CN101880668B - Gene for coding serpin and application thereof - Google Patents

Abstract

The invention relates to a gene spilC for coding serpin activity, which is provided with one of the following nucleotide sequences: 1) DAN sequence of sequence table No.1; 2) amino acid residue sequence of sequence table No.2 with one or more replaced, deficient and/or added amino acid residue and protein having the serpin activity. DAN in sequence 1 in the sequence table is started from one hundred eleventh ribonucleotide from 5' end through a starting codon ATG and is ended with an ending codon TAA to the 755 base, a complete ORF (ribonucleotide 111 to 755), the one hundred eleventh to one hundred thirteenth ribonucleotide from the 5' end is of a starting codon ATG of the spilC gene, and seven hundred fifty third to seven hundred fifty fifth ribonucleotide from the 5' end is of an ending codon TAA of the spilC gene. The novel serpin and the coded gene thereof have wide application on the aspects for playing the active defense effect of the host cell for the infection process of the pathogenic microorganism.

Description

A kind of gene of coding serpin and application thereof

Technical field

The present invention relates to a kind of novel serpin and codase gene and application.

Background technology

To the baroque mankind, various proteinase inhibitor are being brought into play enormous function from virus simple in structure.According to the similarity of its protein structure and aminoacid sequence, proteinase inhibitor generally is divided into four kinds: aspartic acid proteinoid enzyme inhibitors, halfcystine proteinoid enzyme inhibitors, nonmetal proteinase inhibitor and serpin.Wherein serpin is most important, also is to study one of proteinase inhibitor the most widely.Serpin can prevent the unnecessary proteins hydrolysis; Regulate the hydrolysising balance of Tryase; Be to keep the homeostatic important factor of organism; Being present in the intravital serpin of some animal possibly play an important role in the neuronal death that cerebral ischemia or exitotoxicity cause, possibly have neuroprotective.Be that in addition the intravital serpin of some plant-animal can suppress the protease activity of its invasive organism, thereby in being directed against the external source infection processs, take the initiative defence capability.Serpin is generally the single peptide chain protein, and the serpin molecule generally is made up of 29-190 amino acid, and more is about 400.According to the sequence homology of serpin, the number and the three-dimensional structure of disulfide linkage; Serpin can be divided into a plurality of different superfamilies; As pancreatic trypsin inhibitor (English name: Kunitzinhibitor) family, pancreatic secretion tryptase inhibitors (English name: Kazal inhibitor) family, i (streptomyces) subtilisin inhibitor (English name: streptomyces subtilisin inhibitor) family, Bowman-Brik type suppressor factor family, etc.What research was more now is Kunitz type propylhomoserin proteinase inhibitor; Such suppressor factor source is very extensive, from plant-animal such as Pancreas Sus domestica, sea anemone, people's urine, snake, soybean, Semen Phaseoli, Snakegourd Root, Alocasia ordora, Herba Sophorae alopecuroidis, peanut, Taihu Lake blue-green algae, has separated obtaining now.Its three-dimensional structure high conservative: disulfide linkage bridge, three chain beta sheets and a C end alpha-helix by a tangible hydrophobic core, three pairs of high conservatives are formed, and the research of such suppressor factor family is representative with the research of Trypsin inhibitor,Trasylol BPTI.Kazal type serpin is one of comparatively conservative family; It is present in bird egg, insect, small lobsters, mammalian tissues and the body fluid, and they and blood clotting, fibrinolysis, embryo's generation, ontogeny, inflammation and immunne response etc. have substantial connection.Kazal type serpin is made up of the structural domain that one or more repeats to guard usually, and its structural domain sequence is comparatively conservative, and its molecular conformation high conservative.In addition, many Kazal type serpins have a segment signal peptide usually, are positioned at N-terminal, and its length is approximately 20 amino acid.Bowman-Brik type suppressor factor at first equals nineteen forty-four by Bowman, and to utilize acetone be that the insoluble factor is separated from soybean, and molecular weight is 6000-10000, contains a large amount of halfcystines.The single chain polypeptide that Bowman-Brik type suppressor factor is made up of 71 amino acid comprises 7 disulfide linkage, and BBI generally contains 2 active site: one is Methionin 16-Serine 17; Another is a leucine 44-Serine 45, so such suppressor factor can suppress trypsinase and Quimotrase simultaneously.

In the mankind and higher animal, many Tryases are present in the blood, and are relevant with blood coagulation, fibrinolytic.Remove and to be present in the blood China and foreign countries, some proteolytic enzyme also exists and cns, plays an important role for the running balance of cns.And corresponding Serine proteinoid enzyme inhibitors can be regulated the aggegation of blood, and plays a part very important for brain injury and neurodegeneration.In organism, the activity of Tryase depends on its homology serpin (English name: balance serpin inhibitor).These serpins combine closely as the pseudosubstrate of its target protease and the reactive site of enzyme, play an important role at blood coagulation, fibrinolytic, inflammation and immunology.For example serpins can pass through the adjusting of the protein dissolution process of extracellular matrix, in the nerve migration, plays a significant role in the process that axon growth or ripe post-synapse connect.T-PA is the important plasminogen activator in existence and the blood; Belong to the serine stretch protein enzyme; In brain and fetal development; With cell migration, vasculogenesis with to be organized in reinventing of cns relevant, the suppressor factor I type Plasminogen activator (PAI-I) of this enzyme can suppress this enzyme fast, thereby plays a significant role in animal body.Bovine pancreatic trypsin inhibitor (English name: Bovinepancreatic trypsin inhibitor; BPTI) and (English name: Amyloid beta-protein precursor of the amyloid-amyloid protein precursor in the human body; APP) all belong to the serpin of Kunitz family; They not only suppress trypsinase, Chymotrypsin and plasmin; Also suppress Tryases such as plasma kallikrein simultaneously, be widely used in postoperative hemostasis and the concurrent inflammatory reaction of postoperative clinical.In addition BPTI and APP for factor XI, plasma thromboplastin antecedent a,, factor XI, plasma thromboplastin antecedent Ia also has restraining effect, thereby can directly suppress ectogenic blood coagulation system.

Mainly utilize physics, chemistry or biochemical method at present, for example blood of animal and the piece root stem tuber of plant etc. extract all kinds of serpins to collection material in plant-animal and the microbe body.Physical method comprises mechanical means and non-mechanical approach, and mechanical means mainly is through mechanical shear force smudge cells, and commonly used have high speed pearl mill method and a homogenate method, and non-mechanical approach generally is through the physical method smudge cells, and commonly used have freeze-thaw method and a supersonic method.Chemical process generally is to place sample under the low temperature, adds suitable chemical reagent and stirs broken cell membrane, and albumen is discharged.Biochemical method mainly relies on biological enzymolysis albumen, and the enzymolysis process operational condition is gentle, and specificity is high, and the recovery is high.According to the different Physiology and biochemistry character of animal/vegetable protein, also can adopt extraction with aqueous solution method or organic solvent extraction method to obtain crude protein.

As everyone knows, environmental microorganism is a huge genetic resources storehouse.Correlative study points out to have disclosed through DNA-DNA hybridization technique and other independent culture techniques that microbe species trends towards 4 in a 100g pedotheque; 000 kind to 13; Between 000 kind; Just can extract the DNA of tens of micrograms in the earth of average every 500mg, this has wherein comprised a large amount of unknown function genes.For pure culture technigne; Utilize not culturing micro-organisms resource of grand genomic library technological development environment; Can be so that account for can not the culturing micro-organisms development of resources coming true of mikrobe flux 99%, it with the exploration space enlargement of microbial gene hundreds of times.Grand genomic library technology is exactly that clone's target dna imports the host bacterium then and then realizes the screening to the target clone to suitable carriers with direct separation environment culturing micro-organisms macro genome DNA not.Current; Scientist with E.coli or Streptomycin lividans as cloning host; Make up various libraries such as plasmid libraries, cosmid libraries, BAC libraries, fosmid libraries etc., brought the novel active enzyme and the various biologically active substance of enormous amount based on gene function expression and sequence-specific homology screening etc. to people.Therefore, utilize grand genomic library technology, research extreme environment system not culturing micro-organisms with clone not culturing micro-organisms enzyme gene, all kinds of serpins of separation screening reach the meaning that in practical application, all has particularly important in theory.The present invention comes from the grand genomic library of the North Sea, Guangxi ocean environment settling through structure; Adopt the direct order-checking screening strategy of enzyme; Separate the gene that has obtained the inhibition of encoding novel Tryase from the gene library the inside; Can in host cell, be used for the inhibition of serine protease to produce arrestin by this gene of great expression.In invasive organism, has purposes widely to aspects such as the defense reactions of taking the initiative in the external source infection processs.

Summary of the invention

The purpose of this invention is to provide a kind of gene and application thereof of coding serpin.

The gene of a kind of coding serpin provided by the present invention, name is called spi1C, derives from not culturing micro-organisms of the North Sea, Guangxi oceanic sediment, has one of following nucleotide sequences:

1) dna sequence dna of sequence table No.1 and partial sequence thereof;

2) with sequence table No.1 in the amino acid residue sequence of No.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have Tryase and suppress active protein.

Carry the plasmid of this gene; Its classification called after ETEC (Escherichia.coli) BL21 (DE3) pLysS/pGXAG59G is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; Be numbered CGMCC No.3317, date saved is on October 10th, 2009.

The DNA of sequence table No.1 is cloning vector pGEM-3Zf (+) partial dna sequence and the external source DNA of culturing micro-organisms not that is cloned on the carrier; This exogenous dna fragment is by 642 based compositions, comprises complete novel spilC gene in the entrained exogenous dna fragment of recombinant plasmid.GC content is 51.2%.Finish with terminator codon TAA since ATG to 755 base of 5 ' the 111st Nucleotide initiator codon of end; There is a complete ORF (Nucleotide 111-755); From 5 ' end 111-113 position Nucleotide be the initiator codon ATG of spilC gene, from 5 ' end 753-755 position Nucleotide be the terminator codon TAA of spilC gene.This ORF is 642 albumen that the Nucleotide codified is made up of 214 amino acid altogether.Analyze this aminoacid sequence through Blastx and have 56% similarity, 42% consistence with one " proteinase inhibitor I4serpin " coming from Spirosoma linguale DSM 74.

The cloning process of the gene of a kind of coding serpin provided by the invention comprises the following steps:

(1) from the ocean environment sample of the North Sea, Guangxi, extracts not culturing micro-organisms macro genome DNA, make up the grand genomic library of ocean environment;

(2) from the grand genomic library of ocean environment, utilize the directly clone of order-checking screening strategy separation serpin.

The present invention is through the grand genomic library of constructing environment; Utilize directly order-checking screening strategy; Obtained the gene of new coding serpin; Can be in host cell this gene of great expression producing serpin, thereby the protease activity of related diseases pathogenic microorganism is suppressed.

New serpin provided by the present invention and codase gene thereof have purposes widely to aspects such as external source infection processs performance host cell active defense reactions in invasive organism.

Description of drawings

Fig. 1 is the macro genome DNA of the not culturing micro-organisms from the ocean environment sample of the North Sea, Guangxi, extracted.

Fig. 2 is that the grand gene library of the North Sea, Guangxi ocean environment clone's restriction enzyme EcoRI restriction analysis is to judge the library quality.

Fig. 3 cuts detected result for the enzyme of original clone pGXAG59.

Fig. 4 is a spi1C gene DNA sequence analysis.

Fig. 5 is the structure of reorganization cloning by expression E.coli Tuner (DE3) pLacI/pGXAG59G.

Fig. 6 is the recombinant expressed clone's that obtains behind the entrained spi1C gene transformation of recombinant plasmid pGXAG59G intestinal bacteria E.coliTuner (DE3) pLacI SDS-PAGE analytical results.

Fig. 7 is the inhibition effect analysis of serpin.

Embodiment

Used in an embodiment of the present invention main raw comprises: EcoRI restriction enzyme, T4DNA ligase enzyme, calf intestine alkaline phosphatase CIP, pGEM-Teasy carrier be available from Promega company, Tryptones, PVPP (English full name: Polyvinpolypyrrolidone) available from Sigma company.

Embodiment 1

The clone of a kind of gene spi1C of encoding novel serpin; Mainly comprise following key step 1,2,3,4,5.

Key step 1: from the ocean environment sample, extract the purifying macro genome DNA, detailed method steps is described below.

(1) from the ocean environment system of the North Sea, Guangxi, gather the throw out sample, take by weighing the moistening settling of 4-4.5g, add 2 parts of TENS of 10mL, said 2 parts of TENS are: 100mM Tris-HCL, 40mM EDTA, 200mMNaCI, 2% (w/v) SDS, pH8.0; Abundant mixing on vibrator;

(2) add 25mg/mL Protease-K 100 μ L, through 70 ℃ of water-bath dissolving 30min, every 10min stirring and evenly mixing once;

(3) place liquid nitrogen quick-frozen 2.5min then, place-20 ℃ of freezing 5min of cryogenic refrigerator rapidly, immediately dissolve fully after the taking-up through boiling water bath.Step 3 has repetition 3 times altogether;

(4) place the static 3min of room temperature again; At room temperature through centrifugal treating, collect supernatant and place on ice then, add the Buffer A of 10mL in the deposition again, said Buffer A is 50mM Tris-HCL, 25mM EDTA, 3% (w/v) SDS, 1.2% (w/v) PVPP, pH8.0, centrifugal collection supernatant; With all supernatants mixing together, place on ice; Add 0.1g PVPP among supernatant 10～15mL once more, add 1 portion of TE damping fluid of SephadexG-200 and 20mL simultaneously; Said 1 portion of TE damping fluid is: 10mM Tris-HCl, pH8.0,1mM Na ₂EDTA; Abundant mixing; 37 ℃ of insulation 2h, every 30min mixing once.The centrifugal PVPP that removes;

(5) with all supernatants with isopyknic phenol, phenol-chloroform: primary isoamyl alcohol and chloroform extracting are once; Said phenol-chloroform: the volume ratio of primary isoamyl alcohol=25: 24: 1.

(6) deposit D NA, every 5mL add isopyknic 8mol/L ammonium acetate, the liquor kalii acetici of 1/10 volume; The pH of adjustment liquor kalii acetici is 4.8, and adds the above absolute ethyl alcohol of 3 times of volumes, places more than the 15min in subzero 80 ℃;

(7) with 10, after the rotating speed of 000rpm carried out centrifugal collecting precipitation 10min, with 75% (v/v) washing with alcohol 2～3 times, drying was dissolved in 1 portion of an amount of TE damping fluid; Obtain rough macro genome DNA.

Key step 2: adopt electroelution method to reclaim the purification of crude macro genome DNA, detailed method steps is described below.

(1) processing of dialysis tubing

1. cut long 10-20cm dialysis tubing;

2. dialysis tubing is boiled 10min in 2% (w/v) sodium hydrogencarbonate, 1mM EDTA solution;

3. take out dialysis tubing and use the zero(ppm) water cleaning down;

4. in 1mM EDTA solution, boil 10min, do not outwell solution;

5. let the dialysis tubing naturally cooling, 4 ℃ of storages are noted at lay up period, should let dialysis tubing immerse solution all the time, in case dry;

6. before using, with the inside and outside wall of the abundant douche bag of zero(ppm) water.

(2) enzyme is cut an amount of DNA, makes target fragment have about 1 μ g, and the electrophoretic separation poststaining is confirmed the position of target fragment.

(3) downcut with knife blade and contain the segmental agarose adhesive tape of target dna, it is placed on wide flat by on the moistening spoon of 1 part of TAE, described 1 part of Tris-acetate that TAE is 40mmol/L, the EDTA of 1mmol/L, pH8.0.

(4) dialysis tubing one end is sealed tightly, and dialysis tubing is filled 1 part of TAE, with spoon cutting adhesive tape is put into dialysis tubing then.

(5) make glue sink to the bottom of dialysis tubing, remove unnecessary damping fluid, make the agarose adhesive tape just in time be cushioned liquid and encase,, avoid producing bubble with the top that clip is clamped dialysis tubing.

The dialysis tubing that (6) will contain glue is placed on electrophoresis 2-3h in the electrophoresis chamber that 1 part of TAE is housed, and electrophoresis intensity is 4～5V/cm usually, then DNA by electroelution to the wall of sack.

(7) change electrode, counterelectrophoresis 1min makes DNA turn back to the inner chamber of dialysis tubing from the bag wall again.

(8) open dialysis tubing, pour out glue electrophoretic buffer on every side carefully in EP pipe, use a spot of 1 part of TAE then, be added in the bag and clean, and scavenging solution is sucked in the EP pipe in the lump.

(9) whether dye glue, it is complete to detect electroelution.

(10) with phenol, phenol/chloroform and chloroform extracting once, use alcohol precipitation DNA then.After the drying, be dissolved among 1 part of TE of 10 μ l.Described 1 part of TE is 10mM Tris-HCl, pH8.0; 1mMNa ₂EDTA.

Map 1.In implementing key step 1 and 2,1 is λ/HindIII standard, and clip size is followed successively by from big to small: 23.13kb, 9.4kb, 6.6kb, 4.4kb, 2.3kb, 2.0kb; 2 are the macro genome DNA of the not culturing micro-organisms that the extraction separation purifying obtains later on from the ocean environment sample of the North Sea, Guangxi, and running the appearance amount is 1 μ L; 3 is that the 3rd swimming lane is that restriction enzyme EcoRI complete degestion macro genome DNA can carry out the operation of molecular level to judge macro genome DNA purity, and running the appearance amount is 1 μ L.

Key step 3: the structure of the grand genomic library of ocean environment sample, detailed method steps is described below.

At first adopt the alkaline denaturation of revising to extract cloning vector pGEM-3Zf (+) DNA in a large number; Obtain the higher degree DNA; Its principal mode is superhelix CCC, through EcoR1 enzyme single endonuclease digestion, becomes linear DNA molecule; After linear pGEM-3Zf (+) DNA is handled through CIP dephosphorylation enzyme, directly be used for the connection portion enzyme to cut not culturing micro-organisms macro genome DNA of ocean environment.

Carry out partially digested with EcoR1 to the good macro genome DNA of purifying; Agarose electrophoresis is separated enzyme and is cut product; These fragments are connected with pGEM-3Zf (+) DNA that handled through CIP dephosphorylation enzyme; Connect product electricity consumption method for transformation and import bacillus coli DH 5 alpha, detect the white intestinal bacteria bacterium colony that contains recon on the Luria-Bertani solid plate of X-gal, IPTG, penbritin containing.The concentration of X-gal, IPTG, penbritin is respectively: 40 μ g/mL, 40 μ g/mL, 50 μ g/mL.

With the single bacterium colony bridging piece of the positive to the Luria-Bertani solid plate that contains 50 μ g/mL penbritins; 37 ℃ be cultured to bacterium colony and be 2-3mm after; Bridging piece once more; The transformant picking is preserved cultivation based in the 96 well culture plate holes to the library, about 37 ℃ of constant temperature culture 24h, be stored in then in-80 ℃ of cryogenic refrigerators.Finally obtained 29500-30,500 white transformants.12 transformants of picking extract plasmid at random, cut detection with the EcoR1 enzyme then, find that 11 have the exogenous dna fragment that inserts at random, and mean length is 4.0kb.

Map 2, wherein, 1:1kb ladder standard, clip size is followed successively by from big to small: 10.0kb, 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5kb, 2.0kb, 1.5kb, 1.0kb, 750bp, 500bp, 250bp; Other swimming lane 2-11 is respectively the library clone DNA and cuts later result through the EcoR1 enzyme.

Key step 4: separation screening expression Tryase suppresses active clone from grand genomic library, and detailed method steps is described below.

Adopt directly order-checking separating screening method, on ABI 377DNA automatic DNA sequencer DNA, measure dna nucleotide sequence with the dideoxyribonucleoside acid system.Obtain a positive colony that is numbered pGXAG59, carry the gene of a possible encoding serine protein inhibitor.Electrophoresis detection result shows that the entrained exogenous dna fragment size of pGXAG59 clone is 1.1kb.

Map 3, wherein, 1:1kb ladder standard, clip size is followed successively by from big to small: 10.0kb, 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5kb, 2.0kb, 1.5kb, 1.0kb, 750bp, 500bp, 250bp pGXAG59/EcoRI; 2: extract the DNA that purifying obtains positive colony pGXAG59 before enzyme is cut; 3 cut the result of the DNA of positive colony pGXAG59 for the EcoR1 enzyme.

Key step 5: order-checking and ORFs ORF that the last expression of recombinant plasmid E.coli BL21 (DE3) pLysS/pGXAG59G Tryase suppresses active gene analyze, and detailed method steps is described below.

With software DNAStar the resulting sequence of direct order-checking is spliced; With NCBI (NationalCenter for Biotechnology Information; Http:// www.ncbi.nlm.nih.gov) software on is analyzed dna sequence dna; Like ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), Blast (http://www.ncbi.nlm.nih.gov/BLAST).

The entrained foreign DNA of positive colony pGXAG59 is by 1128 based compositions; Map 4; Finish with terminator codon TAA since ATG to 755 base of 5 ' the 111st Nucleotide initiator codon of end; Have a most possible complete ORF (Nucleotide 111-755), from 5 ' end 111-113 position Nucleotide be the initiator codon ATG of spi1C gene, from 5 ' end 753-755 position Nucleotide be the terminator codon TAA of spi1C gene.Through Blastn software relatively, the exogenous DNA array that pGXAG59 carries on dna level, can not find with existing GenBanK DB in any dna sequence dna have homology.The expression Tryase suppresses one of active gene spi1C coding and contains 214 amino acid whose protein; With simple assemblies structural research instrument (English name: Simple Modular Architecture Research Tool; SMART; Http:// smart.embl-heidelberg.de) analyzes, come from not by what dna sequence dna was inferred that the expression Tryase of culturing micro-organisms suppresses active Spi1C albumen unit construction, find a possible independent catalysis region functional structure; This catalyst structure domain is begun by first amino acid ATG, finishes to the 213rd amino acid.The gene of coding target protein comprises 642 Nucleotide altogether; The polypeptide of forming by 214 amino acid of encoding; Analyze this ORF aminoacid sequence with Blastp; Find that this aminoacid sequence and one " proteinase inhibitor I4serpin " coming from Spirosoma linguale DSM 74 exist 56% similarity, 42% consistence.This amino acid sequences encoded unique conservative property putative protein does not have tangible hydrophilic and hydrophobic, does not form and significantly strides the membrane structure zone, does not have the low complex degree structure yet.

Map 4, spi1C gene DNA sequence and the coded amino acid sequence analysis of target ORF.

Embodiment 2

The structure of spi1C dna recombinant expression plasmid.

The plasmid pETBlue-2 that utilizes U.S. Novagen company to provide efficiently expresses as the allos that host cell carries out target gene as expression vector and E.coliTuner (DE3) pLacI; Design forward amplimer FG1:5 '-TTA GGATCCGATGTTCCTTATGAACGCC-3 ' introduces the BamHI restriction enzyme site GGATCCReverse amplimer RG1:5 '-AT AAGCTTCTCCGGCTGCATCACTTTC-3 ' (NotI) introduces the HindIII restriction enzyme site AAGCTTThis ORF (Nucleotide 111-755), 214 Nucleotide altogether increase.With the positive colony that the PCR product is connected back transformed into escherichia coli NovaBlue cell with the pETBlue-2 carrier and the screening forward is connected on the Luria-Bertani solid plate that contains X-gal and IPTG, will comprise the Bacillus coli cells called after pGXAG59G of recombinant expression plasmid DNA.The pGXAG59G plasmid is converted into Tuner (DE3) pLacI gets bacterial strain Tuner (DE3) pLacI/pGXAG59G.The Theoretical pI/Mw (theoretical iso-electric point/molecular weight) that ExPASy website related software is analyzed pGXAG59G is: 4.53/28727.28.

Recombinant plasmid E.coli BL21 (DE3) pLysS/pGXAG59G accomplishes mikrobe in Chinese common micro-organisms culture presevation administrative center and preserves, and preserves and is numbered CGMCC No.3317, and date saved is on October 10th, 2009.

Embodiment 2 please refer to Fig. 5.Fig. 5 is the structure detected result of recombinant plasmid E.coli BL21 (DE3) pLysS/pGXAG59G.1:1kb ladder standard, clip size is followed successively by from big to small: 10.0kb, 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5kb, 2.0kb, 1.5kb, 1.0kb, 750bp, 500bp, 250bp; The DNA detected result before 2:E.coli BL21 (DE3) pLysS/pGXAG59G enzyme is cut, running the appearance amount is 5 μ L; 3: use restriction enzyme BamHI and HindIII enzyme to cut E.coli BL21 (DE3) pLysS/pGXAG59G DNA detected result afterwards, running the appearance amount is 5 μ l.

Embodiment 3

Abduction delivering and the purifying of Spi1C target protein in the intestinal bacteria system.

Contain in the LB substratum of Pyocianil in 10mL being transferred by the correct recombination bacillus coli of checking, cultivate 10-15h with vigor.From 10mL LB bacterium liquid, get 5-10mL and join and fill in the triangular flask of 1000mL that 500mL contains Pyocianil LB liquid nutrient medium, 37 ℃, the 220rpm shaking culture.Take out 1mL bacterium liquid behind the 2-3h and measure OD ₆₀₀, work as OD ₆₀₀Adding IPTG time the 0.4-0.6 is 1.0mmo/L to final concentration, behind 37 ℃, 220rpm shaking culture 10h, and the preparation crude enzyme liquid; Adopt Ni-NTA, the His-Bind Resins of Novagen company that recombinant protein is carried out separation and purification.The target protein that control sample and the separation and purification of preparation obtained carries out the SDS-PAGE protein expression and detects.

Embodiment 3 is please with reference to Fig. 6.Fig. 6 is the recombinant expressed clone's that obtains behind the entrained spi1C gene transformation of recombinant plasmid pGXAG59G intestinal bacteria E.coli Tuner (DE3) pLacI SDS-PAGE analytical results.1: adopt nickel post affinity chromatography technical point from the spring Spi1C Protein Detection result that obtains of China, running the appearance amount is 2 μ L; 2:E.coli BL21 (DE3) pLysS/pETBlue-2 Protein Detection result, running the appearance amount is 2 μ L; 3: protein standard model band, size is followed successively by from top to bottom: 116.0kDa, 66.0kDa, 45kDa, 35kDa, 25.0kDa, 18.8kDa.

Embodiment 4

Spi1C suppresses the mensuration of serine protease.

With reference to pertinent literature and Sigma company activity determination method, carry out part and improve: reaction system is set to 2mL; 0.02mg/mL trypsinase is dissolved in the 1mmol/L HCl of precooling, gets 200 μ L; Get 20-50 μ g and be dissolved in 50mmol/L pH7.6 sodium phosphate buffer and get the Spi1C protein sample, mix 25 ℃ of incubation 10min.Add 1mL 0.5mmol/L BAEE solution, with sterilization ddH ₂O is supplemented to 2mL; Begin reaction, the variation of 253nm photoabsorption in the METHOD FOR CONTINUOUS DETERMINATION 5min.Blank does not add trypsinase, and active control does not add Spi1C albumen fully.

Embodiment 4 sees also Fig. 7, the inhibition effect analysis of serpin.Scarlet 1 graphic representation is not for adding the blank of arrestin Spi1C, the time dependent curve of speed of reaction of complete active BAEE and Tryase; 2 is that green and 3 is that pink graphic representation is to suppress effect analysis, is respectively that Trypsin is a substrate, adds trypsinase and arrestin Spi1C (150 μ L, 180 μ L) speed of reaction time history plot later on respectively.

The expression vector, clone and the product Tryase that contain promotor, ORFs and the partial sequence thereof of gene of the present invention suppress synthetic protection scope of the present invention that all belongs to of enzyme process of active factor.

Sequence table

< 110>Guangxi University

< 120>a kind of gene of coding serpin and application thereof

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<170>PatentIn version 3.3

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atgttcctta tgaacgccct ctacttcaaa ggcacctgga cctggcagtt tgaaaaggac 60

aacaccacca tgaagccctt caccaatgat atcggcggca tcacgagcgt ggaaaccatg 120

tcgggaagca tccctctttg ggcgcactat gaccaggatt tcaatgccat tgaacttttt 180

tacggccaaa gcaacttctc catgatcatc gtcgttcccc gcaccaccat ccaggacctg 240

ctgagccggt tcgacgccga ttcatgggag gagcttaccg gcaaatttga tgcgaagacc 300

ggagatccgg gagagaccga tctgctgatg cccaaattca gctttgaata tgaaaagcaa 360

ctgaaggaca acctttcggc cctggggatg attgatgcct ttgatccggg tctggctgac 420

ctaagtggca tttcagattc ggatatatac gtcaattttg taaaacagaa caccttcgtg 480

aaagtggacg aagaaggcac cgaagcggcg gccgtcacca ccgtcggcat atacgaaact 540

tccgcgggcg aaccttttgt catcaataaa tccttcattt tcgccatccg ggaaaggacc 600

agcaacaccc tgctgttcat cgggaaagtg atgcagccgg agtaa 645

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Met Phe Leu Met Asn Ala Leu Tyr Phe Lys Gly Thr Trp Thr Trp Gln Phe Glu Lys Asp

5 10 15 20

Asn Thr Thr Met Lys Pro Phe Thr Asn Asp Ile Gly Gly Ile Thr Ser Val Glu Thr Met

25 30 35 40

Ser Gly Ser Ile Pro Leu Trp Ala His Tyr Asp Gln Asp Phe Asn Ala Ile Glu Leu Phe

45 50 55 60

Tyr Gly Gln Ser Asn Phe Ser Met Ile Ile Val Val Pro Arg Thr Thr Ile Gln Asp Leu

65 70 75 80

Leu Ser Arg Phe Asp Ala Asp Ser Trp Glu Glu Leu Thr Gly Lys Phe Asp Ala Lys Thr

85 90 95 100

Gly Asp Pro Gly Glu Thr Asp Leu Leu Met Pro Lys Phe Ser Phe Glu Tyr Glu Lys Gln

105 110 115 120

Leu Lys Asp Asn Leu Ser Ala Leu Gly Met Ile Asp Ala Phe Asp Pro Gly Leu Ala Asp

125 130 135 140

Leu Ser Gly Ile Ser Asp Ser Asp Ile Tyr Val Asn Phe Val Lys Gln Asn Thr Phe Val

145 150 155 160

Lys Val Asp Glu Glu Gly Thr Glu Ala Ala Ala Val Thr Thr Val Gly Ile Tyr Glu Thr

165 170 175 180

Ser Ala Gly Glu Pro Phe Val Ile Asn Lys Ser Phe Ile Phe Ala Ile Arg Glu Arg Thr

185 190 195 200

Ser Asn Thr Leu Leu Phe Ile Gly Lys Val Met Gln Pro Glu

205 210 214

Claims (2)

1. the gene spi 1C of an encoding serine proteolytic enzyme arrestin is characterized in that, shown in the dna sequence dna of sequence table No.1.

2. the application of protein in preparation serine protease suppressor factor that gene spi 1C as claimed in claim 1 is coded.

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