried out to subsequent process.
Three, the amplification of Bufo melanostictus cystatin encoding gene
1,95 ℃ of preheating PCR instrument;
2,2 μ l cDNA double-stranded (above-mentioned product), 75.5 μ l deionized waters, 10 μ l PCR damping fluids, 8 μ l dNTP mixtures (each 2.5uM), 2 μ l forward amplimers, the reverse amplimer of 2 μ l and 0.5 μ l e. coli dna polymerase being put into centrifuge tube reacts; Forward amplimer length is 23 Nucleotide, and its sequence is 5 ’ – ATGGATCAAAAAGGAAATGTTCC-3 ' (SEQ ID NO:3), and oppositely amplimer sequence is
5’-CTAAAAATAACAGATGGGGTCTTC-3’ (SEQ ID NO：4)；
3, in PCR instrument, by following program, increase:
(1) denaturation
95 ℃ of 5 minute seconds
(2) 25 circulations:
95 ℃ of 30 second
56 ℃ of 40 second
72 ℃ of 30 second
(3) amplification after
72 ℃ 5 minutes
4, after loop ends, PCR product is carried out to extracting and reclaiming with the DNA product purification test kit of TIANGEN company, step is as follows:
(1) PCR product is combined to liquid with isopyknic film and puts upside down and mix, then will mix liquid and proceed to centrifugal purification post, standing 5 minutes of room temperature, is fully combined DNA with pellosil, and centrifugal minute 1 clock of 12000rpm, outwells the waste liquid in collection tube;
(2), in centrifugal purification post, centrifugal 1 minute of 12000 rpm, outwell the waste liquid in collection tube to add the rinsing liquid (containing ethanol) of 700 μ l;
(3) repeating step 2;
Centrifugal 3 minutes of (4) 12000 rpm;
(5) centrifugal purification post is placed in to new centrifuge tube;
(6) add 30 μ l ultrapure waters, at room temperature standing 5 minutes;
Centrifugal 1 minute of (7) 12000 rpm, pipe end solution is the Bufo melanostictus cystatin encoding gene PCR product that purifying is crossed.
Four, the preparation of bacillus coli DH 5 alpha competent cell
1, the single DH5 α of picking bacterium colony, is inoculated in 3m1 containing in the LB substratum of penbritin, 37 ℃ of overnight incubation, get next day above-mentioned bacterium liquid in proportion 1:100 be inoculated in 50m1 LB substratum, 37 ℃ of vibrations 2 hours.When OD600 value reaches 0.35, results bacterial cultures;
2, bacterium is transferred in the aseptic polypropylene tube of a 50m1 precooling, placed 10min on ice, make culture cooling;
3, the centrifugal 10min of 4100 rpm at 4 ℃, sucking-off nutrient solution, and pipe is inverted to l min so that residual substratum flows to end;
4, the 0.1mol/L CaCl of 30ml precooling for every 50ml initial incubation liquid
2-MgCl
2solution (80mmol/L MgCl
2, 20mmol/L CaCl
2) re-suspended cell precipitation;
5, the centrifugal 10min of 4100 rpm at 4 ℃, sucking-off supernatant liquor, and pipe is inverted to l min so that residual liquid flows to end;
6, the 0.1mol/L CaCl of 2ml precooling for every 50ml initial incubation thing
2the resuspended cell precipitation of solution, standby after packing.
Five, connect and connect the conversion of product
1, the ligase enzyme buffer mixture that adds 1 μ l Takara pMD19-T simple carrier, 4 μ l Bufo melanostictus cystatin encoding gene PCR products and 5 μ l in Eppendorf tube;
2,16 ℃ are reacted 3 hours;
3, full dose (10 μ l) is added in 100 μ l DH5 α competent cells, places 30 minutes in ice;
4,42 ℃ were heated after 90 seconds, then in ice, placed 1 minute;
5, add 37 ℃ of LB substratum 890 μ l of bathing of temperature, 37 ℃ of slow shaking culture 60 minutes;
6, get 200 μ l and coat on the LB substratum that contains X-Gal, IPTG, penbritin, cultivate 16 hours to form single bacterium colony for 37 ℃.
Six, Bufo melanostictus cystatin gene clone screening and evaluation
The above-mentioned single bacterium colony of picking is in containing in the LB substratum of penbritin, and 37 ℃ of slow shaking culture 4 hours, carry out pcr amplification, and amplimer and amplification condition are with the amplification condition of aforementioned Bufo melanostictus cystatin encoding gene; Positive colony through PCR confirmation is carried out after plasmid extraction, with the full-automatic nucleotide sequencing instrument of U.S. Applied Biosystems3730A, carry out the mensuration of nucleotide sequence; Sequencing primer is BcaBESTTM Sequencing Primer M13-47(5 ' CGCCAGGGTTTTCCCAGTCACGAC 3 '), gene sequencing result from 5 ' end to 3 ' terminal sequence is:
atggatcaaa aaggaaatgt tcctggcgga ctgggtgcag aaaagcctgc taccccagaa 60
atacaggcgc tgtgtgacat ggtaaaatgc gcttttgagc aaaaatccgg gactaatgca 120
gcagagttta aggccatctg ttacgcaagt caagttgttg ctggaacaat gtactatgtg 180
aaggtggata ttggaggagg tcagtgctgt catctgaaaa tacttcaacc tctgcctact 240
ggagggaaag tgagtctgag cgactaccag tgcgggaaga agaaggaaga ccccatctgt 300
tatttttag 309
Bufo melanostictus cystatin gene nucleotide series length is 309 bases, sequence type: nucleic acid, chain number: strand, topology: straight chain shape, sequence kind: cDNA, source: Bufo melanostictus skin.
Coding Bufo melanostictus cystatin is 306 Nucleotide of the 1st –, and its aminoacid sequence is as shown in SEQ ID NO:2.
embodiment 2: the prokaryotic expression of Bufo melanostictus cystatin, purifying
One, the structure of Bufo melanostictus cystatin prokaryotic expression carrier
According to the gene design primer of coding Bufo melanostictus cystatin, two of forward nested primers sequences, add Kpn I restriction enzyme to cut site and enteropeptidase restriction enzyme site, F1 sequence is 5 '-GGTACCGACGACGACGACAAGATGGA-3 ' (SEQ ID NO:5); F2 sequence is 5 '-CGACAAGATGGATCAAAAAGG-3 ' (SEQ ID NO:6); Reverse primer R sequence is 5 '-AAGCTTCTAAAAATAACAGATGGG-3 ' (SEQ ID NO:7), adds Hind III restriction enzyme site, after PCR, glue reclaim, carries out double digestion; Step 3 in PCR, the same embodiment 1 of glue reclaimer operation step.
In PCR pipe, prepare following enzyme and cut system:
PCR product/circular vectors 50 ul
ddH
2O 30ul
10×M Buffer 10ul
Kpn I 5ul(15 U )
Hind III 5ul(15 U )
Cumulative volume 100ul
37 ℃ of enzymes are cut 10 hours, add 6 * bromjophenol blue load sample damping fluid, 20 μ l in reaction tubes, after mixing in 1.0% sepharose electrophoresis, according to stripe size, reclaim object fragment, the step 3 in the same embodiment 1 of operation steps.
In PCR pipe, prepare following linked system:
Carrier DNA 200ng
Insert Fragment DNA 30ng
Ligase enzyme 10X damping fluid 2 ul
T4 DNA ligase enzyme 2U
Add nuclease free water to 20ul
15 ℃ connect 18 hours, connect product and proceed to e. coli bl21 competent cell screening positive clone, the step 3 in the same embodiment 1 of operation steps.
Two, the abduction delivering of Bufo melanostictus cystatin
1,37 ℃ of shaking tables are cultured to OD600 to 0.4-1 (suggestion OD600 is 0.6);
2, add 100mM IPTG liquid storage to final concentration 0.4mM, continue to cultivate 2-3 hour;
3, shaking flask is placed in to 5 minutes on ice, 4 ℃ of centrifugal 5 minutes collection thalline of 5000g;
4, re-suspended cell is in the 20mM of 0.25 times of volume precooling sodium phosphate, in 20mM imidazoles (pH7.4), centrifugal;
5, remove supernatant, thalline is stored in-70 ℃ of refrigerators or continues purifying.
Three, the purifying of Bufo melanostictus cystatin
1, albumen is extracted in ultrasonication
With 20mM sodium phosphate, after 20mM imidazoles (pH7.4) re-suspended cell, in ice bath, carry out ultrasonic disruption, power 100-200W, each ultrasonic 3s, 7s intermittently, total time 15min, centrifuging and taking supernatant can be used for affinity chromatography;
2, the preparation of affinity column
After assembling chromatography column, bubble with distilled water flushing with removal end, close chromatographic column effluent mouth, keep chromatography column lower end to have part water to exist, after resuspended Ni Sepharose 6 Fast Flow fillers, single is packed into chromatography column continuously, opens chromatography column outlet, constant flow pump is set to after required flow rate, completes the wash-out, standby of at least 3 column volumes;
3, affinity chromatography
Binding buffer liquid (20mM sodium phosphate with the linear rate of flow of 150cm/h with 5 to 10 column volumes, 20mM imidazoles, pH7.4) balance chromatography column, adds aforementioned sample, with binding buffer liquid, wash pillar, until absorbing, 280nm place returns back to baseline place, with elution buffer (20mM sodium phosphate, 0.5M NaCl, 0.5M imidazoles, pH7.4) carry out linear gradient elution, collect 60%-100% elution fraction, be Bufo melanostictus cystatin fusion rotein;
4, ultrafiltration
Use the ultra-filtration centrifuge tube of molecular weight cut-off 3kD to carry out ultrafiltration, under normal temperature, 4000 leave heart 30min, add equal-volume ddH
2repeated centrifugation step twice after O, the liquid of collecting centrifuge tube top carries out subsequent operations;
5, enteropeptidase cutting and purifying
Get above-mentioned albumen 5mg, add recombinant enterokinase 20U, 10 * reaction buffer 100ul, cumulative volume 1ml; 25 ℃ of reactions, after 12 hours, are carried out affinity chromatography, collect chromatography column can not in conjunction with component, again carry out ultrafiltration, obtain purity and surpass 99% Bufo melanostictus cystatin.
embodiment 3: the application of Bufo melanostictus cystatin
The cathepsin B of detecting Bufo melanostictus cystatin suppresses active.
The chromogenic substrate Z-Arg-Arg-pNA (Benzyloxycarbonyl-L-arginyl-L-arginine-p-nitroani-lide) of take is reaction substrate, Jiang Niu cathepsin B (0.2ng/ reaction) and reaction buffer (0.1 M sodium acetate, pH 5.5, containing 1mM DTT, 2mM EDTA and 4mM halfcystine), the Bufo melanostictus cystatin of different concns (final concentration 2 – 8 μ M), mix and be placed on room temperature 5min, add Z-Arg-Arg-pNA to final concentration be 400uM, 37 ℃ are detected 410nm place absorbancy (being designated as Abo I) after hatching 30min, what setting added restructuring Bufo melanostictus cystatin (is designated as Abo C) in contrast, calculate inhibiting rate, method of calculation are (Abo C-Abo I)/Abo C * 100%.
Result shows as Fig. 1, Bufo melanostictus cystatin has powerful cathepsin B and suppresses active, when 2uM concentration, can suppress cathepsin B's activity of 75%, 8uM concentration Bufo melanostictus cystatin can suppress cathepsin B's activity of 90%, and this restraining effect is very rapid, in 15 seconds, 2uM Bufo melanostictus cystatin can suppress cathepsin B's activity of 60%, experimental results show that it can be used as cathepsin inhibitors and uses.
SEQUENCE LISTING
<110> Kunming University of Science and Technology
<120> Bufo melanostictus cystatin and gene thereof and application
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 309
<212> DNA
<213> Bufo melanostictus
<400> 1
atggatcaaa aaggaaatgt tcctggcgga ctgggtgcag aaaagcctgc taccccagaa 60
atacaggcgc tgtgtgacat ggtaaaatgc gcttttgagc aaaaatccgg gactaatgca 120
gcagagttta aggccatctg ttacgcaagt caagttgttg ctggaacaat gtactatgtg 180
aaggtggata ttggaggagg tcagtgctgt catctgaaaa tacttcaacc tctgcctact 240
ggagggaaag tgagtctgag cgactaccag tgcgggaaga agaaggaaga ccccatctgt 300
tatttttag 309
<210> 2
<211> 102
<212> PRT
<213> Bufo melanostictus
<400> 2
Met Asp Gln Lys Gly Asn Val Pro Gly Gly Leu Gly Ala Glu Lys Pro
1 5 10 15
Ala Thr Pro Glu Ile Gln Ala Leu Cys Asp Met Val Lys Cys Ala Phe
20 25 30
Glu Gln Lys Ser Gly Thr Asn Ala Ala Glu Phe Lys Ala Ile Cys Tyr
35 40 45
Ala Ser Gln Val Val Ala Gly Thr Met Tyr Tyr Val Lys Val Asp Ile
50 55 60
Gly Gly Gly Gln Cys Cys His Leu Lys Ile Leu Gln Pro Leu Pro Thr
65 70 75 80
Gly Gly Lys Val Ser Leu Ser Asp Tyr Gln Cys Gly Lys Lys Lys Glu
85 90 95
Asp Pro Ile Cys Tyr Phe
100
<210> 3
<211> 23
<212> DNA
<213> artificial sequence
<400> 3
atggatcaaa aaggaaatgt tcc 23
<210> 4
<211> 24
<212> DNA
<213> artificial sequence
<400> 4
ctaaaaataa cagatggggt cttc 24
<210> 5
<211> 26
<212> DNA
<213> artificial sequence
<400> 5
ggtaccgacg acgacgacaa gatgga 26
<210> 6
<211> 21
<212> DNA
<213> artificial sequence
<400> 6
cgacaagatg gatcaaaaag g 21
<210> 7
<211> 24
<212> DNA
<213> artificial sequence
<400> 7
aagcttctaa aaataacaga tggg 24
Claims (3)
1. a Bufo melanostictus cystatin, is characterized in that: the aminoacid sequence of described Bufo melanostictus cystatin is as shown in SEQ ID NO:2.
2. a gene for Bufo melanostictus cystatin described in the claim 1 of encoding, is characterized in that: nucleotide sequence is as shown in SEQ ID NO:1.
3. the application of Bufo melanostictus cystatin claimed in claim 1 in preparing cathepsin B inhibitors.
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| CN201310085704.9A CN103172730B (en) | 2013-03-18 | 2013-03-18 | Bufo melanostictus schneider cystatin as well gene and application thereof |
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| US5935817A (en) * | 1997-01-30 | 1999-08-10 | Incyte Pharmaceuticals, Inc. | cDNA encoding a human cystatin-like protein (CSTIN) |
| CN1194092C (en) * | 2003-05-15 | 2005-03-23 | 中山大学 | Medusa cystatin gene, and expression and use thereof |
| CN101851627B (en) * | 2010-03-10 | 2012-11-14 | 中国科学院海洋研究所 | Gene encoding protein of eriocheir sinensis cysteine protease inhibitor Escystatin and application |
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