CN105820239A - Duttaphrynus melanostictus serine protease inhibitor and gene and application thereof - Google Patents
Duttaphrynus melanostictus serine protease inhibitor and gene and application thereof Download PDFInfo
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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
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
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- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
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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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Abstract
The invention discloses a duttaphrynus melanostictus serine protease inhibitor and a gene thereof. The amino acid sequence of the duttaphrynus melanostictus serine protease inhibitor is as shown in SEQ ID NO:2. The amino acid sequence of the gene encoding the duttaphrynus melanostictus serine protease inhibitor is as shown in SEQ ID NO:1. The duttaphrynus melanostictus serine protease inhibitor obtained through the prokaryotic expression means has high trypsin inhibition activity, has the advantages of being convenient to produce in vitro and high in trypsin inhibition activity, and can be used as a trypsin inhibitor.
Description
Technical field
The present invention relates to Bufo melanostictus research field, particularly to a kind of Bufo melanostictus serpin and gene thereof and application.
Background technology
Bufo melanostictus, Anura, Bufonidae, Bufo, is one of the main source of Chinese medicine material " Bufo siccus ", typically catches in summer, season in autumn two；Acrid in the mouth, cool, poisonous, return liver, spleen, lung meridian, there is effect of subduing swelling and detoxicating, pain relieving, diuresis, be usually used in the disease treatments such as chronic tracheitis, carbuncle furuncle furuncle, laryngopharynx swelling and pain, edema and dysuria；Also the proved recipe having for cancer (especially breast carcinoma) etc. among the people, but the most less about the Study on Molecular Mechanism of its effect.
Serine protease (serineprotease) be a class using serine as the proteolytic enzyme in active center, belong to proteolytic enzyme, be present in the various biology of nature.Serine protease has a unique reactivity serine residue, it plays the effect of regulatory factor by activating pepsinogen or suppressing in biologic artifact, and plays an important role during the biochemical reactions such as tissue reconstruction, cell differentiation, fetal development, poisoning intrusion and angiogenesis.Serpin (serpin) is the material that a class has suppression serine protease hydrolysing activity, and it is widely present in the various organism of nature.Serine protease is as the homeostatic important regulatory factor of organism, its the most many important physiological and biochemical activities play an important role, such as cellular matrix structure, protein folding, complement activation, blood clotting, cell differentiation, cell migration, immunity and inflammatory reaction and tumor suppression etc..There is not been reported for Bufo melanostictus serpin at present.
Bufo melanostictus serpin overall amino acid sequence and the encoding gene thereof of the present invention are carried out comparison search to Protein Data Bank and gene database respectively, all finds no any identical sequence information.
Summary of the invention
For solving the problem that above-mentioned prior art exists, it is an object of the invention to provide a kind of Bufo melanostictus serpin and gene thereof and application.
For reaching above-mentioned purpose, the technical scheme is that
A kind of Bufo melanostictus serpin, the aminoacid sequence of this Bufo melanostictus serpin is as shown in SEQIDNO:2, and it is a kind of single chain polypeptide, molecular weight 11604.5Da dalton, isoelectric point, IP 7.33.
Another object of the present invention is to provide a kind of gene encoding described Bufo melanostictus serpin, and its nucleotide sequence is as shown in SEQIDNO:1.
The cloning process of Bufo melanostictus serpin gene is as follows:
Bufo melanostictus skin Total RNAs extraction, reverse transcription construction cDNA library, with Bufo melanostictus cDNA as template, degenerate primer is designed according to conserved domain, by nested PCR amplification serpin partial sequence, primer is 5 ' PCRprimerF1 and 5 ' PCRprimerF2 and 3 ' primerR1.According to partial sequence design primer (3 ' primerR2 and 3 ' primerR3) recorded, again carrying out nest-type PRC with building storehouse 5 ' PCRprimerIIA, correlated response condition is the same, final acquisition Bufo melanostictus serpin total length ORF.
1, primer:
5’PCRprimerF1:5’-TGYGGNWSNGCNTGYCCN-3’
5’PCRprimerF2:5’-CCNTGYGTNGARGGNTGYGTNTGY-3’
5’PCRprimerIIA:5’-AAGCAGTGGTATCAACGCAGAGT-3’
3’primerR1:5’-ATTGACTCGAGTCGACATCGA-3’
3’primerR2:5’-ATGGACATTCTTCCTTTGGGA-3’
3’primerR3:5’-AGGACCTATAACATATCC-3’
2, by 0.5 μ lrTaq, 10 μ lPCR buffer, 8 μ ldNTP mixture (each 2.5 μMs), 6 μ lMgCl2, 2 μ lRNA, primer 5 ' PCRprimerF1,5 ' PCRprimerF2 and 3 ' primerR1: each 2 μ l, 65.5 μ l deionized waters, put in centrifuge tube and react.
3, expand by following procedure in PCR instrument:
1. 95 DEG C 5 minutes
2. 30 circulations:
95 DEG C 30 seconds
55 DEG C 30 seconds；
72 DEG C 30 seconds；
3. 72 DEG C extend 5 minutes.
4. PCR, reaction system and PCR reaction condition are proceeded ibid with product for template.
According to the serpin partial sequence design primer amplified above
Its sequence is: 5 ' ATGCATCCTTGTTCTGGTTGTG-3 ',
Reversely amplimer sequence is 5 '-CTATTTCACCTTTGGACATTCCT-3 '；
Obtained positive monoclonal carries out gene nucleotide series mensuration, gene sequencing result show encode Bufo melanostictus serpin gene be made up of 333 nucleotide, its sequence as shown in SEQIDNO:1,
Bufo melanostictus maturation serpin is made up of 110 amino acid residues, and its aminoacid sequence is for as shown in SEQIDNO:2.
The prokaryotic expression of Bufo melanostictus serpin, purification process are as follows:
Prokaryotic expression:
1. by the positive colony pET32a-Hyl inoculation selected to the LB fluid medium containing Amp resistance, 37 DEG C, 180rpm overnight incubation.
2. the bacterium solution after cultivating by the inoculum concentration of 1% is transferred in the LB culture medium that 100ml contains Amp resistance, and 37 DEG C, 180rpm cultivates about 4h, is between 0.4-0.6 to OD600, takes out 10ml's
Bacterium solution is put into and is continued in aseptic test tube to cultivate, and is used as not inducing comparison.Remaining bacterium solution adds the IPTG (Isopropyl β-D-1-Thiogalactopyranoside of final concentration of 0.4mM；IPTG), 30 DEG C, 160rpm carries out abduction delivering.
3. continuing to cultivate 4-5h, take out the bacterium solution after 10ml induction, 12000rpm is centrifuged 5min, and abandoning supernatant collects bacterial sediment.
4. determine that recombiant protein is to be present in host bacterial with soluble status or with inclusion body state.Concrete detection method is: be resuspended in the 20mM phosphate buffer (pH=7.4) of 15ml by the bacterial sediment before and after induction, use ultrasonic disruption instrument that it is carried out cell breakage, power is 200W, super 6s stops 4s, net cycle time is 15min, crushes thalline in ice bath.4 DEG C, 12000g is centrifuged 20min.It is precipitated and dissolved in 8M carbamide, is positioned over 4 DEG C of Refrigerator stores together with supernatant.
5. supernatant after taking cell breakage and the precipitation after again dissolving carry out little peptide polyacrylamide gel electrophoresis (tricinesodi μm dodecyls μ lfatepolyacrylamidegelelectrophoresis；Tricine-SDS-PAGE), recombiant protein location in cell is observed.
Purification:
1. 100ml bacterium solution 10000g after induction being centrifuged 5min, collect bacterial sediment, add the 20mM phosphate buffer (pH7.4) of 15ml, vortex concussion makes it fully mix, and 10000g is centrifuged 5min, is repeated twice.
2. adding the phosphate buffer (pH7.4) of 15ml20mM, vortex concussion makes it fully mix, crushes thalline with ultrasonic cell disruption instrument after ice bath 30min, 4 DEG C, 12000g is centrifuged 20min, is repeated twice centrifugal, collect supernatant, supernatant is joined Ni2+On post, stand 30min, enable the fusion protein with His label to be attached to Ni as far as possible2+On post, in conjunction with removing filtrate after 30min.
3. to Ni2+The combination buffer (Bingingb μ ffer) being slowly added into 5-6 times of column volume in post cleans.
4. add the elution buffer (El μ tionb μ ffer, containing 200mM imidazoles) of 5-6 times of column volume, collect eluent.
5. the eluent of collection is joined (3000MWCO) in 50ml super filter tube, 4 DEG C, 4000rpm is centrifuged 40-50min, then the filtrate in sleeve pipe is outwelled, the 20mM phosphate buffer adding 12ml again in super filter tube carries out changing liquid (being repeated twice), finally collects the protein concentrated solution on filter membrane.
6. Coomassie Brilliant Blue measures the concentration of protein concentrated solution.
Build prokaryotic expression carrier pET32a-Hyl, be transformed into BL21 competent cell, be incubated in the LB culture medium containing ampicillin, after IPTG abduction delivering, centrifugal collection thalline, ultrasonication centrifuging and taking supernatant, carry out affinity chromatograph and obtain fusion Bufo melanostictus serpin；After digesting with enterokinase after ultrafiltration, purification i.e. obtains restructuring Bufo melanostictus serpin, and this serpin has strong suppression tryptic activity.
The present invention another object is that and applied in preparing trypsin inhibitor by Bufo melanostictus serpin.
Described Bufo melanostictus serpin is the Trypsin inhibitors thing of active component.
The application in terms of preparing Trypsin inhibitors thing of the described Bufo melanostictus serpin.
In the present invention, Bufo melanostictus used derives from Xishuangbanna Prefecture, Yunnan Province, buys from the market of farm produce.
Relative to prior art, the invention have the benefit that
The present invention is derived its amino acid structure by Bufo melanostictus serpin encoding gene, and carry out prokaryotic expression, Bufo melanostictus serpin after purification has the tryptic activity of strong suppression, and this Bufo melanostictus serpin has the advantages that produced in vitro is convenient, suppression tryptic activity is powerful.
The Bufo melanostictus serpin that the present invention provides can suppress the tryptic activity of 62% 2 μMs of concentration, and 8 μMs of concentration Bufo melanostictus serpins can suppress the tryptic activity of about 80%.
Accompanying drawing explanation
Fig. 1 is that Bufo melanostictus serpin is to trypsin inhibition activity and response time graph of a relation.
Detailed description of the invention
Below by drawings and Examples, the present invention is described in further detail, but present disclosure is not limited thereto, method operating the most according to a conventional method if no special instructions in the present embodiment, agents useful for same if no special instructions use conventional reagent or the reagent configured according to a conventional method.
Embodiment 1: the clone of Bufo melanostictus serpin gene
One, Bufo melanostictus skin Total RNAs extraction
A, live body Bufo melanostictus use water is cleaned up, put into quick-freezing 4 hours in liquid nitrogen, bark fetching skin tissue, weigh, take 300mg skin histology, add 10ml Total RNAs extraction buffer (Trizol solution, American I nvitrogen Products), it is homogenized 10 minutes in 20ml glass homogenizer；
B, addition equal-volume phenol/chloroformic solution, vibration mixing, room temperature placement 10 minutes, 4 DEG C, 12000rpm is centrifuged 10 minutes, draws upper strata aqueous phase liquid；
C, the isopropanol of addition supernatant 1/2 volume, room temperature placement 10 minutes in supernatant, at 4 DEG C, 7500g is centrifuged 10 minutes, and precipitation is washed once with 75% ethanol, dries, and precipitate at the bottom of pipe is Bufo melanostictus skin total serum IgE.
Two, Bufo melanostictus skin cDNA library synthesis
Using CLONTECH company CreatorTMSMARTTMcDNALibraryConstr μ ctionKit Construction of Plasmid cDNA Library test kit, the operation of reference reagent box description method is as follows:
A, cDNA first chain synthesis (mRNA reverse transcription), specifically comprise the following steps that
1, add 1 μ l Bufo melanostictus skin total serum IgE, 1 μ lSMARTIV oligonucleotide, 1 μ lCDSIII/3 ' PCR primer at the centrifuge tube that 0.5ml is aseptic, add 2 μ l deionized waters and make cumulative volume reach 5 μ l；
2, the reagent in mixing centrifuge tube of short duration centrifugal, 72 DEG C are incubated 2 minutes；
3, centrifuge tube is hatched on ice 2 minutes；
4, in centrifuge tube, add following reagent 2.0 μ l5 × the first chain buffering, 1.0 μ l20mM dithiothreitol, DTTs, 1.0 μ l10mMdNTP mixture, 1.0 μ lPowerScript reverse transcription；
5, reagent of short duration centrifugal in mixing centrifuge tube, is incubated 1 hour at 42 DEG C；
6, centrifuge tube is placed in stops the synthesis of the first chain on ice；
7, cDNA the first chain taken synthesized by 2 μ l from centrifuge tube is standby.
B, using long end polymeric polymerase chain reaction (LD-PCR) method to expand the second chain, particular content is as follows:
1,95 DEG C of preheating PCR instrument；
2,2 μ lcDNA the first chains (mRNA reverse transcription), 80 μ l deionized waters, 10 μ l10 × Advantage2PCR buffering, 2 μ l50 × dNTP mixture, 2 μ l5 ' PCR primer, 2 μ lCDSIII/3 ' PCR primer and 2 μ l Escherichia coli polymerase centrifuge tubes are reacted；
3, expand by following procedure in PCR instrument:
1. 95 DEG C 20 seconds
2. 22 circulations:
95 DEG C 5 seconds
68 DEG C 6 minutes；
4, after loop ends, the cDNA double-strand synthesized in centrifuge tube is carried out subsequent process.
Three, the amplification of Bufo melanostictus serpin encoding gene
1,95 DEG C of preheating PCR instrument；
2,2 μ lcDNA double-strands (above-mentioned product), 75.5 μ l deionized waters, 10 μ lPCR buffer, 8 μ ldNTP mixture (each 2.5 μMs), 2 μ l forward amplimers, the 2 reverse amplimers of μ l and 0.5 μ l e. coli dna polymerase are put in centrifuge tube and reacted；A length of 23 nucleotide of forward amplimer, its sequence is 5 ' ATGCATCCTTGTTCTGGTTGTG-3 ' (SEQIDNO:3), and reverse amplimer sequence is 5 '-CTATTTCACCTTTGGACATTCCT-3 ' (SEQIDNO:4)；
3, expand by following procedure in PCR instrument:
(1) denaturation
95 DEG C of 5 every minute and second clock
(2) 25 circulations:
95 DEG C 30 seconds
56 DEG C 40 seconds
72 DEG C 30 seconds
(3) expand afterwards
72 DEG C 5 minutes
4, after loop ends, being stripped the DNA product purification kit of PCR primer TIANGEN company reclaiming, step is as follows:
(1) PCR primer being combined with isopyknic film the reverse mixing of liquid, then mixed liquor proceeds to centrifugal purification post, room temperature stands 5 minutes, makes DNA fully be combined with pellosil, centrifugal point of 1 clock of 12000rpm, outwells the waste liquid in collecting pipe；
(2) adding the rinsing liquid (containing ethanol) of 700 μ l in centrifugal purification post, 12000rpm is centrifuged 1 minute, outwells the waste liquid in collecting pipe；
(3) step 2 is repeated；
(4) 12000rpm is centrifuged 3 minutes；
(5) centrifugal purification post is placed in new centrifuge tube；
(6) add 30 μ l ultra-pure waters, at room temperature stand 5 minutes；
(7) 12000rpm is centrifuged 1 minute, and solution at the bottom of pipe is the Bufo melanostictus serpin encoding gene PCR primer that purification is crossed.
Four, the preparation of bacillus coli DH 5 alpha competent cell
1, picking single DH5 α bacterium colony, is inoculated in the LB culture medium that 3ml does not contains ampicillin, 37 DEG C of overnight incubation, takes above-mentioned bacterium solution 1:100 in proportion next day and be inoculated in 50mlLB culture medium, and 37 DEG C vibrate 2 hours.When OD600 value reaches 0.35, gather in the crops bacterial cultures；
2, transfer to antibacterial, in the sterile polypropylene tube of a 50ml pre-cooling, place 10min on ice, make culture cool down；
3, at 4 DEG C, 4100rpm is centrifuged 10min, sucking-off culture fluid, and pipe is inverted 1min so that the culture medium of residual is flow to end；
4, the 0.1mol/LCaCl of every 50ml initial incubation liquid 30ml pre-cooling2-MgCl2Solution (80mmol/LMgCl2, 20mmol/LCaCl2) re-suspended cell precipitation；
5, at 4 DEG C, 4100rpm is centrifuged 10min, sucking-off supernatant, and pipe is inverted 1min so that the liquid of residual flows to end；
6, the 0.1mol/LCaCl of every 50ml initial incubation thing 2ml pre-cooling2Solution re-suspended cell precipitates, standby after subpackage.
Five, connect and connect the conversion of product
1, in microcentrifugal tube, add 1 μ lTakarapMD19-Tsimple carrier, 4 μ l Bufo melanostictus serpin encoding gene PCR primer and the ligase buffer mixtures of 5 μ l；
2,16 DEG C are reacted 3 hours；
3, full dose (10 μ l) adds to 100 μ lDH5 α competent cells, places 30 minutes in ice；
4,42 DEG C heating 90 seconds after, then in ice place 1 minute；
5, the LB culture medium 890 μ l that bathed of 37 DEG C of temperature is added, 37 DEG C of slow shaken cultivation 60 minutes；
6, take 200 μ l coat containing X-Gal (the bromo-4-of 5-chloro-3-indole-β-D-galactoside), IPTG (isopropyl-beta D-thio galactopyranoside), ampicillin LB culture medium on, cultivate 16 hours to form single bacterium colony for 37 DEG C.
Six, Bufo melanostictus serpin gene colony screening and qualification
The above-mentioned single bacterium colony of picking in the LB culture medium containing ampicillin, 37 DEG C of slow shaken cultivation 4 hours, carry out PCR amplification, amplimer and the amplification condition amplification condition with aforementioned Bufo melanostictus serpin encoding gene；After the positive colony confirmed through PCR is carried out plasmid extraction, carry out the mensuration of nucleotide sequence with U.S. AppliedBiosystems3730A full-automatic nucleotide sequencing instrument；Sequencing primer is BcaBESTTMSeq μ encingPrimerM13-47 (5 ' CGCCAGGGTTTTCCCAGTCACGAC3 '), and its sequence is as shown in SEQIDNO:1
A length of 333 bases of Bufo melanostictus serpin gene nucleotide series, sequence type: nucleic acid, chain number: strand, topology: straight-chain, sequence kind: cDNA, source: Bufo melanostictus skin.
Coding Bufo melanostictus serpin is the 1st 333 nucleotide, encodes 110 its aminoacid sequences of amino acid residue as shown in SEQIDNO:2.
Embodiment 2: the prokaryotic expression of Bufo melanostictus serpin, purification
One, the structure of Bufo melanostictus serpin prokaryotic expression carrier
Gene design primer according to coding Bufo melanostictus serpin, forward nested primers sequence two, adding KpnI restriction enzyme enzyme action site and enterokinase cleavage site, F1 sequence is 5 '-GGTACCGACGACGACGACAAGATGC-3 ' (SEQIDNO:5)；F2 sequence is 5 '-ACAAGATGCATCCTTGTTCTGGTTG-3 ' (SEQIDNO:6)；Reverse primer R sequence is 5 '-AAGCTTCTATTTCACCTTTGGACATTC-3 ' (SEQIDNO:7), adds HindIII restriction enzyme site, after PCR, glue reclaim, carries out double digestion；Step 3 in PCR, the same embodiment of glue reclaimer operation step 1.
Following enzyme action system is prepared in PCR pipe:
37 DEG C of enzyme action 10 hours, add 6 × bromjophenol blue loading buffer 20 μ l in reaction tube, after mixing in 1.0% agarose gel electrophoresis, reclaim purpose fragment, the step 3 in the same embodiment of operating procedure 1 according to stripe size.
Following linked system is prepared in PCR pipe:
15 DEG C connect 18 hours, connect product and proceed to e. coli bl21 competent cell screening positive clone, the step 3 in the same embodiment of operating procedure 1.
Two, the abduction delivering of Bufo melanostictus serpin
1,37 DEG C of shaking tables are cultivated to OD600 to 0.4-1 (suggestion OD600 is 0.6)；
2, add 100mMIPTG liquid storage to cultivate 2-3 hour to final concentration 0.4mM, continuation；
3, shaking flask is placed in 5 minutes on ice, within centrifugal 5 minutes, collects thalline for 5000g4 DEG C；
4, re-suspended cell is in the 20mM sodium phosphate of 0.25 times of volume pre-cooling, 20mM imidazoles (pH7.4), centrifugal；
5, removing supernatant, thalline is stored in-70 DEG C of refrigerators or continues purification.
Three, the purification of Bufo melanostictus serpin
1, albumen is extracted in ultrasonication
With 20mM sodium phosphate, after 20mM imidazoles (pH7.4) re-suspended cell, carrying out ultrasonic disruption, power 100-200W, each ultrasonic 3s, intermittently 7s, total time 15min in ice bath, centrifuging and taking supernatant i.e. can be used for affinity chromatograph；
2, the preparation of affinity column
After assembling chromatographic column, with distilled water flushing to remove the bubble of end, close chromatographic column flow export, keep chromatographic column lower end with the presence of part water, after resuspended NiSepharose6FastFlow filler, continuous single is loaded into chromatographic column, opens chromatographic column outlet, after constant flow pump is set to required flow rate, complete the eluting of at least 3 column volumes, standby；
3, affinity chromatograph
Linear flow rate combination buffer (the 20mM sodium phosphate of 5 to 10 column volumes with 150cm/h, 20mM imidazoles, pH7.4) balance chromatographic column, add aforementioned sample, pillar is washed with combining buffer, until absorption returns back at baseline at 280nm, with elution buffer (20mM sodium phosphate, 0.5MNaCl, 0.5M imidazoles, pH7.4) carry out linear gradient elution, collect 60%-100% elution fraction, be Bufo melanostictus serpin fusion albumen；
4, ultrafiltration
The ultra-filtration centrifuge tube using molecular cut off 3kD carries out ultrafiltration, and under room temperature, 4000 leave heart 30min, add equal-volume ddH2Repeated centrifugation step twice after O, collects the liquid above centrifuge tube and carries out subsequent operation；
5, enterokinase cutting and purification
Take above-mentioned albumen 5mg, add recombinant enterokinase 20 Μ, 10 × reaction buffer 100 μ l, cumulative volume 1ml；After 25 DEG C of reactions 12 hours, carry out affinity chromatograph, collect chromatographic column can not in conjunction with component, again carry out ultrafiltration, obtain the purity Bufo melanostictus serpin more than 99%.
Embodiment 3: the application of Bufo melanostictus serpin
The trypsin inhibition activity of detection Bufo melanostictus serpin.
nullWith chromogenic substrate N α-Benzoyl-L-arginine4-nitroanilidehydrochloride as reaction substrate，By bovine trypsin (0.2ng/ reaction) and reaction buffer、The Bufo melanostictus serpin (final concentration 28 μMs) of variable concentrations，Mixing is placed on room temperature 5min，Add N α-Benzoyl-L-arginine4-nitroanilidehydrochloride to final concentration of 400 μMs，37 DEG C hatch 30min after detect absorbance (being designated as AboI) at 410nm，Conduct comparison (being designated as AboC) adding restructuring Bufo melanostictus serpin is set，Calculate suppression ratio，Computational methods are (AboC-AboI)/AboC × 100%.
Result is as shown in Figure 1, Bufo melanostictus serpin has powerful trypsin inhibition activity, the tryptic activity of 62% can be suppressed when 2 μMs of concentration, 8 μMs of concentration Bufo melanostictus serpins can suppress the tryptic activity of 78%, and this inhibitory action is the rapidest, in 60 seconds, 2 μMs of Bufo melanostictus serpins can suppress the tryptic activity of 50%, experiment to prove that it can use as serpin.
Claims (7)
1. a Bufo melanostictus serpin, it is characterised in that: the aminoacid sequence of described Bufo melanostictus serpin is as shown in SEQIDNO:2.
2. one kind encodes the gene of Bufo melanostictus serpin described in claim 1, it is characterised in that: its nucleotide sequence is as shown in SEQIDNO:1.
3. the cloning process of Bufo melanostictus serpin gene described in a claim 2, it is characterized in that, it concretely comprises the following steps: Bufo melanostictus skin Total RNAs extraction, reverse transcription and design primer and utilize PCR method screen Bufo melanostictus serpin gene, wherein a length of 22 nucleotide of forward amplimer
Its sequence is: 5 ' ATGCATCCTTGTTCTGGTTGTG-3 ',
Reversely amplimer sequence is 5 '-CTATTTCACCTTTGGACATTCCT-3 '.
The cloning process of Bufo melanostictus serpin gene the most according to claim 3, it is characterized in that, it concretely comprises the following steps: Bufo melanostictus skin Total RNAs extraction, reverse transcription construction cDNA library, with Bufo melanostictus cDNA as template, degenerate primer is designed according to conserved domain, by nested PCR amplification serpin partial sequence, primer is 5 ' PCRprimerF1 and 5 ' PCRprimerF2 and 3 ' primerR1；Partial sequence design primer according to recording: 3 ' primerR2 and 3 ' primerR3, carries out nest-type PRC with building storehouse again with 5 ' PCRprimerIIA, final acquisition Bufo melanostictus serpin total length ORF.
The cloning process of Bufo melanostictus serpin gene the most according to claim 4, it is characterised in that wherein, 5 ' PCRprimerF1:5 '-TGYGGNWSNGCNTGYCCN-3 '
5’PCRprimerF2:5’-CCNTGYGTNGARGGNTGYGTNTGY-3’
5’PCRprimerIIA:5’-AAGCAGTGGTATCAACGCAGAGT-3’
3’primerR1:5’-ATTGACTCGAGTCGACATCGA-3’
3’primerR2:5’-ATGGACATTCTTCCTTTGGGA-3’
3’primerR3:5’-AGGACCTATAACATATCC-3’。
6. the Trypsin inhibitors thing with Bufo melanostictus serpin described in claim 1 as active component.
7. the application in terms of preparing Trypsin inhibitors thing of the Bufo melanostictus serpin described in claim 1.
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| CN108840918B (en) * | 2018-06-14 | 2021-07-23 | 浙江农林大学 | Cloning method of PLA2 inhibitor encoding gene |
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