CN1294988C - Buckwheat trypsase inhibitor, expression and application thereof - Google Patents
Buckwheat trypsase inhibitor, expression and application thereof Download PDFInfo
- Publication number
- CN1294988C CN1294988C CNB2005100123854A CN200510012385A CN1294988C CN 1294988 C CN1294988 C CN 1294988C CN B2005100123854 A CNB2005100123854 A CN B2005100123854A CN 200510012385 A CN200510012385 A CN 200510012385A CN 1294988 C CN1294988 C CN 1294988C
- Authority
- CN
- China
- Prior art keywords
- trypsin inhibitor
- bti
- buckwheat
- trypsin
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention discloses a buckwheat trypsin inhibitor and the expression thereof and the appliaction of a product during inhibiting trypsin hydrolysis. The trypsin inhibitor of the present invention is small-molecular albumen composed of 69 amino-acid molecules, and the molecular weight is about 10kD. The trypsin inhibitor has obvious inhibiting effect on trypsin and forms compounds of 1: 1 with the trypsin through a competitive inhibiting mode to inhibit the hydrolysis of the trypsin. The buckwheat trypsin inhibitor has certain effect on promoting tumor cells to wither, and can be used for the preparation of new antitumor drug.
Description
Technical field
The present invention relates to biology field, specifically belong to a kind of buckwheat Trypsin Inhibitor (buckwheat TrypsinInhibitor is called for short BTI) and expression, purification and application.
Technical background
Protease inhibitor is that a class has inhibiting protein to specificity protease.This proteinoid is prevalent in animal, plant and the microorganism, is one of the abundantest albumen kind of nature content.Their effects in vivo are the hydrolysis that suppresses some protease, thus the proteic meaningless degraded of protection body function.Nineteen ninety-five Kennedy has reported that soybean trypsin inhibitor has in various degree inhibitory action to colon tumor, liver tumor, oral epithelium tumor etc.He had confirmed further that Bowman-Birk type trypsin inhibitor from Semen sojae atricolor can prevent to cause the activation of the specific gene of canceration in 1996, and protective tissue is avoided the damage of radiation and free radical etc.At present, the effect of protease inhibitor aspect medical science is subjected to attracting attention of common people, and further research finds that also protease inhibitor has certain curative effect in that resisting HIV (HIV) is caught.
Semen Fagopyri Esculenti belongs to polygonaceae plant, contains multiple bioactive substance in the seed, comprises biological species pyrite, trypsin inhibitor etc.In recent years, people such as Belozersky and Dunaevsky is separated to several trypsin inhibitors from Semen Fagopyri Esculenti, and adopt technology such as mass spectrum, high pressure liquid chromatography and protein sequencing, the biochemical property of buckwheat Trypsin Inhibitor, structure etc. are studied.But the preparation of relevant reorganization buckwheat Trypsin Inhibitor and biological function report thereof are seldom, and its inhibitory action to tumor cell is not appeared in the newspapers as yet.Therefore, prepare a kind of novel trypsin inhibitor, and disclose its possible in vivo biological function,, prepare new protide antitumor drug and have important significance for theories and realistic meaning for the sphere of action of Abundant protein enzyme inhibitor.
Summary of the invention
The purposes that the purpose of this invention is to provide a kind of buckwheat Trypsin Inhibitor and expression thereof and this trypsin inhibitor.
A kind of buckwheat Trypsin Inhibitor provided by the invention (Buckweat Trypsin Inhibitor is called for short BTI) is the small molecule protein in the organism, contains 69 amino acid residues, can the tryptic activity of specific inhibition.Its aminoacid sequence is as follows:
LRQCSGKQEW?PELVGERGSK?AAKIIENENE?DVRAIVLPEG?SAVPRDLRCD 50
RVWVFVDERG?VVVDTPVVM 69
The nucleotide sequence of BTI provided by the invention is made up of 210 nucleotide, comprises the reading frame and the termination codon TAA of a smoothness.Its nucleotide sequence is as follows:
CTGCGTCAGT?GCTCCGGTAA?ACAAGAATGG?CCAGAGCTCG?TTGGAGAGAG 50
AGGGTCCAAG?GCTGCCAAGA?TCATCGAAAA?CGAGAACGAA?GACGTGCGAG 100
CTATCGTCTT?GCCTGAGGGT?AGCGCGGTGC?CTAGAGACCT?CCGATGTGAC 150
CGTGTGTGGG?TTTTCGTAGA?CGAGCGAGGA?GTTGTTGTTG?ATACTCCTGT 200
TGTTATGTAA 210
A kind of carrier that contains the nucleotide sequence of the BTI that encodes, it be with the coding BTI nucleotide fragments be cloned into the recon pQE31-BTI that obtains behind the prokaryotic expression carrier.Described carrier is a plasmid, contains the T7 promoter.
A kind of BTI engineering bacteria, it contains foregoing carrier.Be that described pQE31-BTI recon is converted in a kind of bacterium, for example in the escherichia coli M15 cell, be built into engineering bacteria, for example pQE31-BTI/M15.
The expression of BTI, preferably the pQE31-BTI expression vector is transformed into escherichia coli M15, make up colibacillus engineering pQE31-BTI/M15, engineering bacteria is seeded in the LB culture medium that contains ampicillin, 37 ℃, the 165r/min shaken cultivation to O.D 600nm be 0.5-0.7, it is 0.5mmol/L that adding IPTG makes final concentration, abduction delivering 2-4 hour, collect thalline, smudge cells, with supernatant through affinitive layer purification, merge the destination protein that each post eluting obtains, obtain product after the lyophilization.
BTI is a newcomer in the trypsin inhibitor family, and narrow spectrum inhibition experiment shows: this inhibitor forms 1: 1 complex with the mode and the trypsin of competitive inhibition, suppresses tryptic hydrolysis.
Adopt mtt assay and flow cytometry biological activity research to reorganization BTI, experimental results show that: when reorganization BTI acts on myelomatosis multiplex people's IM-9 cell, can effectively suppress the growth of IM-9 cell, when BTI concentration during, promptly demonstrate certain effect (see figure 5) of inducing the IM-9 natural death of cerebral cells at 0.2g/L.This inhibitor can be used for the preparation of new type antineoplastic medicine.
Description of drawings
The aminoacid sequence of accompanying drawing 1BTI.
The nucleotide sequence of accompanying drawing 2BTI.
The SDS-PAGE electrophoretogram of accompanying drawing 3BTI.
The SDS-PAGE purity of accompanying drawing 4BTI is identified electrophoretogram.
Accompanying drawing 5BTI induces the accent of the IM-9 tumor cell aspect graph (A. contrast, B.0.2g/L BTI) of dying
Embodiment 1:
(1) structure of BTI expression vector
PGEM-T-BTI and pQE31 empty carrier that laboratory is preserved are used BamH I and Hind III double digestion respectively, use the glue of promega company to reclaim test kit recovery genes of interest, spending the night at 4 ℃ with the pQE31 prokaryotic expression carrier by the T4 dna ligase is connected.Connect product transformed into escherichia coli BL21, the picking positive colony, carry out bacterium colony PCR and to expression plasmid order-checking identify.Show that through dna sequence analysis the buck wheat protein enzyme inhibitor gene has been cloned among the pQE31, with this recombiant plasmid called after pQE31-BTI.
(2) prokaryotic expression of BTI
With recombiant plasmid pQE31-BTI transformed into escherichia coli M15, make up colibacillus engineering strain pQE-31-BTI/M15, engineering bacteria is seeded in the LB culture medium that contains ampicillin, select different induction time (1h-7h) for use, different IPTG concentration (0.1-1.0) and 16 ℃ of low temperature inductions and 37 ℃ of room temperature are induced and are expressed destination protein respectively.By the optimization expression condition, the final concentration of selected IPTG is 0.2mmol/L, and induction time is 4h, and the expression of BTI was the highest when inducing temperature was 37 ℃.Further collect thalline, ultrasonication cell, cleer and peaceful precipitation in the 13000r/min centrifugalize.To go up cleer and peaceful precipitation and show that through the SDS-PAGE detection a new protein band appears in about 10.0kD place on gel pattern, this proteic molecular weight and the identical (see figure 3) of expectation destination protein molecular weight size.Illustrate that prokaryotic expression carrier that this law makes up can the great expression destination protein in M15, and the overwhelming majority expresses in soluble mode, expression accounts for about 40% of bacterial protein.
(3) purification of BTI
The solubility expression protein sample of above-mentioned acquisition is passed through Hitrap chelating affinity column, with 0.5ml0.1mol/L nickel salt solution eluting, collect destination protein after the balance, lyophilization is reorganization buck wheat protein enzyme inhibitor product.This product shows single band (see figure 4) on electrophoresis pattern after SDS-PAGE detects, the analysis showed that through protein content, the destination protein response rate of Ni metal-chelate and affinity column purification reaches 79%, the affinity chromatograph method post effect height that proof this law is set up utilizes this law only to need single step purification can obtain the pure product of the higher destination protein of output.
The destination protein of purification adopts the BApNA method to carry out the inhibitor activity analysis, concrete grammar is: the buck wheat protein enzyme inhibitor that successively adds substrate B ApNA, trypsin and this law preparation in the 3ml reaction system, effect is 10 minutes under 37 ℃ of conditions, add 33% acetic acid cessation reaction, under the 410nm wavelength, carry out colorimetric analysis afterwards.With unconstrained dose system is contrast, residual enzyme activity more separately.Suppress active being defined as: make every milliliter of reactant liquor in the per minute at A
410nmAbsorption value reduce .0.01 and be one and suppress unit of activity (UI) that rejection ratio vigor (UI/mg) be the inhibition vigor of every milligram of albumen generation.The concrete method of measuring with reference to Erlanger.Experimental result explanation, the reorganization buckwheat Trypsin Inhibitor of this law preparation can the tryptic activity of specific inhibition, and form 1: 1 complex in the mode of competitive inhibition with trypsin, and the inhibition trypsin is to the hydrolysis of its substrate B ApNA.
Further adopt mtt assay and flow cytometry that the biological activity research of reorganization BTI is proved in addition: when reorganization BTI acts on myelomatosis multiplex people's IM-9 cell, can effectively suppress the growth of IM-9 cell, when BTI concentration during, promptly demonstrate certain effect (see figure 5) of inducing the IM-9 natural death of cerebral cells at 0.2g/L.This inhibitor can be used for the preparation of new type antineoplastic medicine.
The present invention is method construction recombination plasmid and the engineered strain that adopts prokaryotic expression by such scheme, express destination protein in batches, affinity purification obtains the higher BTI of purity, and this albumen can the tryptic activity of narrow spectrum inhibition, induces the accent of IM-9 tumor cell to die.It is a kind of new type antineoplastic medicine that has potentiality and application prospect.
Claims (8)
1. buckwheat Trypsin Inhibitor is characterized in that having following aminoacid sequence:
LRQCSGKQEW?PELVGERGSK?AAKIIENENE?DVRAIVLPEG?SAVPRDLRCD 50
RVWVFVDERG?VVVDTPVVM 69。
2. the nucleotide sequence of the buckwheat Trypsin Inhibitor of the claim 1 of encoding:
CTGCGTCAGT?GCTCCGGTAA?ACAAGAATGG?CCAGAGCTCG?TTGGAGAGAG 50
AGGGTCCAAG?GCTGCCAAGA?TCATCGAAAA?CGAGAACGAA?GACGTGCGAG 100
CTATCGTCTT?GCCTGAGGGT?AGCGCGGTGC?CTAGAGACCT?CCGATGTGAC 150
CGTGTGTGGG?TTTTCGTAGA?CGAGCGAGGA?GTTGTTGTTG?ATACTCCTGT 200
TGTTATGTAA 210。
3. carrier, it contains the described nucleotide sequence of claim 2.
4. carrier as claimed in claim 3 is a plasmid, contains the T7 promoter.
5. engineering bacteria is characterized in that it contains the described carrier of claim 3.
6. the described engineering bacteria of claim 5 is prokaryotic expression system.
7. the expression of the described buckwheat Trypsin Inhibitor of claim 1 is characterized in that, it is as follows to express step:
(a) make up the pQE31-BTI expression vector; (b) according to standard technique transformed into escherichia coli M15, make up colibacillus engineering strain pQE31-BTI/M15, engineering bacteria is seeded in the LB culture medium that contains ampicillin, 37 ℃, the 165r/min shaken cultivation to O.D 600nm be 0.5-0.7, it is 0.5mmol/L that adding IPTG makes final concentration, abduction delivering 2-4 hour.
8. the application of the described buckwheat Trypsin Inhibitor of claim 1 in the preparation antitumor drug.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB2005100123854A CN1294988C (en) | 2005-03-12 | 2005-03-12 | Buckwheat trypsase inhibitor, expression and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB2005100123854A CN1294988C (en) | 2005-03-12 | 2005-03-12 | Buckwheat trypsase inhibitor, expression and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1718241A CN1718241A (en) | 2006-01-11 |
CN1294988C true CN1294988C (en) | 2007-01-17 |
Family
ID=35930270
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB2005100123854A Expired - Fee Related CN1294988C (en) | 2005-03-12 | 2005-03-12 | Buckwheat trypsase inhibitor, expression and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1294988C (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102727873B (en) * | 2012-06-15 | 2013-07-10 | 山西大学 | Applications of buckwheat trypsin inhibitor |
CN105801690B (en) * | 2016-05-30 | 2019-01-29 | 山西大学 | A kind of trypsin inhibitor and its preparation method and application |
CN108181370A (en) * | 2018-01-08 | 2018-06-19 | 太原科技大学 | Preparation, determination of activity and the inhibition constant method for measuring of Fructus Sophorae trypsin inhibitor |
CN109942700B (en) * | 2019-03-21 | 2020-12-25 | 山西大学 | Recombinant buckwheat trypsin inhibitor mutant and trypsin affinity material |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN86104618A (en) * | 1986-07-02 | 1988-01-13 | 日本化学研究株式会社 | Concentrate with separate Urina Hominis in trypsin inhibitor and the method for kallidinogenase |
CN87108349A (en) * | 1986-12-19 | 1988-07-13 | 农业遗传工程公司 | The dna molecular that is used for plant protection |
-
2005
- 2005-03-12 CN CNB2005100123854A patent/CN1294988C/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN86104618A (en) * | 1986-07-02 | 1988-01-13 | 日本化学研究株式会社 | Concentrate with separate Urina Hominis in trypsin inhibitor and the method for kallidinogenase |
CN87108349A (en) * | 1986-12-19 | 1988-07-13 | 农业遗传工程公司 | The dna molecular that is used for plant protection |
Also Published As
Publication number | Publication date |
---|---|
CN1718241A (en) | 2006-01-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Mori et al. | Isolation and structure of the bacterial sex pheromone, cAD1, that induces plasmid transfer in Streptococcus faecalis | |
CN101519446A (en) | Method for preparing recombinant human insulin and analogs of recombinant human insulin | |
EP1502949B1 (en) | Antimicrobial polypeptide and utilization thereof | |
CN105061603B (en) | Antibacterial peptide-lysozyme fusion protein preparation method and applications | |
CN1294988C (en) | Buckwheat trypsase inhibitor, expression and application thereof | |
CA1340841C (en) | Cecropin-like polypeptides with activity against gram-positive and gram-negative bacteria | |
CN107312095A (en) | A kind of Melittin of antibacterial peptide LL 37 and its application and the preparation method in bacillus subtilis | |
JP2011072294A (en) | New antibacterial peptide | |
CN101220092B (en) | Human keratinized cell growth factor-1 analogue preparation method and application thereof | |
WO2004020461A1 (en) | A group of novel synthetic antibiotic peptides | |
CN100432230C (en) | Fusion expression method for metallothionein and use thereof | |
CN104328131A (en) | Recombinant expression, separation and purification method of human ribonuclease 4 protein | |
CN1936000A (en) | Fennero penaeus chinensis antibacterial protein gene and recombinant expression and use | |
CN100420751C (en) | Pesticidal gene and its use | |
CN111057144A (en) | Gene recombination high-stability collagen oligopeptide MYS-1 and preparation method and application thereof | |
CN109971776A (en) | Method for purifying proteins based on light cutting motif | |
CN113201074B (en) | PKEK fusion protein and preparation method and application thereof | |
CN109305996A (en) | Fusarium graminearum secreted protein exciton FgHrip1 and its application | |
CN101622269A (en) | Expression of proteins in e. coli | |
CN109776653B (en) | Human serum albumin adhesion peptide and application thereof | |
CN1306030C (en) | Human interleukin-10 gene sequenc and E coli containing the said gene sequence | |
CN113214409B (en) | Melittin-mortiferin hybrid peptide mutant MTM and application thereof | |
CN113666996B (en) | Tachypleus tridentatus antibacterial peptide and preparation method and application thereof | |
CN108314728A (en) | A kind of alfalfa trypsin inhibitor MT-mth2-89i19 and its encoding gene and application | |
CN100523172C (en) | Gene engineering bacteria of high efficiency expression of human alpha 1-thymulin and its construction method and use |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
C17 | Cessation of patent right | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070117 Termination date: 20120312 |