CN1294988C - Buckwheat trypsase inhibitor, expression and application thereof - Google Patents

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

A kind of buckwheat Trypsin Inhibitor and expression thereof and application

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.

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