CN106047892B - The purification process of Rhizoma Chuanxiong alpha-amylase/subtilisin inhibitor gene and expression product - Google Patents
The purification process of Rhizoma Chuanxiong alpha-amylase/subtilisin inhibitor gene and expression product Download PDFInfo
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Abstract
The invention discloses the purification process of the Protein S EQ ID NO.2 and expression product of a kind of Rhizoma Chuanxiong alpha-amylase/subtilisin inhibitor genes of SEQ ID NO.1 and its coding.It is 31.60% with rice (Oryza sativa (L.) Genbank:P29421) alpha-amylase/subtilisin inhibitor similitude.LASI recombinant protein can not only effectively inhibit the activity of the alpha-amylase of diamondback moth, inhibiting rate 33%, additionally it is possible to inhibit the activity of subtilopeptidase A, inhibiting rate 56.28%.Present invention discloses one section in Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong new alpha-amylase/subtilisin inhibitor gene orders, this provides effective candidate gene for pest-resistant/antibacterial plant genetic engineering.
Description
Technical field
The invention belongs to molecular biology and field of biotechnology, are related to a new Rhizoma Chuanxiong alpha-amylase/hay bacillus
Protease inhibitor gene sequence.
Background technique
Alpha-amylase/subtilopeptidase A (α-amylase/subtilisin inhibitor, ASI) is a kind of source
In the natural bioactive peptide of plant, it is able to suppress the activity of alpha-amylase in lepidopterous insects enteron aisle of ingesting, hinders Lepidoptera
Digestion and absorption of the insect to starch in food and other saccharide compounds, influences the growth and development of lepidopterous insects, to play
Pest-resistant effect.Since the molecular weight of ASI is smaller, and activity is clear, therefore it is the candidate gene of engineering of insect-resistant plant.Separately
Outside, ASI can also inhibit the activity of microbe-derived subtilopeptidase A, it is considered to be plant against harmful microorganism
Critical function gene.Therefore, obtaining new ASI genetic resources seems significant.
Rhizoma Chuanxiong is the rhizome of Umbelliferae rhizoma ligustici platymiscium Rhizoma Chuanxiong (Ligusticum chuanxiong Hort.), is Sichuan
Save famous genunie medicinal materials, record be in Shennong's Herbal hypoglycemic, antilipemic Chinese herbal medicine compound and Chinese patent drug important component.
Clone obtains Rhizoma Chuanxiong alpha-amylase/subtilopeptidase A (Ligusticum to this project group from Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong for the first time
Chuanxiong α-amylase/subtilisin inhibitor, LASI) gene cDNA sequence, building contain the gene
Recombinant organism has successfully obtained purity by IPTG inducing expression, Ni-NTA His Bind column chromatography
For 95% LASI recombinant protein.The LASI recombinant protein can effectively inhibit the alpha-amylase activity of diamondback moth, and inhibiting rate can
Up to 33%.And LASI recombinant protein can also inhibit the activity of subtilopeptidase A, inhibiting rate 56.28%.This project
Innovative research achievement formed LASI gene with independent intellectual property rights, this be utilize pest-resistant/antibacterial plant gene work
Journey provides effective candidate gene.
Summary of the invention
In view of the deficiency of existing investigative technique, the present invention, which provides one, new has anti-insect activity from Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong
Alpha-amylase/subtilisin inhibitor (LASI) gene order, specific achievement embodies are as follows:
One section of Rhizoma Chuanxiong alpha-amylase/subtilisin inhibitor LASI gene order, feature such as sequence table SEQ
Shown in ID NO.1.
The LASI recombinant protein of the gene order, amino acid is as shown in sequence table SEQ ID NO.2.
It is a further object to provide the expression of the coli expression carrier containing LASI gene, LASI gene
And the purification process of expression product.Using means below: obtaining LASI weight using SEQ ID NO.1 building pronuclear recombination carrier
The method of histone, conversion Escherichia coli form genetic engineering bacterium, are chromatographed by IPTG inducing expression, Ni-NTA His Bind
Column purification, obtains the LASI recombinant protein that purity is 95%, and the LASI recombinant protein can effectively inhibit α-shallow lake of diamondback moth
The activity of powder enzyme and subtilopeptidase A, comprising the following steps:
A) umbelliferae Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong alpha-amylase/subtilisin inhibitor LASI gene acquisition；
B) LASI Prokaryotic expression vector construction and expression and purification.
A) the step includes following concrete technology:
1) Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong total serum IgE is extracted, Rhizoma Chuanxiong total serum IgE quality is detected；
2) synthesis of cDNA；
3) Rhizoma Chuanxiong is gone out by PCR amplification from Rhizoma Chuanxiong cDNA with the primer for being respectively provided with BamHI and EcoRI double enzyme site
LASI gene order.
B) the step includes following concrete technology:
1) the LASI gene order come will be amplified and pET28-a carrier uses BamHI and EcoRI double digested respectively,
The segment that both ends have cohesive end is obtained, two segments is connected with T4DNA ligase, obtains the recombinant plasmid connected；
2) 10 μ l recombinant plasmid transformed E.coli Rosetta competent cells are taken；It is converted into the recombination E.coli of function
Rosetta genetic engineering bacterium, which is coated in the plate containing (10mg/L) kanamycins and chloramphenicol (25mg/L), screens positive gram
It is grand；
3) recombinant bacterial strain for coming out preliminary screening is sequenced and is read with determination with after bacterium colony PCR and the identification of plasmid double digestion
Frame it is correct, obtain expected recombinant bacterial strain；
4) fermentation expands recombinant bacterial strain
(1) .LB fermentation medium: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L；After dissolution, high temperature and pressure
Sterilizing；
(2) fermentation condition: 37 DEG C, 200rpm is by recombinant bacterial strain culture to OD600 ≈ 0.6；IPTG is added, makes its final concentration
For 1mM, 37 DEG C of culture 8-10h.
Compared with current technology, the present invention mainly has the advantages that LASI gene provided by the invention is from umbrella
The new sequence extracted in shape section plant Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong.By genetic engineering LASI recombinant protein obtained not only to pickles
The alpha-amylase activity of moth has inhibiting effect, and also has inhibitory activity to subtilopeptidase A, is pest-resistant/antibacterial plant
Genetic engineering provides candidate gene.
Detailed description of the invention
Fig. 1 is experimental technique flow chart.
Fig. 2 is the plasmid map of recombinant plasmid pET28a-LASI.
Specific embodiment
Embodiment 1: umbelliferae Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong alpha-amylase/subtilisin inhibitor (LASI) gene obtains
?
Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong total serum IgE is extracted using the plant RNA extraction kit that OMEGA company provides, it is total to the Rhizoma Chuanxiong of extraction
RNA carries out 1.2% agarose gel electrophoresis and detects its extracted amount and concentration.
The synthesis of cDNA: using obtained Rhizoma Chuanxiong RNA as template, OligodT (18) is primer, according to M-MLV reverse transcriptase
Specification reverse transcription obtains cDNA.
Two primers are designed, are separately added into protection base and corresponding restriction site, primer sequence B1:
CGCGGATCCGATGCATCGCCTGATGCT(BamHI)；B2:CCGGAATTCTCAAACCTTCAAGAACATAACCA
(EcoRI)。
For the cDNA obtained using reverse transcription as template, B1 and B2 are that two primers carry out PCR reaction.Reaction system is as follows:
25 211 21 μ l of μ l, ddH2O of μ l, B2 of μ l, B1 of μ l, cDNA of Prime Star Mix.PCR response parameter are as follows: 94 DEG C of reactions
5min, initial denaturation；94 DEG C of 1min, 66 DEG C of 1min, 72 DEG C of 2min are expanded, and are recycled 35 times；72 DEG C of 10min renaturation；4 DEG C of reactions
Stop.PCR product carries out 1.2% agarose gel electrophoresis detection.Glue recycles the amplified band of 600bp or so and adds at its end
The segment for adding PolyA to complete is connected on pMD19-T carrier by PolyA tail, and Transformed E .coli DH5 α competent cell will be positive
Property clone bacterium send company to be sequenced to determine the correctness of extension increasing sequence.LASI- is extracted with Omega Plasmid Mini Kit I
PMD19-T recombinant plasmid.
Embodiment 2:LASI Prokaryotic expression vector construction and expression and purification
Obtained LASI-pMD19-T recombinant plasmid will be extracted and carry out double enzyme digestion reaction with restriction enzyme BamH1 and EcoRI, instead
4 hours between seasonable, 37 DEG C of reaction temperature, reaction system is 0.5 0.5 μ l, 10*K Buffer of μ l, EcoRI of BamHI, 2 μ l,
LASI-pMD19-T carrier 9 μ l, ddH2O 8μl.1.2% agarose gel electrophoresis detection separation digestion piece is carried out after reaction
Section, with the clear DNA band of Gel Extraction Kit recycling 600bp or so, obtaining both ends has digestion cohesive end
LASI genetic fragment.
The pET28a plasmid being stored in E.coli DH5 α bacterial strain is extracted with Omega Plasmid Mini Kit I, is led to
It crosses restriction enzyme BamHI and EcoRI and carries out the linear plasmid that double enzyme digestion reaction acquisition both ends are cohesive end, double digestion system is with before
It is consistent in section.Endonuclease bamhi is separated through agarose gel electrophoresis after double enzyme digestion reaction, with Gel Extraction Kit
Recycle the correct linear carrier segment of length.
With T4DNA ligase by the above-mentioned LASI genetic fragment with BamHI and EcoRI restriction enzyme site and linear pET28a
Plasmid completes splicing, re-forms cyclic plasmid, as shown in Figure 2.It is converted and enters E.coli Rosetta competence bacterial strain.
In LB solid medium (peptone 10g/L, the yeast extract of (10mg/L) containing kanamycin and chloramphenicol (25mg/L)
5g/L, NaCl 10g/L) on screening obtain positive restructuring Escherichia coli, picking single colonie send to Qing Ke Bioisystech Co., Ltd
The correctness for determining recombinant plasmid splicing is sequenced.
The LB solid in (10mg/L) containing kanamycin and chloramphenicol (25mg/L) is being added in positive colony recombinant bacterium
It crosses in culture medium, 37 DEG C of cultures are to there is single colonie appearance.Picking single colonie is inoculated in 50ml and is added containing kanamycin
In the LB liquid medium of (10mg/L) and chloramphenicol (25mg/L), 200rmp shaking table culture 8-10h.By above-mentioned bacterium solution according to 1:
20 ratio expands culture to OD600When ≈ 0.6, add IPTG (1mM), 200rpm, 37 DEG C are incubated overnight (8-10h), induce large intestine
Bacillus high efficient expression LASI recombinant protein.
The bacterium solution for taking 200ml to express is centrifuged 20 minutes collection thallus in 5000 × g, and 10ml PBS buffer solution is added
(pH7.5) piping and druming precipitating is allowed to be resuspended repeatedly.Ultrasonic disruption thalline discharges LASI albumen (ice-bath ultrasonic 30min smudge cells
Albumen is discharged, ultrasound procedures are 45% power, ultrasonic 6s, interval 3s.Lysate frozen-thawed is primary after the completion of ultrasound).It splits
It solves liquid to remove the impurity such as cell fragment in 13000 × g centrifugation 10min, retains supernatant, 4 DEG C spare.Take 2ml Ni-NTA
His Bind affine resin fills column, washes column with 10ml deionization, rear in conjunction with liquid (20mM imidazoles, 10mM Tris-HCl
PH7.9,500mM NaCl) wash column 3-5 times, each 1ml.4ml supernatant is taken to be added to Ni- after 0.45 μm of aperture membrane filtration
On NTA His Bind chromatographic column, reserve it naturally by gravity.With combining liquid to wash by the foreign protein in chromatographic column, every time
2ml is washed 4 times.The elution LASI recombinant protein for being later 200mM with 2ml imidazole concentration.To contain LASI recombinant protein
Eluent dialyse, dialyzate group becomes 0.05mM Tris-HCl (pH7.4), 10% glycerol, and bag filter molecular weight is
8000-14000.Every 12h changes a not good liquor and collects the protein liquid in bag filter, -20 DEG C save backup after dialysis 2 days.
Embodiment 3:LASI recombinant protein inhibits the alpha-amylase of diamondback moth and the activity of subtilopeptidase A.
1) inhibitory activity of the LASI to diamondback moth alpha-amylase
(1) the preparation of diamondback moth alpha-amylase crude extract
Diamondback moth is purchased from Henan white clouds biological pesticide company.Liquid nitrogen grinding is added in diamondback moth larvae (4-5 age), is added
0.15M NaCl is homogenized, afterwards use 10000rpm, be centrifuged within 10 minutes supernatant is diamondback moth alpha-amylase crude extract.
(2) inhibiting effect of the LASI to diamondback moth alpha-amylase activity
1. 25 μ l diamondback moth alpha-amylase crude extracts (protein concentration is calculated with 10mg/ml) and 50 μ l LASI recombinant proteins
(protein concentration is calculated with 0.783mg/ml) after mixing, 37 DEG C of processing 10min；
2. mixed liquor 1., which is added to 0.15% soluble starch solution of 10ml, (includes the soluble shallow lake 0.15% (v/w)
Powder, the sodium-acetate buffer (pH5.0) of 50mM, 1mM CaCl2) in, 37 DEG C of processing 10min；
3. taking 200 μ l reaction solutions, 750 μ l reaction terminating liquids are added and (include 0.05M HCl, 180mg KI/ml, 18mg
I2/ ml), terminate reaction；The absorbance under 640nm wavelength is measured after mixing；
4. to be added without the control of the reaction tube of LASI.
2) inhibitory activity of the LASI to subtilopeptidase A
(1) 10 μ l subtilopeptidase As (1mg/ml, CAS number 9014-01-1) and 10 μ l LASI recombinant protein (albumen
Concentration is 0.783mg/ml calculating) in 37 DEG C of mixing, heat preservation 10min.Mixed liquor is added to 1ml bovine serum albumin(BSA) (1mg/
Ml) in solution, 37 DEG C of reaction 10min use BCA kit measurement residual protein content.
(2) compareed with the reaction tube for being added without LASI.
<110>Southwest Jiaotong University
<120>purification process of Rhizoma Chuanxiong alpha-amylase/subtilisin inhibitor gene and expression product
<160> 2
<170> Patent In Version 2.1
<210> 1
<211> 543
<212> DNA
<213>Rhizoma Chuanxiong (Ligusticum chuanxiongHort)
<400> 1
GATGCATCGCCTGATGCTGTACGCGACATGGACGGAGACATACTCCGAGCAGGTGTTCATTACTATATC
TTGCCTGGTTTACGAGGTATGGGAGGTGGTGTAACACTAGGTAGCACCAGAAATGAATCTTGCCCCTTAGACGTTGT
CCAAGAAACATTTGAAACAGACAACGGTAACCTTCCCTTAACATTCACAATGGTTGATCCGAAAAAAGGTGTTATCC
GCGAATCAACTGATTTGAACATCGAGTTCAATGGTGTAACGATTTGCATTCAATCATTGGTTTGGAAACTTGATAAC
TATGATGGAGAGTACGTTGTTAGTACTCGGGGAGTTAAAGGAAACCCGGGAATTGAAACTCTAGACAGTTGGTTTAA
GATTGAAAAATATTCGAATAACTACAAGTTTGTTTTTGTTTTCTGCCCAACCGTATGCGATTTCTGCAAACCAATAT
GCGGAGATATCGGCATCTCGATCAAGAATGGTGTTCGAAGATTGGTTCTTAGCGATGAACCCTTCATGGTTATGTTC
TTGAAGGTTTGA
<210> 2
<211> 180
<212>amino acid sequence
<213>Rhizoma Chuanxiong (Ligusticum chuanxiong)
<400> 2
DASPDAVRDMDGDILRAGVHYYILPGLRGMGGGVTLGSTRNESCPLDVVQETFETDNGNLPLTFTMVDP
KKGVIRESTDLNIEFNGVTICIQSLVWKLDNYDGEYVVSTRGVKGNPGIETLDSWFKIEKYSNNYKFVFVFCPTVCD
FCKPICGDIGISIKNGVRRLVLSDEPFMVMFLKV
Claims (5)
1. a kind of coding Rhizoma Chuanxiong alpha-amylase/subtilisin inhibitor LASI gene order, it is characterised in that: the gene
Sequence is as shown in sequence table SEQ ID NO.1.
2. the LASI recombinant protein of the coding of gene order described in claim 1, amino acid sequence such as sequence table SEQ ID NO.2
It is shown.
3. the method for obtaining LASI recombinant protein using SEQ ID NO.1 building pronuclear recombination carrier, including conversion Escherichia coli
Genetic engineering bacterium is formed, by IPTG inducing expression, Ni-NTA His Bind column chromatography, obtaining purity is 95%
LASI recombinant protein；The LASI recombinant protein can effectively inhibit the alpha-amylase of diamondback moth and the work of subtilopeptidase A
Property, steps are as follows for specific method:
A) umbelliferae Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong alpha-amylase/subtilisin inhibitor LASI gene acquisition；
B) LASI Prokaryotic expression vector construction and expression and purification.
4. according to the method described in claim 3, it is characterized in that, a) step includes following concrete technology:
1) Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong total serum IgE is extracted, Rhizoma Chuanxiong total serum IgE quality is detected；
2) synthesis of cDNA；
3) Rhizoma Chuanxiong LASI is gone out by PCR amplification from Rhizoma Chuanxiong cDNA with the primer for being respectively provided with BamHI and EcoRI double enzyme site
Gene order.
5. according to the method described in claim 3, it is characterized in that, b) step includes following concrete technology:
1) the LASI gene order come will be amplified and pET28-a carrier uses BamHI and EcoRI double digested respectively, obtained
Both ends have the segment of cohesive end, connect two segments with T4 DNA ligase, obtain the recombinant plasmid connected；
2) 10 μ l recombinant plasmid transformed E.coli Rosetta competent cells are taken；It is converted into the recombination E.coli of function
Rosetta genetic engineering bacterium, which is coated in the plate containing 10mg/L kanamycins and containing 25mg/L chloramphenicol, screens positive gram
It is grand；
3) recombinant bacterial strain for coming out preliminary screening determines reading frame through being sequenced with after bacterium colony PCR and the identification of plasmid double digestion
It is correct, obtain expected recombinant bacterial strain；
4) fermentation expands recombinant bacterial strain:
(1) .LB fermentation medium: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L；
After dissolution, autoclave sterilization；
(2) fermentation condition: 37 DEG C, 200rpm is by recombinant bacterial strain culture to OD600 ≈ 0.6；IPTG is added, keeps its final concentration of
1mM, 37 DEG C of culture 8-10h.
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| CN1317052A (en) * | 1998-07-07 | 2001-10-10 | 宝洁公司 | Proteases fused with variants of streptomyces subtilisin proteases inhibitor |
| CN102370681A (en) * | 2007-08-07 | 2012-03-14 | 北京北大维信生物科技有限公司 | Application of extracts of Chinese herbal medicine rhizoma ligustici wallichii in preparation of weight-losing and lipid-lowering medicines or medicines with function of inhibiting lipase activity |
| CN102532305A (en) * | 2012-03-12 | 2012-07-04 | 西南大学 | Protease inhibitor BmSPI38 and preparation method and application thereof |
| CN102787130A (en) * | 2012-07-17 | 2012-11-21 | 天津科技大学 | Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method |
| CN103740673A (en) * | 2014-01-17 | 2014-04-23 | 北京科为博生物科技有限公司 | Low-temperature acidic alpha-amylase AMY-L27, and gene and application thereof |
| CN104480087A (en) * | 2015-01-06 | 2015-04-01 | 天津科技大学 | Novel high-temperature resistant alpha-amylase, preparing method of novel high-temperature resistant alpha-amylase and application of novel high-temperature resistant alpha-amylase |
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2016
- 2016-07-14 CN CN201610552488.8A patent/CN106047892B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1317052A (en) * | 1998-07-07 | 2001-10-10 | 宝洁公司 | Proteases fused with variants of streptomyces subtilisin proteases inhibitor |
| CN102370681A (en) * | 2007-08-07 | 2012-03-14 | 北京北大维信生物科技有限公司 | Application of extracts of Chinese herbal medicine rhizoma ligustici wallichii in preparation of weight-losing and lipid-lowering medicines or medicines with function of inhibiting lipase activity |
| CN102532305A (en) * | 2012-03-12 | 2012-07-04 | 西南大学 | Protease inhibitor BmSPI38 and preparation method and application thereof |
| CN102787130A (en) * | 2012-07-17 | 2012-11-21 | 天津科技大学 | Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method |
| CN103740673A (en) * | 2014-01-17 | 2014-04-23 | 北京科为博生物科技有限公司 | Low-temperature acidic alpha-amylase AMY-L27, and gene and application thereof |
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