CN102127552B - CPTI (Cowpea Trypsin Inhibitor) gene, applications thereof and method for preparing great amount of CPTI - Google Patents
CPTI (Cowpea Trypsin Inhibitor) gene, applications thereof and method for preparing great amount of CPTI Download PDFInfo
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- CN102127552B CN102127552B CN 201010590327 CN201010590327A CN102127552B CN 102127552 B CN102127552 B CN 102127552B CN 201010590327 CN201010590327 CN 201010590327 CN 201010590327 A CN201010590327 A CN 201010590327A CN 102127552 B CN102127552 B CN 102127552B
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- trypsin inhibitor
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Abstract
The invention relates to genetic engineering, in particular to a CPTI (Cowpea Trypsin Inhibitor), the applications thereof and a method for preparing a great amount of CPTIs. The inventor of the invention firstly directly obtains a CPTI gene (without introne) from a cowpea gene group DNA (Deoxyribonucleic Acid) by utilizing a PCR (Polymerase Chain Reaction) and then optimizes and modifies the CPTI sequence so that the expression quantity and the expression specific activity of the modified sequence in colon bacillus are remarkably improved.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of cowpea trypsin inhibitor gene and use, prepare in a large number the method for Cowpea Trypsin Inhibitor.
Background technology
Cowpea Trypsin Inhibitor (cowpea trypsin inhibitor, CPTI) be the serpin of a kind of Bowman-Birk of belonging to family, can with Agricultural pests (mainly being lepidopteran, Orthoptera, coleopteron) digestive tube in serine protease form the mixture of enzyme-inhibitor, thereby weaken or the digestion hydrolytic action of blocks protein enzyme, and then it is malnutritive that insect is produced, and disorder even the phenomena of mortality occur.Compare with traditional Bt albumen, CPTI albumen have press down worm or insecticidal spectrum is wider, to advantages such as person poultry safeties, be a kind of potential development object of biological pesticide, thereby have very high application and promotional value in agroforestry.
Coli expression system is present most widely used prokaryotic expression system, mainly is comprised of expression vector and Host Strains two portions.Expression vector generally has following characteristics: stable genetic replication, ability goes down to posterity; Has selection markers; Regulatable promotor, the background transcriptional level is lower during inhibition; Possesses the multiple clone site that foreign gene inserts.Host Strains is generally selected the bacterial strain of Deficient In Extracellular Proteases, is exactly lon and ompT protease-deficient such as the serial bacterial strain of the BL21 of classics.
But be not that all foreign genes can both efficiently express in escherichia expression system, the present inventor finds that the Cowpea Trypsin Inhibitor that the clone obtains from cowpea can not be at E. coli, its expression amount is lower, and the protein-active of expression is lower.
Summary of the invention
In order to address the above problem, the present inventor proposes and has finished the present invention.
The purpose of this invention is to provide a kind of cowpea trypsin inhibitor gene.
A further object of the present invention provides a kind of Cowpea Trypsin Inhibitor.
Another object of the present invention provides a kind of Cowpea Trypsin Inhibitor prokaryotic expression carrier.
Another object of the present invention provides a kind of method of efficient, great expression Cowpea Trypsin Inhibitor.
Another object of the present invention provides a kind of engineering strain of efficient, great expression Cowpea Trypsin Inhibitor.
Another object of the present invention provides the application of above-mentioned cowpea trypsin inhibitor gene.
The present inventor at first utilizes the PCR reaction directly to obtain CPTI gene (the CPTI gene does not contain intron) from the cowpea genomic dna as mentioned above, 106 amino acid of the open reading frame of CPTI gene coding, this proteins encoded N end with 18 amino acid whose signal peptides (SignalP:http: //www.cbs.dtu.dk/services/SignalP/).This gene at expression in escherichia coli, is found that its expression amount is lower, and the protein-active of expression is lower.
Because the function that prokaryotic expression system does not usually have identification eukaryotic cell signal peptide and this albumen is successfully located, and ripe activated albumen always could form behind signal peptidase excision signal peptide, the synchronous signal peptide mainly contains hydrophobic amino acid, usually forms inclusion body in prokaryotic expression system during great expression.Therefore, the present inventor has removed the part signal peptide sequence when expressing above-mentioned CPTI gene, optimized simultaneously sequence according to removing as much as possible signal peptide sequence, guaranteeing not destroy the principle of the secondary structure of albumen again, front 30 deoxyribonucleotides of gene are removed, be beneficial to the recombinant protein that goes out this gene at expression in escherichia coli.Then the encoding sequence with the CPTI gene of transformation and optimization is building up among the prokaryotic expression carrier PGEX-6p-1; Subsequently the CPTI expression vector that builds is transformed among the expression strain BL21 (DE3), induces the expression of fusion rotein with inductor IPTG (isopropyl-β-d-1-thiogalactopyranoside, sec.-propyl-β-d-thiogalactoside); Utilize fusion rotein the GST label can with the affine characteristic of Glutathione Sephrose 4B, can purifying obtain purity and reach fusion rotein GST-CPTI more than 90%.
The sequence of CPTI gene is shown in SEQ ID No.1 after optimizing:
cttgtagggg ttactactgc agccatggat ctgaaccacc tcggaagtaa tcatcatgat 60
gactcaagcg atgaaccttc tgagtcttca gaaccatgct gcgattcatg catctgcact 120
aaatcaatac ctcctcaatg ccattgtaca gatatcaggt tgaattcgtg tcactcggct 180
tgcaaatcct gcatgtgtac acgatcaatg ccaggcaagt gtcgttgcct tgacattgct 240
gatttctgtt acaaaccttg caagtccagg gatgaagatg atgag 285
According to Cowpea Trypsin Inhibitor of the present invention, it is by the genes encoding of nucleotide sequence shown in SEQ ID No.1, and particularly, it has the aminoacid sequence shown in SEQ ID No.2.
LVGVTTAAMD LNHLGSNHHD DSSDEPSESS EPCCDSCICT KSIPPQCHCTDIRLNSCHSA 60
CKSCMCTRSM PGKCRCLDIA DFCYKPCKSR DEDDE 95
Therefore, according to Cowpea Trypsin Inhibitor prokaryotic expression carrier of the present invention, it comprises above-mentioned Cowpea Trypsin Inhibitor CPTI gene.
Method efficient, the great expression Cowpea Trypsin Inhibitor according to the present invention is included in expresses the above-mentioned step of stating cowpea trypsin inhibitor gene in the prokaryotic organism, preferably at expression in escherichia coli.
According to the engineering strain of efficient, great expression Cowpea Trypsin Inhibitor of the present invention, it has above-mentioned cowpea trypsin inhibitor gene, is preferably the intestinal bacteria recombinant bacterial strain.
Although Cowpea Trypsin Inhibitor content in some beans is abundanter, but the technique of natural extract and purifying CPTI is loaded down with trivial details, yield poorly and cost high, be not suitable for large-scale promotion and application, restricted the application at agricultural field control lepidoptera pest of CPTI albumen.
Optimization CPTI gene of the present invention can be at E. coli, and expression amount significantly improves, and the specific activity of expressing protein also improves, when inhibitor concentration is 0.05mg/mL, suppress activity and reach 0.204, when inhibitor concentration is 0.2mg/mL, suppresses activity and reach 0.807.
Intestinal bacteria (E.coli) expression system is a present most widely used exogenous gene expression system.The advantage of coli expression system is that genetics and physiology background are clear; Cultivate easily, particularly high density fermentation; Foreign gene often can reach and efficiently express.Used coli strain BL21 (DE3).BL21 (DE3) is the lysogenic bacteria of a λ DE3 (λ DE3 is the mutant strain of lambda particles phage), namely is integrated with one section karyomit(e) of λ DE3 at the karyomit(e) of BL21.Expression vector used in the present invention be PGEX-6p-1 (GE, Healthcare) with the derivable strong promoter of Ptac, give expression to N end with the fusion rotein of the GST label of a 26kDa.GST is as a kind of label of purifying, the very high target protein of purifying purity easily, and GST also has the effect of the less CPTI albumen of stable molecule amount simultaneously.
Description of drawings
Fig. 1 is the pcr amplification product of cowpea CPTI gene, M:DNA molecular weight standard, 1: negative control, 2:CPTI gene.
Fig. 2 is the double digestion checking of PGEX-6p-1-CPTI expression vector, 1: the product of recombinant plasmid double digestion, M:DNA molecular weight standard.
Fig. 3 is that fusion rotein GST-CPTI abduction delivering detects M: the bacterium liquid that albumen Marker, 1:IPTG induce, 2: do not add the bacterium liquid that IPTG induces.
Fig. 4 is fusion rotein GST-CPTI affinitive layer purification, M: albumen Marker, 1: the fusion rotein GST-CPTI behind the purifying.
Fig. 5 is that trypsin inhibitor is active to tryptic inhibition.
Embodiment
The extraction of embodiment 1 cowpea genomic dna and the amplified production of CPTI gene
Do not contain intron in Cowpea Trypsin Inhibitor (cowpea trypsin inhibitor, the CPTI) gene, by extracting the cowpea genomic dna, according to the primers of delivering out, amplify the CPTI gene.
Cowpea seed (available from the Chinese Academy of Agricultural Sciences) is planted in flowerpot, gets its blade after about 30 days and extracts genomic dna.Adopt The chargeswitch gDNA plant kit (available from invitrogen company) to extract the cowpea genomic dna.Take above-mentioned cowpea genomic dna as template, utilize the PCR reaction to amplify CPTI gene (Fig. 1).The PCR product of CPTI gene is sent to Shanghai and gives birth to the order-checking of worker's biotechnology company limited.Sequencing result shows: the encoding sequence of CPTI gene is comprised of the 321bp base, and 106 amino acid of encoding contain a terminator codon, the very high homology (more than 90%) with delivering sequence.
106 amino acid of the open reading frame of CPTI gene coding, this proteins encoded N end with 18 amino acid whose signal peptides (SignalP:
Http:// www.cbs.dtu.dk/services/SignalP/).The function that prokaryotic expression system does not usually have identification eukaryotic cell signal peptide and this albumen is successfully located, and ripe activated albumen always could form behind signal peptidase excision signal peptide, the synchronous signal peptide mainly contains hydrophobic amino acid, usually forms inclusion body in prokaryotic expression system during great expression.According to removing as much as possible signal peptide sequence, assurance does not destroy the principle of the secondary structure of albumen again, and front 30 deoxyribonucleotides of gene are removed.The expressed CPTI of the present invention is from 106 amino acid of the 11st amino acid to the.Design upstream and downstream primer is also introduced respectively restriction enzyme site BamH I and Xho I.
Upstream primer CPTI-BamH I-F:gaa att ggatcc ctt gta ggg gtt act act
Downstream primer CPTI-Xho I-R:gaa att ctc gag ctc atc atc ttc atc
The PCR response procedures is: 95 ℃ of thermally denatures 5 minutes; 95 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds; 30 circulations, 72 ℃ were extended 10 minutes.The PCR product is cut glue and is reclaimed the goal gene band through 1% agarose gel electrophoresis.
The structure of embodiment 3GST-CPTI fusion protein expression vector
BamH I and Xho I double digestion CPTI gene band and PGEX-6p-1 expression vector, 16 ℃ of connections of spending the night under T4 ligase effect.To connect product and transform DH5 α competent cell next day, and it is dull and stereotyped that coating contains ammonia benzyl microbiotic solid LB, 37 ℃ of incubated overnight.The clone that bacterium colony PCR is positive extracts plasmid and cuts checking (Fig. 2) as enzyme.Plasmid is sent to Shanghai gives birth to the order-checking of worker company, the result shows that the structure of expression vector is entirely true.
The expression of embodiment 4GST-CPTI fusion rotein
With the expression vector Plasmid Transformation expression strain BL21 (DE3) that builds.Picking list bacterium colony is to containing in the antibiotic liquid LB of the 100 μ g/ml ammonia benzyls substratum, 37 ℃ of cultivations when treating that OD600 reaches 0.5 left and right sides, are taken out 500 μ l bacterium liquid and are done contrast, residue bacterium liquid adds IPTG to final concentration 0.5mmol/L abduction delivering, and 37 ℃ are continued to cultivate 3 hours.Get 500 μ l bacterium liquid, centrifugal 1 minute of 12000rpm collects thalline, with the resuspended thalline of 80 μ lPBS, adds 20 μ l 5x SDS-PAGE loading buffer, and boiling water boiling 5 minutes is got 10 μ l and carried out SDS-PAGE gel electrophoresis (electrophoresis result is seen Fig. 3).
The purifying of embodiment 5GST-CPTI fusion rotein
The picking mono-clonal contains in the antibiotic liquid LB of the ammonia benzyl substratum in 20ml, 37 ℃ of incubated overnight.The bacterial classification of activation is inoculated in 1L by 1: 100 ratio contains in the antibiotic fresh LB substratum of ammonia benzyl, 37 ℃, 200rpm cultivates two hours to OD600=0.5, and adding IPTG is 0.2mmol/L to final concentration, and 16 ℃ are spent the night and induce.4 ℃ of inferior daily large capacity refrigerated centrifuges, 4000rpm, 30 minutes collection thalline.Then use 30ml PBS resuspended, ultrasonic degradation under the ice bath.4 ℃ of high speed freezing centrifuges of cell lysate, centrifugal 30 minutes of 15000rpm.The supernatant that obtains is splined on Glutathione Sephrose 4B chromatography column with the PBS balance with the flow velocity of 1ml/min, behind end of the sample, continues to wash with the PBS of 20 times of column volumes the albumen of non-specific binding.Then use elutriant (pH 8.0 for 10mmol/L reduced glutathion, 50mmol/L Tris-HCl) the wash-out target protein of 3 times of column volumes.The purity (Fig. 4) of polyacrylate hydrogel electrophoresis detection fusion rotein adds the target protein that elutes in the concentrated centrifuge tube of 10kDaMillipore, change Glutathione Sephrose 4B elutriant into 20mmol/L Tris pH 8.0,100mmol/LNaCl, 3mmol/L DTT also is concentrated into 1.5ml.-20 ℃, preserve.Utilize the 280nm absorbance of this albumen of spectrophotometer measurement, calculate in the 1L bacterium liquid and can obtain 25mg GST-CPTI albumen.
Embodiment 6 fusion rotein GST-CPTI are to the tryptic mensuration that suppresses activity
(1) enzyme is lived and is suppressed the determination of activity principle:
Trypsinase can act on synthetic substrate benzoyl-DL-arginine p-Nitroaniline (BAPNA), discharges yellow p-Nitroaniline, and this material has obtained the maximum absorption under 410nm.Trypsin inhibitor can suppress this reaction, and the 410nm absorbance is descended, and its decline degree is directly proportional with trypsin inhibitor activity.With the variation of spectrophotometer at 410nm place mensuration absorbance, can carry out quantitative analysis to trypsin inhibitor activity.
(2) preparation of solution
Take by weighing trypsinase 30.0mg (Trypsin 1:250, porcine pancrease) and be dissolved in 5mmol/L CaCl
2With 1mmol/LHCl solution (pH3.0), and be settled to 100ml.Be stored in 4 ℃ of refrigerators.
Take by weighing the BAPNA of 60mg with 1ml dimethyl sulfoxide (DMSO) (DMSO) dissolving, then use 50mmol/LTris-HCl (pH8.1) and 5mmol/L CaCl
2Solution is settled to 100ml.
(3) fusion rotein GST-CPTI suppresses the measuring method of tryptic activity:
Control group:
Get the Protein G ST-CPTI (different concns) of 200 μ l purifying in 750 μ l BAPNA solution (37 ℃ of preheatings), 37 ℃ were reacted 10 minutes, stopped this reaction with 100 μ l acetums immediately, then added 200 μ l trypsinase, mixed.Take water as blank, 410nm measures absorbance A1.
The inhibition group:
The Protein G ST-CPTI (different concns) that gets 200 μ l purifying joins in the 750 μ l BAPNA solution (37 ℃ of preheatings), then adds 200 μ l trypsin solutions, and 37 ℃ were reacted 10 minutes, and stopped this reaction with 100 μ l acetums immediately.Take water as blank, 410nm measures absorbance A2.
The unrestraint group:
Getting 200 μ l trypsin solutions joins in the 750 μ l BAPNA solution (37 ℃ of preheatings), 37 ℃ were reacted 10 minutes, add 100 μ l acetums and stop this reaction, and then add the Protein G ST-CPTI (different concns) of 200 μ l purifying, mix.Take water as blank, 410nm measures absorbance A3.
Fusion rotein GST-CPTI is to the inhibition of tryptic activity
The results are shown in Figure five.
Above-mentioned experimental data shows along with the rising of inhibitor (GST-CPTI) concentration is just higher to tryptic inhibition degree.The Cowpea Trypsin Inhibitor (GST-CPTI) of the method preparation kept and tryptic higher bioconjugation active, this lays a good foundation for the development and use of trypsin inhibitor and popularization.
Claims (7)
1. a cowpea trypsin inhibitor gene is characterized in that, its nucleotide sequence is shown in SEQ ID No.1.
2. a Cowpea Trypsin Inhibitor is characterized in that, described Cowpea Trypsin Inhibitor is by the genes encoding of nucleotide sequence shown in SEQ ID No.1.
3. a Cowpea Trypsin Inhibitor is characterized in that, its aminoacid sequence is shown in SEQ ID No.2.
4. Cowpea Trypsin Inhibitor prokaryotic expression carrier PGEX-6p-1-CPTI, it is characterized in that described carrier links to each other the described cowpea trypsin inhibitor gene of claim 1 by restriction enzyme site BamH I with prokaryotic expression carrier PGEX-6p-1 with Xho I.
5. the method for efficient, a great expression Cowpea Trypsin Inhibitor, it is characterized in that, described method is included in the step that expression in escherichia coli comprises the described cowpea trypsin inhibitor gene of claim 1, wherein, use Cowpea Trypsin Inhibitor prokaryotic expression carrier PGEX-6p-1-CPTI to express, described Cowpea Trypsin Inhibitor prokaryotic expression carrier PGEX-6p-1-CPTI by with the described cowpea trypsin inhibitor gene of claim 1 and prokaryotic expression carrier PGEX-6p-1 by the restriction enzyme site BamH I structure that links to each other with Xho I.
6. the engineering strain of efficient, a great expression Cowpea Trypsin Inhibitor is characterized in that described bacterial strain is the intestinal bacteria that contain the described Cowpea Trypsin Inhibitor prokaryotic expression carrier of claim 4 PGEX-6p-1-CPTI.
7. the described cowpea trypsin inhibitor gene of claim 1 is used for suppressing the application of tryptic activity.
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| CN107449905A (en) * | 2017-08-09 | 2017-12-08 | 华中农业大学 | A kind of artificial microballoon for detecting rice Cowpea Trypsin Inhibitor |
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| US20070006351A1 (en) * | 2004-02-13 | 2007-01-04 | The Scotts Company | Method for regenerating and transforming St. Augustinegrass from embryogenic callus |
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| US20070006351A1 (en) * | 2004-02-13 | 2007-01-04 | The Scotts Company | Method for regenerating and transforming St. Augustinegrass from embryogenic callus |
Non-Patent Citations (2)
| Title |
|---|
| Wang X et al.EU088405 Vigna unguiculata trypsin inhibitor (CpTI) gene.《GenBank》.2007, * |
| 杨丽琛等.CpTI 蛋白在大肠杆菌中的高效融合表达及其纯化与活性测定.《生物工程学报》.2003,第19卷(第1期), * |
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