CN104292328A - Chymotrypsin inhibitor from urechis unicincts as well as application of gene and recombinant protein of chymotrypsin inhibitor - Google Patents
Chymotrypsin inhibitor from urechis unicincts as well as application of gene and recombinant protein of chymotrypsin inhibitor Download PDFInfo
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- CN104292328A CN104292328A CN201410549248.3A CN201410549248A CN104292328A CN 104292328 A CN104292328 A CN 104292328A CN 201410549248 A CN201410549248 A CN 201410549248A CN 104292328 A CN104292328 A CN 104292328A
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Abstract
The invention relates to a chymotrypsin inhibitor from urechis unicincts as well as an application of a gene and a recombinant protein of the chymotrypsin inhibitor, belonging to the biomedical field. The protein of the chymotrypsin inhibitor has an amino acid sequence as shown in SEQ ID No.1; the coded urechis unicincts chymotrypsin inhibitor UCI01 is single strand polypeptide, has molecular weight of about 7516Da and an isoelectric point of 4.38, contains 67 amino acid residues and does not contain a disulfide bond and an obvious signal peptide area. The gene has a base sequence as shown in SEQ ID No.2 in a sequence table, the overall length of the cDNA sequence is 697bp, the cDNA sequence contains a 80bp 5' non-translated region, a 413bp 3' non-translated region and a polyA tail, and the length of an open coding frame is 201bp. A prokaryotic expression technology is utilized to perform recombinant expression on the gene, so that the obtained recombinant protein has activity of the chymotrypsin inhibitor, and IC50 of the recombinant protein is 53nM. The recombinant protein has relatively good pH stability and a certain temperature stability, and has antibacterial effects on staphylococcus aureus.
Description
Technical field
The present invention relates to a kind of derive from Urechis uniconctus chymotrypsin inhibitor and the application of gene and its recombinant protein, belong to biomedical sector.
Background technology
Proteolytic enzyme system in organism is one of key substance of maintaining life, but the abnormal expression of proteolytic enzyme or release also can cause body to produce serious pathology.One of main path that organism regulates these enzymes to live accumulates certain specific protease inhibitor, and chymotrypsin inhibitor is exactly wherein a kind of.
Chymotrypsin inhibitor (Chymotrypsin Inhibitor, CI) be extensively present in the body of microorganism, leguminous plants, grass, arthropods and Mammals etc., Main Function is stopping, stops or reduce chymotrypsin activity.Quimotrase (chymotrypsin) be Chymotrypsin again, it is a kind of typical serine protease, in the Various Tissues and organ of organism, play very important effect, be also the important pathogen factor of some causal organisms or tumour cell.
According to the difference of structure, molecular weight etc., serpin is divided into dissimilar, and wherein potato proteinase inhibitor I (PI-1) family is wherein subspecies.The source of potato proteinase inhibitor I family is not limited to potato, is also found in other biological body (as: barley, tomato), animal (as: leech).This proteinoid generally has the activity suppressing Quimotrase or elastoser, and in structure, the molecular weight (60 ~ 90 amino-acid residues) of this kind of proteolytic enzyme, not containing disulfide linkage in structure.
Urechis uniconctus (Urechis uniconctus) is commonly called as extra large intestines, and being northern China coastal distinctive a kind of ocean Yi insects animal, is the unique class distributed at China coast without pipe Yi order.Mainly be distributed in the ground such as China's Bohai and Yellow Seas, Russia, the Korea peninsula, Hokkaido, Japan, area, Jiangzhou, Shandong is the maximum place of production of China's sea intestines.Its individuality is thick, and meat is tender delicious, and body wall protein content up to 22.84%, and is rich in multiple essential amino acid, has high edibleness, also known as " nude sea cucumber ".Simultaneously, Urechis uniconctus inhabits in coastal hurst tideland inferior segment and subtidal zone shoaling water " U-shaped " tunnel, can survive in the inviable rich sulphur environment of other animals, in the raw material as extraction Ocean Medicinal active substance, healthcare products, also there is very high potential.
Summary of the invention
The object of the invention is, based on above-mentioned theory and prior art basis, to provide a kind of application utilizing chymotrypsin inhibitor that is prepared by recombination and expression techniques, that derive from Urechis uniconctus and gene and its recombinant protein.
Technical scheme of the present invention is:
Derive from a chymotrypsin inhibitor for Urechis uniconctus, the protein of this chymotrypsin inhibitor has aminoacid sequence SEQ ID No.1:
MATKEQWPELVGKSGEEAKAAIIAERPELEKVEICNELSPMTMDYRTDRVRIFVNNDGNVVNPPQTG。
The described chymotrypsin inhibitor deriving from Urechis uniconctus, the Urechis uniconctus chymotrypsin inhibitor UCI01 of coding is single chain polypeptide, containing 67 amino-acid residues, molecular weight is about 7516Da, iso-electric point is 4.38, not containing disulfide linkage, does not have obvious signal peptide region.
Derive from a chymotrypsin inhibitor gene for Urechis uniconctus, this gene has the base sequence in sequence table SEQ IDNo.2.
The described chymotrypsin inhibitor gene deriving from Urechis uniconctus, this gene cDNA sequence overall length is 697bp, the 5` non-translational region wherein containing 80bp, the 3` non-translational region of 413bp and polyA tail, and open encoder block is 201bp.
Derive from an application for the chymotrypsin inhibitor gene recombinant protein of Urechis uniconctus, the recombinant expressed engineering strain EUCI01 of this chymotrypsin inhibitor gene, realizes the solubility expression of described gene.
The described application deriving from the chymotrypsin inhibitor gene recombinant protein of Urechis uniconctus, the recombinant protein that EUCI01 expresses, have stronger chymotrypsin inhibitor active, its IC50 is 53nM.
The described application deriving from the chymotrypsin inhibitor gene recombinant protein of Urechis uniconctus, chymotrypsin inhibitor is active, and recombinant protein is stablized pH, has temperature stability simultaneously.
The described application deriving from the chymotrypsin inhibitor gene recombinant protein of Urechis uniconctus, the recombinant protein that EUCI01 expresses, can produce streptococcus aureus and suppress.
Advantage of the present invention and beneficial effect are:
1, the present invention clone obtains the cDNA sequence of a Urechis uniconctus chymotrypsin inhibitor, and utilizes recombination method purification to have the protein of chymotrypsin protein enzyme inhibition activity.Thus, for subsequent applications is laid a good foundation.
2, the present invention utilizes prokaryotic expression technology to carry out recombinant expressed to this gene, and the recombinant protein of acquisition has the activity of chymotrypsin inhibitor, and its IC50 is 53nM.This recombinant protein has good pH stability and certain temperature stability, has fungistatic effect to streptococcus aureus.
Accompanying drawing explanation
Fig. 1: the SDS-PAGE analysis chart of recombinant protein.Wherein, 1 albumen marker; 2 EUCI01 do not induced; EUC01 after 3 inductions; UCI01 after 4 purifying.
Fig. 2: UCI01 inhibition analysis to proteolytic enzyme.Wherein, Trypsin is trypsinase, and Chymotrypsin is Quimotrase, and X-coordinate Inhibition Concentration represents the concentration of UCI01, and ordinate zou Residual enzyme activity represents the activity of proteolytic enzyme.
Fig. 3: temperature, pH are on the impact of UCI01 activity.Wherein, a figure is the impact of pH on UCI01 activity; B is the impact of temperature on UCI01 activity, and ordinate zou Residual enzyme activity represents the activity of institute's test proteins enzyme.
Embodiment
Be below the embodiment of content of the present invention, but content of the present invention is not limited to this, concrete summary of the invention comprises:
1, Urechis uniconctus chymotrypsin inhibitor UCI01 gene
The present invention utilizes sea farming pathogenic bacteria post-stimulatory Urechis uniconctus tissue construction cDNA library, by a large amount of order-checkings, obtains the gene fragment of an albuminoid enzyme inhibitors, obtains its full-length gene by RACE technology.Urechis uniconctus chymotrypsin inhibitor encoding gene of the present invention is carried out search comparison through NCBI gene database, does not find the report of any homologous genes or gene fragment or gene expression product.By finding the compare of analysis of its possible coded product, the similarity that the supposition in itself and a kind of leech (Helobdella robusta), Anthocidaris crassispina (Strongylocentrotus purpuratus) and some bacteriums may have the protein of proteinase inhibitor function is 42 ~ 59%.Find that it may belong to potato proteinase inhibitor I family by protein results analysis, called after UCI01.
The cDNA sequence of Urechis uniconctus chymotrypsin inhibitor UCI01 has 80bp 5` non-translational region, 413bp 3` non-translational region and polyA tail, and the open encoder block of 201bp, this gene has the base sequence in sequence table SEQ ID No.2.
The Urechis uniconctus chymotrypsin inhibitor UCI01 of coding is single chain polypeptide, containing 67 amino-acid residues, molecular weight is about 7516Da, and iso-electric point is 4.38, not containing disulfide linkage, the protein of this chymotrypsin inhibitor has aminoacid sequence SEQ ID No.1.
2, the recombinant strains of UCI01
The present invention is according to the UCI01 gene design primer obtained, and after pcr amplification, subclone is in prokaryotic expression plasmid pET25 (a), is converted in corresponding Host Strains, successfully builds engineering strain EUCI01.
According to the operation instruction of pET system, by optimizing inductive condition, carry out the abduction delivering of recombinant protein.By induction after EUC01 ice bath after ultrasonication, based on expression vector with 6 histidine-tagged, utilize Ni affinity column to carry out the purifying of recombinant protein.Analyze through SDS-PAGE, the restructuring UCI01 after purifying only has a band, and size is about 7.6kDa, in the same size with expection.Recombinant protein concentration through coomassie brilliant blue R_250 colorimetric method for determining is 4mg/ml.
3, the application of restructuring UCI01
The protease inhibitory activity of UCI01 measures and adopts colorimetry, utilize BCTR and people's trypsinase, wherein chymotryptic substrate is Nsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma, USA), tryptic substrate adopts BAPNA ((Sigma, USA).After tested, UCI01 shows more weak trypsin inhibition capacity.But UCI01 has the inhibit activities of stronger Quimotrase, its IC50 is 53nM.Find that the activity of pH to UCI01 does not have much affect subsequently, UCI01 has certain temperature stability simultaneously, preserves the activity that 3.5h still can keep about 20% at 80 DEG C.
Below by drawings and Examples, the present invention is described in more detail.
Embodiment 1: the acquisition of Urechis uniconctus chymotrypsin inhibitor gene UCI01
(1) total tissue RNA is extracted: get commercially available Urechis uniconctus and support 2 days temporarily in laboratory conditions, select the healthy individuals without any illness, with about 1 × 10
6individual incubated overnight is injected in Urechis uniconctus body cavity to seawater pathogenic bacterium Vibrio parahaemolyticus (Vibrio parahaemdyticus) CGMCC 2164 of logarithmic phase.After 2h, Urechis uniconctus is put to death in anesthesia, gets the tissues such as the breathing intestines of Urechis uniconctus, body lumen wall, body cavity blood cell, weigh, get 200 ~ 300mg tissue, add 8 ~ 10m1 Total RNAs extraction damping fluid (Trizol solution, TAKARA), homogenate in 20m1 glass homogenizer.Add equal-volume phenol/chloroform solution, acutely mix, room temperature place 10 ~ 15min, 4 ~ 6 DEG C in the centrifugal 10 ~ 15min of 10000 ~ 12000rpm, reject precipitate.Supernatant adds isopyknic Virahol, room temperature place 10 ~ 15min, 4 ~ 6 DEG C in the centrifugal 10 ~ 15min of 10000 ~ 12000rpm, precipitation 75wt% ethanol is washed once, dries, and at the bottom of pipe, throw out is total serum IgE.
(2) purifying of mRNA: PROMEGA company of the employing U.S.
mRNA Isolation Systems test kit carries out mRNA separation and purification.Concrete operations are: get total serum IgE 300 ~ 500 μ g and be dissolved in the DEPC water of 300 ~ 500 μ l, put into 65 DEG C of water-bath 8 ~ 10min, add Oligo (dT) probe of 3 ~ 5 μ l and 20 × SSC solution (sodium citrate buffer solution) of 13 ~ 15 μ l, mixing, placement room temperature cools, and is called A liquid, magnetic bead (SA-PMP) is flicked mixing, to magnetic frame adsorption time 30 ~ 40s, abandons supernatant, add 0.5 × SSC (sodium citrate buffer solution) 0.3 ~ 0.5m1, to magnetic frame adsorption time 30 ~ 40s, the 0.5 × SSC finally adding 0.1 ~ 0.2ml suspends, and is referred to as B liquid, A liquid is added in B liquid, room temperature places 10 ~ 15min, to magnetic frame absorption 30s, abandon supernatant, 4 times are washed with 0.1 × SSC, finally abandon supernatant, add the DEPC aqueous suspension of 0.1 ~ 0.2ml, 30s is adsorbed to magnetic frame, supernatant is moved to new test tube, add the DEPC water Eddy diffusion of 0.15m1 again, to magnetic frame absorption 30s, move supernatant to above-mentioned test tube, add 1/10 volume 3M (mol/L) sodium acetate and isopyknic Virahol, room temperature places 30 ~ 60min, 4 DEG C, centrifugal 10 ~ 15 minutes of 12000rpm, abandon supernatant, be precipitated and dissolved in the DEPC water of 10 ~ 20 μ l.
(3) structure of Urechis uniconctus cDNA library: adopt CLONTECH company Creator
tMsMART
tMcDNA Library Construction Kit.Specific as follows:
A, cDNA first chain synthesis (mRNA reverse transcription): in the centrifuge tube of DEPC process, add 1 μ l Urechis uniconctus mRNA, the SMART IV oligonucleotide of 1 μ l, the CDS III/3 ' PCR primer of 1 μ l, the water adding the DEPC process of 2 μ l makes cumulative volume reach 5 μ l.Reagent in mixing centrifuge tube is also of short duration centrifugal, and 72 DEG C are incubated 2 minutes.Centrifuge tube is hatched 2 minutes on ice, in centrifuge tube, adds the first chain damping fluid in the test kit of 2.0 μ l, 20mM (mmol/L) dithiothreitol (DTT) of 1.0 μ l, the 10mM dNTP mixture of 1.0 μ l, the PowerScript ThermoScript II of 1.0 μ l.Reagent in mixing centrifuge tube was also of short duration centrifugal, 42 DEG C of insulations 1 hour.Centrifuge tube is placed in the synthesis stopping the first chain on ice, cDNA first chain got synthesized by 2 μ l from centrifuge tube is for subsequent use.
The amplification of b, the second chain: adopt long end polymeric polymerase chain reaction (LD-PCR) method: the Escherichia coli polymerase centrifuge tube of 50 × dNTP mixture of 10 × Advantage 2 PCR damping fluid of cDNA first chain (mRNA reverse transcription) of 2 μ l, 80 μ l deionized waters, 10 μ l, 2 μ l, the 5 ' PCR primer of 2 μ l, the CDS III/3 ' PCR primer of 2 μ l and 2 μ l is reacted.In 95 DEG C of denaturation 20 seconds, 95 DEG C, 5 seconds, 68 DEG C, 6 minutes, circulate 20 times, the cDNA double-strand of synthesis is placed in-20 DEG C of preservations.
The screening of c, gene fragment: the cDNA library built is connected to
vertor (TAKARA), transforms DH5 α, blue hickie screening positive clone, and use Applied Biosystems DNA sequencer, model ABI PRISM 377 carries out nucleotide sequencing.
The acquisition of d, global cDNA sequence: to the Partial Fragment two ends design primer of the UCI01 gene that order-checking obtains, utilize the global cDNA sequence of RACE technical limit spacing gene.The cDNA sequence obtaining UCI01 has 80bp 5` non-translational region, 413bp 3` non-translational region and polyA tail, the open encoder block of 201bp.Its DNA sequence dna is SEQ ID No.2:
5`-GCAGTGGTATCAACGCAGAGTACAGGGGACTCTCTGTCCAGCAGCACACGGCGGTGATCAGACTTCGTACAGATATCAAA
ATGGCAACCAAGGAACA ATGGCCCGAGTTGGTTGGCAAGAGTGGCGAGGAAGCCAAGGCTGCTATCA TTGCAGAAAGACCTGAGTTGGAAAAGGTTGAAATCTGCAACGAGTTGAG TCCGATGACCATGGACTACCGCACCGACAGAGTTCGCATTTTTGTCAACAA TGACGGCAATGTTGTGAACCCTCCCCAGACCGGTTAGATGTACATCTACATGTAGAATGCTGGAGATGGATTGATTGGAAGTGTCATGTTACTTTAAGAACTGCAATCTATCTGTGAATGGTGCTGCAAGCGACAAGAAAGTGGTCAATGTTATCTGTTACGTATGTGAACAATGCTGTGAGCCAGTAATTATTGTGTACTGTCATCTGGATATGTTACCATTCCAACATGTTAACTCAAGTTTGGACCCAATTTTTTTCAAAATTTCTGGGGGACCCTATCTTCCAACAATTTGATATGGCTGCGTTTCTTATATTTCGATGGAAGTCATCATTTGTCCAAACAAATTTTGATTGTGTCAGATAAGTTGACGTATGCATTTCTATATGTTGGTAATAACAAAGTAAACTTATATAAAACTGATTTGTTAAAAAAAAAAAAAAAAAAAAA-3`
E, by finding gene encoding production analysis, the Urechis uniconctus chymotrypsin inhibitor UCI01 of coding is single chain polypeptide, and containing 67 amino-acid residues, molecular weight is about 7516 Da, and iso-electric point is 4.38, not containing disulfide linkage.Its aminoacid sequence is SEQ ID No.1:
MATKEQWPELVGKSGEEAKAAIIAERPELEKVEICNELSPMTMDYRTDRVRIFVNNDGNVVNPPQTG
The structure of the recombinant strains of embodiment 2:UCI01
According to the cDNA sequence obtained, design primer carries out the clone of UCI01 gene.Primer sequence is:
Forward primer 5`-
cATATGgCAACCAAGGAACAATGGC-3` (NdeI);
Reverse primer 5`-
cTCGAGaCCGGTCTGGGGAGGGT-3` (XhoI).
PCR reaction is carried out under the following conditions: 94 DEG C, 2 minutes, 94 DEG C, 30 seconds, 68 DEG C, 30 seconds and 72 DEG C, 40 seconds, 30 circulations.The rear glue that increased reclaims the recovery that test kit (sky root is biological) carries out object fragment.By the DNA fragmentation of recovery after NdeI and XhoI double digestion, being transformed in BL21 (DE3) bacterium after connecting with adopting the pET25 (a) of identical double digestion process, building recombinant strains EUCI01.
The abduction delivering of embodiment 3:UCI01 and purifying
(1) abduction delivering: conveniently colibacillary culture condition (LB substratum, 37 DEG C of incubated overnight), EUCI01 is cultured to logarithmic growth in earlier stage, add isopropyl-β-D-thiogalactoside(IPTG) (IPTG) induction that final concentration is 0.1 ~ 10mmol/L respectively, 18 ~ 37 DEG C, 100 ~ 350rpm abduction delivering, 4 ~ 12h, bacterium liquid is transferred in 5ml centrifuge tube, 4 ~ 6 DEG C, centrifugal 2 ~ the 5min of 6000 ~ 8000r/min, the Lysis butter of precipitation with 2 ~ 5ml precooling is rinsed 1 ~ 2 time, after recentrifuge, the Lysis butter of precipitation 5ml precooling is resuspended, be placed in 0 ~ 4 DEG C of ultrasonic disruption (working hour 3 ~ 5s, intermittent time 6 ~ 8s, power 350 ~ 400W, 150 ~ 300 circulations).Transferred to by solution after fragmentation in new 5ml off-line pipe, 4 ~ 6 DEG C, the centrifugal 20 ~ 30min of 12000 ~ 15000r/min, collects upper cleer and peaceful precipitation, the expression of SDS-PAGE analysis purposes albumen respectively.Found that, when the IPTG of interpolation 0.01 ~ 0.2mmol/L makees inductor, 18 ~ 25 DEG C, 100 ~ 200rpm induce 4 ~ 8h time, target protein achieves solubility expression.
(2) protein purification preparation: the IPTG EUCI01 being cultured to logarithmic phase being added 0.01mmol/L, after 18 DEG C of induction 8h, supernatant is got in ultrasonication.In addition on the Ni-NTA pillar balance supernatant with Lysis Buffer in advance, foreign protein is washed off with the Wash Buffer of 5 ~ 6 times of column volumes, with the Elution Buffer of 2 ~ 3 times of column volumes by fusion rotein wash-out.By the PBS damping fluid dialyzed overnight of the solution after wash-out, retain centrifugal column in 4 ~ 6 DEG C with the protein molecular that minimum molecular weight is 5kDa, the centrifugal 20 ~ 30min of 6000 ~ 8000rpm, obtain recombinant protein solution.
(3) protein concentration and purity testing: the concentration utilizing coomassie brilliant blue R250 method to measure recombinant protein is 4mg/ml.Utilize SDS-PAGE to analyze and find that recombinant protein only has the band of an entry, size is about 7.6kDa, with expection consistent (Fig. 1).
Embodiment 4: protein-active measures
(1) rejection ability of proteolytic enzyme is tested: the damping fluid of the present embodiment reaction is the Tris-HCl damping fluid of 0.2mmol/L, the CaCl of 0.02mmol/L
2the aqueous solution, pH=8.0.Get bovine trypsin or Quimotrase (BBI) that 300 μ l concentration are 5.0mg/ml, add isopyknic containing different UCI01 concentration (0,160,320,480,640,800,1000 μ g) sample, 37 DEG C of water bath heat preservation 30min, add trypsin substrate BAPNA (Sigma, USA) or the chymotryptic substrate Nsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma, USA) of 500 μ l, 1.5mg/ml.37 DEG C of insulation 5min, add the acetic acid termination reaction of 600 μ l, concentration 30wt%.10000 ~ 12000rpm, centrifugal 3 ~ 5min, get supernatant and measure 405nm place light absorption value.The activity influence of result display UCI01 to Quimotrase is little, when the concentration of UCI01 is increased to maximum concentration, tryptic activity is only produced to the suppression of 20%, but significantly can suppress the activity of Quimotrase, its IC50 is 53nM (Fig. 2).
(2) pH is on the impact of UCI01: the reaction buffer preparing different pH (4,5,6,7,8,9), according to the method for step 2 in embodiment 4, is determined at the impact of pH change on the Quimotrase rejection ability of UCI01.As shown in Figure 3 a, the activity influence of pH to UCI01 is not obvious for result, illustrates that UCI01 has wider pH adaptive faculty.
(3) temperature is on the impact of UCI01: UCI01 solution is placed in differing temps (-20,4,10,20,30,40,50,70 DEG C) and preserves 3.5 ~ 4h, utilize the method for embodiment 4 step 2 subsequently, measure the change of UCI01 activity under different heat-retaining condition.As shown in Figure 3 b, the activity influence of temperature to UCI01 is larger.Along with the rising activity of storage temperature reduces gradually.But still there is after preserving 4h at 70 DEG C the activity of 20%.
(4) UCI01 bacteriostasis measures: the bacteriostasis adopting filter paper In Vitro Bacteriostasis method test UCI01.The test strain preserved in laboratory to logarithmic phase, gets 10 according to the culture condition incubated overnight of recommending
7individual test strain is spread evenly across on corresponding solid medium flat board, and the aseptic filter paper sheet containing finite concentration UCI01 is carefully attached at media surface, is just putting cultivation 18 ~ 24 hours for 37 DEG C, observes inhibition zone and is formed.Result shows UCI01 to tested Gram-negative bacteria as Klebsiella Pneumoniae (klebsiella pneumoniae), intestinal bacteria (Escherichia coil), Pseudomonas aeruginosa (Pseudomonas aeruginosa), first paratyphosum Bacterium (B paratyphoid coil) do not have obvious fungistatic effect; More weak bacteriostatic action (antibacterial circle diameter <3mm) is had to Candida albicans (Monilia albican); To gram-positive microorganism streptococcus aureus (staphylococcus aeruginosa) ATCC25923 fungistatic effect better (inhibition zone >10mm).
Claims (8)
1. derive from a chymotrypsin inhibitor for Urechis uniconctus, it is characterized in that, the protein of this chymotrypsin inhibitor has aminoacid sequence SEQ ID No.1:
MATKEQWPELVGKSGEEAKAAIIAERPELEKVEICNELSPMTMDYRTDRVRIFVNNDGNVVNPPQTG。
2. according to the chymotrypsin inhibitor deriving from Urechis uniconctus according to claim 1, it is characterized in that, the Urechis uniconctus chymotrypsin inhibitor UCI01 of coding is single chain polypeptide, containing 67 amino-acid residues, molecular weight is about 7516Da, iso-electric point is 4.38, not containing disulfide linkage, does not have obvious signal peptide region.
3. derive from a chymotrypsin inhibitor gene for Urechis uniconctus, it is characterized in that, this gene has the base sequence in sequence table SEQ ID No.2.
4. according to the chymotrypsin inhibitor gene deriving from Urechis uniconctus according to claim 1, it is characterized in that, this gene cDNA sequence overall length is 697bp, the 5` non-translational region wherein containing 80bp, the 3` non-translational region of 413bp and polyA tail, open encoder block is 201bp.
5. derive from an application for the chymotrypsin inhibitor gene recombinant protein of Urechis uniconctus, it is characterized in that, the recombinant expressed engineering strain EUCI01 of this chymotrypsin inhibitor gene, realizes the solubility expression of described gene.
6. according to the application deriving from the chymotrypsin inhibitor gene recombinant protein of Urechis uniconctus according to claim 5, it is characterized in that, the recombinant protein that described EUCI01 expresses, have stronger chymotrypsin inhibitor active, its IC50 is 53nM.
7. according to the application deriving from the chymotrypsin inhibitor gene recombinant protein of Urechis uniconctus according to claim 6, it is characterized in that, described chymotrypsin inhibitor is active, and recombinant protein is stablized pH, has temperature stability simultaneously.
8. according to the application deriving from the chymotrypsin inhibitor gene recombinant protein of Urechis uniconctus according to claim 5, it is characterized in that, the recombinant protein that described EUCI01 expresses, can produce streptococcus aureus and suppress.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117164690A (en) * | 2023-10-25 | 2023-12-05 | 济宁医学院 | Antibacterial protein, encoding gene, application method and product thereof |
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| US6294648B1 (en) * | 1999-07-20 | 2001-09-25 | Bayer Corporation | Protein having proteinase inhibitor activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6294648B1 (en) * | 1999-07-20 | 2001-09-25 | Bayer Corporation | Protein having proteinase inhibitor activity |
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| KIMJ.Y.等: "Protease Inhibitors from Plants with Antimicrobial Activity", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 * |
| 张莉等: "豆类植物蛋白酶抑制剂研究进展", 《大豆科学》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117164690A (en) * | 2023-10-25 | 2023-12-05 | 济宁医学院 | Antibacterial protein, encoding gene, application method and product thereof |
| CN117164690B (en) * | 2023-10-25 | 2024-01-30 | 济宁医学院 | Antibacterial protein, encoding gene, application method and product thereof |
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