CN103319607B - CBM fragment and application thereof in specific binding of plant-source crystallized cellulose - Google Patents
CBM fragment and application thereof in specific binding of plant-source crystallized cellulose Download PDFInfo
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- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a CBM fragment and an application thereof in specific binding of plant-source crystallized cellulose. The invention protects the CBM fragment or fusion protein therewith; the CBM fragment is as follows: (a) or (b) or (c), wherein (a) is protein consisting of 36th-223rd amino acid residue at the tail end of N of a sequence 3 in a sequence table; (b) is protein consisting of 36th-223rd amino acid residue at the tail end of N of a sequence 5 in the sequence table; (c) is protein consisting of 36th-223rd amino acid residue at the tail end of N of a sequence 6 in the sequence table. The invention also protects an application of the CBM fragment or the fusion protein therewith in preparing a product for binding the crystallized cellulose and an application in preparing a product for recognizing the crystallized cellulose. The invention can be used for separating, purifying or identifying the crystallized cellulose.
Description
Technical field
The present invention relates to CBM fragment and the application in specific combination plant-sourced crystallization Mierocrystalline cellulose thereof.
Background technology
Plant cell wall is a kind of fibrillar meshwork structure of complexity, is made up of in an orderly manner different structural polysaccharides, aromatic essence and Protein high, and its structure is to maintenance cellular form, and the mechanical support power maintaining erect plants growth has vital role.Plant cell wall constitutes natural resource the abundantest on the earth.
Mierocrystalline cellulose is the main composition of plant cell wall, is about 33% of plant gross weight, and its economic worth can be summarized with food, fiber, paper and bioenergy etc., all closely bound up with the life of the mankind.Therefore, Mierocrystalline cellulose is the cell wall constituent having economic worth most.Its structure is very simple, but synthesis mechanism is but extremely complicated.Current research is thought, Mierocrystalline cellulose is made up of unbranched β-Isosorbide-5-Nitrae-dextran glycosides chain, and every bar chain includes 15 at most, 000 glucosyl residue.Dextran glycosides interchain relies on hydrogen bond and Van der Waals force to combine and arranged in parallel, and every 36 dextran glycosides chains form a micro-fibril, and micro-fibril arranges the cellulosic higher structure of formation of deposits further.
The controlling element of resolving cellulosic synthesis mechanism and structure is basis content of cellulose and crystalline structure being realized to genetic improvement.Plant is while synthetic cellulose chain, and Mierocrystalline cellulose can be carried out outside direct secretion to born of the same parents assembling, depositing.The Mierocrystalline cellulose of crystallization, closely, acid, alkali or enzyme etc. are not easy to infiltrate and wherein destroy its structure in arrangement.And the Mierocrystalline cellulose of amorphization, arrange loose, be easier to be degraded by chemistry or biological enzyme reagent.Therefore, cellulosic crystal degree and cellulosic quality have close relationship.
Summary of the invention
The object of this invention is to provide CBM fragment and the application in specific combination plant-sourced crystallization Mierocrystalline cellulose thereof.
The present invention protects CBM fragment or has the fusion rotein of described CBM fragment; Described CBM fragment is following (a) or (b) or (c) or (d) or (e): the protein that (a) is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 3; B protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 5; C protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 6; (d) by (a) or (b) or (c) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have with plant crystallization Mierocrystalline cellulose specific binding activity by its derivative protein; (e) by (a) or (b) or (c) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have with plant crystallization Mierocrystalline cellulose specific binding activity by its derivative protein.
The described fusion rotein with described CBM fragment specifically can be following (f) or (g) or (h) or (i) or (j): the protein that (f) is made up of the aminoacid sequence shown in sequence in sequence table 3; G protein that () is made up of the aminoacid sequence shown in sequence in sequence table 5; H protein that () is made up of the aminoacid sequence shown in sequence in sequence table 6; By (f) or (g) or (h) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have with plant crystallization Mierocrystalline cellulose specific binding activity by its derivative protein; (j) by (f) or (g) or (h) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have with plant crystallization Mierocrystalline cellulose specific binding activity by its derivative protein.
The gene (abbreviation antigen-4 fusion protein gene) described in the gene of described CBM fragment of encoding (being called for short CBM gene) or coding with the fusion rotein of described CBM fragment also belongs to protection scope of the present invention.
Described CBM gene can be (1) or (2) or (3) or (4) or the DNA molecular described in (5) as follows: the sequence 7 of (1) sequence table is from the DNA molecular shown in 5 ' end 109-669 position Nucleotide; (2) sequence 9 of sequence table is from the DNA molecular shown in 5 ' end 109-669 position Nucleotide; (3) sequence 10 of sequence table is from the DNA molecular shown in 5 ' end 109-669 position Nucleotide; (4) DNA sequence dna limited with (1) or (2) or (3) is under strict conditions hybridized and is encoded and has the DNA molecular with the albumen of plant crystallization Mierocrystalline cellulose specific binding activity; (5) DNA sequence dna limited with (1) or (2) or (3) at least has more than 90% homology and encodes and has the DNA molecular with the albumen of plant crystallization Mierocrystalline cellulose specific binding activity.Above-mentioned stringent condition can be in the solution of 6 × SSC, 0.5%SDS, hybridizes under 65 ° of C, then uses 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively to wash film once.
Described antigen-4 fusion protein gene can be (6) or (7) or (8) or (9) or the DNA molecular described in (10) as follows: the DNA molecular shown in sequence 7 of (6) sequence table; (7) DNA molecular shown in sequence 9 of sequence table; (8) DNA molecular shown in sequence 10 of sequence table; (9) DNA sequence dna limited with (1) or (2) or (3) is under strict conditions hybridized and is encoded and has the DNA molecular with the albumen of plant crystallization Mierocrystalline cellulose specific binding activity; (10) DNA sequence dna limited with (1) or (2) or (3) at least has more than 90% homology and encodes and has the DNA molecular with the albumen of plant crystallization Mierocrystalline cellulose specific binding activity.Above-mentioned stringent condition can be in the solution of 6 × SSC, 0.5%SDS, hybridizes under 65 ° of C, then uses 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively to wash film once.
Expression cassette containing described CBM gene or described antigen-4 fusion protein gene, recombinant vectors, transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.
The present invention also protect described CBM fragment or described in there is the application of fusion rotein in the product for the preparation of binding crystal cellulose of described CBM fragment.
The present invention also protects a kind of product for binding crystal cellulose, its activeconstituents for described CBM fragment or described in there is the fusion rotein of described CBM fragment.
The present invention also protect described CBM fragment or described in there is described CBM fragment fusion rotein for the preparation of the application identified in the cellulosic product of crystallization.
The present invention also protects a kind of for identifying the cellulosic product of crystallization, its activeconstituents for described CBM fragment or described in there is the fusion rotein of described CBM fragment.
Arbitrary described Mierocrystalline cellulose specifically can be plant-sourced crystallization Mierocrystalline cellulose above.Cellulosic concrete preparation method is as follows for described plant-sourced crystallization: plant is dried the stem stalk after not changing to weight and be polished into powder, 70% ethanol aqueous wash once, wash once with the chloroform-methanol mixed solution of equal-volume mixing, gained precipitation washing with acetone post-drying also removes coarse grain with 200 mesh sieve sieves, obtains the alcohol insoluble matter component (Alcohol insolubleresidue/ is called for short AIR) based on cell wall constituent; The AIR of 20mg is cleared up through the alpha amylose enzyme of 1500Units and 10Units β Starch debranching enzyme in the sodium acetate soln of pH5.0,0.1M, the centrifugal 10min of 10000g collecting precipitation, cross 100 mesh sieves by washing with acetone post-drying, obtain powder; Take 2mg powder, add TFA containing 2M trifluoroacetic acid in 121 DEG C of process 90 minutes, centrifugally abandon supernatant, add 1mL Updegraff reagent (HNO
3: HOAC:H
2o=1:8:2; Volume ratio) rear 100 DEG C process 30 minutes, centrifugal removing supernatant, with water and acetone cleaning post-drying, obtain plant-sourced crystallization Mierocrystalline cellulose.Described plant can be paddy rice (as japonica rice variety " gold is fine "), Arabidopis thaliana (as Columbia ecotype Arabidopis thaliana), corn (as corn variety " neat 319 ") or willow (as Poplar Varieties " Populus hopeiensis ").
The present invention can be used for separation and purification or plant identification source crystal cellulose.
Accompanying drawing explanation
Fig. 1 is the structural representation of BC1 albumen.
Fig. 2 is fusion rotein His-CBM solution, fusion rotein His-CBM
y46Asolution, fusion rotein His-CBM
w66Asolution and fusion rotein His-CBM
w72Athe electrophorogram of solution.
Fig. 3 is the In vitro Binding Characteristics analysis of fusion rotein His-CBM.
Fig. 4 is the result that ELISA tests the Sugar-binding activitv of quantitative analysis CBM fragment.
Fig. 5 is the binding kinetics curve of the external binding crystal cellulose of fusion rotein His-CBM.
Fig. 6 is fusion rotein His-CBM
y46A, fusion rotein His-CBM
w66Aor fusion rotein His-CBM
w72Awith the Relative binding capacity compared with fusion rotein His-CBM.
Fig. 7 is the Immunochemical staining assay result in embodiment 4.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
Japonica rice variety " gold is fine " (WT, also known as WT lines): buy in rice in China institute.PET28a (+) carrier: buy in Merck company, this carrier has the encoding sequence of His label, expresses the foreign protein with His label.Intestinal bacteria Rosetta (DE3): buy in Merck company.Carrier pCAMBIA1300: purchased from CAMIA company, Australia.Agrobacterium EHA105: purchased from CAMIA company, Australia.Carrier pDONR207:LifeTechnologies company, article No. 12213-013.Carrier pDEST17:Life Technologies company, article No. 11803-012.
PBS damping fluid used in embodiment, is following PBS damping fluid: pH7.4, if no special instructions containing NaCl137mmol, KCl2.7mmol, Na
2hPO
410mmol, KH
2pO
42mmol.
The sequential analysis of embodiment 1, BC1 albumen
Paddy rice fragile straw mutant brittle culm1 is the spontaneous mutation material of japonica rice variety " gold is fine ".Mutant bc1 is compared with japonica rice variety " gold is fine ", and its main manifestations is: (1) cauline leaf becomes fragile (physical strength of stem stalk and blade significantly declines, and stem stalk sclerenchymatous cell secondary thickening is thinning, the remarkable decline of content of cellulose in secondary wall); (2) plant becomes short.
By the protein called after BC1 albumen shown in the sequence 1 of sequence table.Be BC1 gene by the unnamed gene of coding BC1 albumen, as shown in the sequence 2 of sequence table.The structural representation of BC1 albumen is shown in Fig. 1.SP representation signal peptide, ω site represents phosphatidylinositols anchor link position, and (after protein maturation, ω site protein sequence is below by specific enzyme identification and excision, and connect upper GPI), CBM represents carbohydrate land (Carbohydrate bindingmodule), and AG represents the sero-fast antigen fragment of BC1.BC1 gene generation base deletion in mutant bc1 causes BC1 protein translation frameshit and premature termination.
The preparation of embodiment 2, CBM fragment and each mutant fragments
One, the structure of recombinant plasmid
1, extract japonica rice variety " gold is fine " total serum IgE and reverse transcription is cDNA.
2, with step 1 extract cDNA for template, with F2 and R2 composition primer pair carry out pcr amplification, reclaim pcr amplification product.
F2：5'-C
GAATTCGTTGCAGTGGCGTATGATCCGCTGG-3'；
R2：5'-C
CTCGAGTCAGGTGACGGTCCACGTCATGAGC-3’。
3, use the pcr amplification product of restriction enzyme EcoRI and XhoI double digestion step 2, reclaim digestion products.
4, with restriction enzyme EcoRI and XhoI double digestion pET28a (+) carrier, the carrier framework of about 5335bp is reclaimed.
5, the digestion products of step 3 is connected with the carrier framework of step 4, obtains recombinant plasmid pET28a-CBM.According to sequencing result, structrual description carries out to recombinant plasmid pET28a-CBM as follows: between EcoRI and the XhoI restriction enzyme site of pET28a (+) carrier, insert the sequence 7 of sequence table from the double chain DNA molecule shown in 5 ' end 109-672 position Nucleotide.In recombinant plasmid pET28a-CBM, the fusion gene His-CBM shown in sequence 7 of the partial nucleotide formation sequence table on external source insertion sequence and carrier framework, the fusion rotein His-CBM shown in sequence 3 of expressed sequence table.
Two, the preparation of CBM fragment
1, recombinant plasmid pET28a-CBM is imported intestinal bacteria Rosetta (DE3), obtain recombinant bacterium.
2, the recombinant bacterium that step 1 obtains is seeded to the LB liquid nutrient medium containing 50 μ g/mL penbritins, 50 μ g/mL paraxin and 5g/100mL glucose, 37 DEG C, 200rpm shaking culture is to OD
600nmadd IPTG when=0.6 and make its concentration be 0.5mM, 16 DEG C, 200rpm shaking culture 12 hours, then 4 DEG C, the centrifugal 4min of 8000rpm, collecting bacterial sediment.
3, the precipitation that step 2 obtains is got, with binding buffer, (solvent is water, containing 20mM sodium phosphate, 0.5M sodium-chlor and 20mM imidazoles, pH7.4) suspend, put ultrasonication on ice (70w, ultrasonic 90 times, each 10s, 15s interval) centrifugal 30 minutes of 10000rpm afterwards, collects supernatant liquor.
4, the supernatant liquor that step 3 obtains is splined on affinity column (the Glutathione Ni Sepharose6Fast Flow4B of GE company) and carries out affinity chromatography.Wash-out is carried out successively with the binding buffer containing 200mM imidazoles of the bindingbuffer containing 75mM imidazoles of the binding buffer containing 50mM imidazoles of the binding buffer containing 35mM imidazoles of 5 column volumes, 5 column volumes, 5 column volumes, binding buffer and 5 column volume containing 100mM imidazoles of 5 column volumes, solution after the post excessively of the binding buffer of collection containing 100mM imidazoles and the binding buffer containing 200mM imidazoles, is the fusion rotein His-CBM solution that purifying obtains.
Three, CBM
y46Afragment, CBM
w66Afragment and CBM
w72Athe preparation of fragment
Recombinant plasmid pET28a-CBM
y46A: between EcoRI and the XhoI restriction enzyme site of pET28a (+) carrier, insert the sequence 8 of sequence table from the double chain DNA molecule shown in 5 ' end 109-672 position Nucleotide.By the double chain DNA molecule called after fusion gene His-CBM shown in the sequence 8 of sequence table
y46A, the fusion rotein His-CBM shown in sequence 4 of expressed sequence table
y46A.
Recombinant plasmid pET28a-CBM
w66A: between EcoRI and the XhoI restriction enzyme site of pET28a (+) carrier, insert the sequence 9 of sequence table from the double chain DNA molecule shown in 5 ' end 109-672 position Nucleotide.By the double chain DNA molecule called after fusion gene His-CBM shown in the sequence 9 of sequence table
w66A, the fusion rotein His-CBM shown in sequence 5 of expressed sequence table
w66A.
Recombinant plasmid pET28a-CBM
w72A: between EcoRI and the XhoI restriction enzyme site of pET28a (+) carrier, insert the sequence 10 of sequence table from the double chain DNA molecule shown in 5 ' end 109-672 position Nucleotide.By the double chain DNA molecule called after fusion gene His-CBM shown in the sequence 10 of sequence table
w72A, the fusion rotein His-CBM shown in sequence 6 of expressed sequence table
w72A.
Use recombinant plasmid pET28a-CBM respectively
y46A, recombinant plasmid pET28a-CBM
w66Awith recombinant plasmid pET28a-CBM
w72Areplace recombinant plasmid pET28a-CBM to carry out step 2, obtain fusion rotein His-CBM successively
y46Asolution, fusion rotein His-CBM
w66Asolution and fusion rotein His-CBM
w72Asolution.
Fusion rotein His-CBM solution, fusion rotein His-CBM
y46Asolution, fusion rotein His-CBM
w66Asolution and fusion rotein His-CBM
w72Athe electrophorogram of solution is shown in Fig. 2.
The In vitro Binding Characteristics analysis of embodiment 3, CBM fragment
One, the sugared bound substrates specificity of CBM fragment
1, polysaccharide sample is respectively:
EPG-Rice represent japonica rice variety " gold is fine " stalk powder end by pectinase solution, (Pectinase is purchased from Sigma company, article No. is P4716, be dissolved in pH7.0,50mM NaAc damping fluid, concentration 30ul/ml) component of dissolving that obtains after 12 hours of 37 DEG C of process, namely get after supernatant liquor rotary evaporation obtains dry-matter and be dissolved in PBS damping fluid, make its concentration be 10mg/mL;
The stalk powder end that 1N-KOH-soluble-Rice, 4N-KOH-soluble-Rice represent japonica rice variety " gold is fine " is successively processed by the 1M KOH aqueous solution or the/4M KOH aqueous solution 37 DEG C the dissolved constituent obtained for 12 hours respectively, namely get after supernatant liquor rotary evaporation obtains dry-matter and be dissolved in PBS damping fluid, make its concentration be 10mg/mL;
Pectin-Citrus represents the pectin (article No.: P9135 is purchased from Sigma company) in oranges and tangerines, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
PGA-Citrus represents the polygalacturonic acid (article No.: P0853 is purchased from Sigma company) in oranges and tangerines, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
MLG-Barley represents the dextran (article No.: G6513 is purchased from Sigma company) in barley, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
XyG-Tamarind represents the xyloglucan (article No.: P-XYGLN is purchased from Megazyme company) in tamarind, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
Galactomannan-Guar represents the polygalactomannan (article No.: E-AGLGU is purchased from Megazyme company) in guar-bean, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
AX-Wheat represents the araboxylan (article No.: 89887 is purchased from Sigma company) in wheat, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
RG-Soybean represents the poly-rhamno-galacturonic acid (article No.: P-RHAGN is purchased from Megazyme company) in soybean, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
Xylan-Wood represents the xylan (article No.: X0502 is purchased from Sigma company) in timber, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
Galactan-Lupin represents the Polygalactan (article No.: P-PGALU is purchased from Megazyme company) in lupine, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
RG I-Potato represents the poly-rhamno-galacturonic acid I(article No. in potato :-RHAM1, is purchased from Megazyme company), be dissolved in PBS damping fluid, make its concentration be 10mg/mL;
Arabinan-Sugar beet represents the arabinan (article No.: 07681 is purchased from Sigma company) in beet, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
The stalk powder end that 4N-insoluble-Rice represents japonica rice variety " gold is fine " is processed by the 4M KOH aqueous solution 37 DEG C the indissolvable component obtained for 12 hours, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL;
TFA insoluble-Rice represents the indissolvable component that Culm of Rice powder is obtained after 120 minutes by 2M trifluoroacetic acid aqueous solution 121 DEG C process, is dissolved in PBS damping fluid, makes its concentration be 10mg/mL.
2, by 5 microlitre polysaccharide soln point samples to nylon fiber element film on, room temperature leave standstill 1 hour; Then in the PBS damping fluid containing 5g/100mL skimmed milk, room temperature leaves standstill 1-2 hour (blockading), uses PBS buffer solution; Then add fusion rotein His-CBM solution (protein content is 1 microgram) prepared by 10 microliter Examples 2,4 DEG C of hold over night, use PBS buffer solution; Then add Anti-His primary antibodie (sigma, 1:1000 dilute), room temperature leaves standstill 2 hours, uses PBS buffer solution; Then add the goat anti-mouse antibody (ancient cooking vessel state, 1:1000 dilutes) of HRP mark, room temperature 50rpm vibrates 2 hours, uses PBS buffer solution; Take a picture after developing the color 5 minutes with horseradish peroxidase nitrite ion (Pierce).Fusion rotein His-CBM solution is replaced, as negative control with isopyknic PBS damping fluid.
Take negative control as background signal 1, by gel signal quantitative VisionWorks LS software operation, calculate the signal value of binding ability, it is EPG-Rice that all signal values obtain histogram 3(1 compared with background signal value; 2 is 1N-KOH-soluble-Rice; 3 is 4N-KOH-soluble-Rice; 4 is Pectin-Citrus; 5 is PGA-Citrus; 6 is MLG-Barley; 7 is XyG-Tamarind; 8 is Galactomannan-Guar; 9 is AX-Wheat; 10 is RG-Soybean; 11 is Xylan-Wood; 12 is Galactan-Lupin; 13 to represent the poly-rhamno-galacturonic acid I in potato for RGI-Potato; 14 is Arabinan-Sugar beet; 15 is 4N-insoluble-Rice; 16 is TFA insoluble-Rice).The specific paddy rice component insoluble in conjunction with trifluoroacetic acid of fusion rotein His-CBM.Containing a large amount of crystallization Mierocrystalline celluloses and other a small amount of polysaccharide in the rice cell wall fraction that trifluoroacetic acid is insoluble.
Two, ELISA tests the Sugar-binding activitv of quantitative analysis CBM fragment
1, Mierocrystalline cellulose sample
Updefraff reagent is adopted to prepare plant (japonica rice variety " gold is fine ", Columbia ecotype Arabidopis thaliana, corn variety " neat 319 " and Poplar Varieties " Populus hopeiensis ") the crystallization Mierocrystalline cellulose in different plant species source.Concrete steps are as follows:
Powder is polished between second section maturation plant being dried the stem stalk after not changing to weight, 70% ethanol aqueous wash once, wash once with the chloroform-methanol mixed solution of equal-volume mixing, gained precipitation washing with acetone post-drying also removes coarse grain with 200 mesh sieve sieves, obtains the alcohol insoluble matter component (Alcohol insolubleresidue/ is called for short AIR) based on cell wall constituent.The AIR powder of 20mg is cleared up through the alpha amylose enzyme of 1500Units and 10Units β Starch debranching enzyme in the sodium acetate soln of pH5.0,0.1M, the centrifugal 10min of 10000g collecting precipitation, cross 100 mesh sieves by washing with acetone post-drying, obtain powder.Take 2mg powder in the glass test tube of drying, add TFA containing 2M trifluoroacetic acid in 121 DEG C of process 90 minutes, centrifugally abandon supernatant, add 1mL Updegraff reagent (HNO
3: HOAC:H
2o=1:8:2; Volume ratio) rear 100 DEG C process 30 minutes, centrifugal removing supernatant, respectively cleans 3 post-dryings with distilled water and acetone, obtains crystallization Mierocrystalline cellulose.
Commercial Mierocrystalline cellulose: cellulose powder(Sigma, catalog number is 22183), cellulose20 μm powder(Sigma, catalog number is S3504), Avicel pH101cellulose powder(Fluka, catalog number is 11365).
2, ELISA tests the Sugar-binding activitv of quantitative analysis CBM fragment
Fusion rotein His-CBM solution (containing 1 μ g albumen) prepared by Example 2, add Mierocrystalline cellulose to be measured, then add PBS damping fluid to 200 μ L, cellulosic concentration to be measured is 200 μ g/mL; Total amount contrast does not add Mierocrystalline cellulose to be measured, and blank does not add fusion rotein His-CBM solution.On ice in conjunction with after 1 hour, centrifugal 10 minutes of 12000rpm, gets 100 μ L supernatants and adds in the elisa plate (Greiner) of 96 hole high-bonds.After 100rpm room temperature rotates 2 hours, 4 DEG C are spent the night, abandon supernatant, wash 5 times with 200 μ L PBS damping fluids.Add 300 μ L containing the PBS damping fluid of 5g/100mL skimmed milk and room temperature blockade 2 hours, abandon supernatant, wash 5 times with 200 μ L PBS damping fluids.Add Anti-His primary antibodie (sigma, 1:1000 dilute), 100rpm rotates room temperature temperature bath 2 hours, abandons supernatant, washes 5 times with 200 μ L PBS damping fluids.Add the goat anti-mouse antibody (ancient cooking vessel state, 1:2000 dilutes) of HRP mark, 100rpm room temperature rotates 2 hours, abandons supernatant, washes 5 times with 200 μ L PBS damping fluids.Add 150 μ L tetramethyl biphenyl amine aqueous solution (tetramethylbenzidine/TMB, sigma) develop the color 5 minutes, then add 35 μ L1M sulfuric acid with termination reaction, room temperature left standstill after 30 minutes, adopted Origin microplate reader under 450nm wavelength, read value (Abs450).With the fusion rotein His-CBM solution production standard curve adding 0 μ g, 0.2 μ g, 0.4 μ g, 0.6 μ g, 0.8 μ g, 1 μ g, 1.5 μ g and 2.5 μ g respectively, try to achieve the regression equation y=0.0342x+0.1179 that Y becomes with X, wherein x value is the light absorption value of optical density(OD) Abs450nm, y is the content of fusion rotein His-CBM, and unit is microgram.The content of fusion rotein His-CBM is obtained by regression equation, then by following formulae discovery binding ability: binding ability (Binding activity)=(protein content after total protein concentration-combination)/total protein concentration according to the light absorption value recorded.
The results are shown in Figure 4.Found that, fusion rotein His-CBM really can in conjunction with the crystallization Mierocrystalline cellulose in Culm of Rice source, also higher binding ability is had to the crystallization Mierocrystalline cellulose that the crystallization Mierocrystalline cellulose in Arabidopis thaliana source, the crystallization Mierocrystalline cellulose of corn source and willow are originated, and have certain binding activities to other business-like Mierocrystalline cellulose (cellulose powder, cellulose20 μm powder), but the cellulosic binding activities of crystallization of the plant-sourced do not obtained in conjunction with Updegraff process is high.
3, the kinetic curve combined
Protein concentration in the fusion rotein His-CBM solution prepared by PBS damping fluid adjustment embodiment 2.Getting protein concentration is that 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, 2 μMs, 4 μMs, 6 μMs, 8 μMs or 10 μMs of fusion rotein His-CBM solution join in EP pipe respectively, add the crystallization Mierocrystalline cellulose in the paddy rice source of preparation in 100 μ g steps 1 again, mend to 200 μ L with PBS damping fluid; Total amount contrast does not add Mierocrystalline cellulose, and blank does not add fusion rotein His-CBM solution.Subsequent processes is with step 2.Numerical value Origin v8.0 software (Origin) fitting of a curve.
The binding kinetics curve of the external binding crystal cellulose of fusion rotein His-CBM is shown in Fig. 5.Found that, the cellulosic combination of crystallization that fusion rotein His-CBM and paddy rice are originated meets kinetic curve, improving constantly namely along with fusion rotein His-CBM concentration, the fusion rotein His-CBM be attached on crystallization Mierocrystalline cellulose can reach capacity, free fusion rotein His-CBM can become many, meets the binding kinetics reaction of one-level.
Three, the CBM of point mutation is to the effect of Mierocrystalline cellulose binding characteristic
Fusion rotein His-CBM solution, fusion rotein His-CBM prepared by Example 2
y46Asolution, fusion rotein His-CBM
w66Asolution or fusion rotein His-CBM
w72Asolution (all containing 1 μ g albumen), adds 100 μ g cellulose20 μm of powder(Sigma), then add PBS damping fluid to 200 μ L; Total amount contrast does not add Mierocrystalline cellulose, and blank does not add CBM fragment solution.Subsequent processes is with 2 of step 2.
The binding ability of fusion rotein His-CBM is 58%, fusion rotein His-CBM
y46A, fusion rotein His-CBM
w66Aor fusion rotein His-CBM
w72Arelative binding capacity compared with fusion rotein His-CBM is shown in Fig. 6.Found that, the sudden change of Y46A and W72A significantly reduces the binding activities of fusion rotein His-CBM to commercial fibers element, and the sudden change of W66A is not on fusion rotein His-CBM substantially affecting in conjunction with commercial fibers element.This shows that the Mierocrystalline cellulose of die aromatischen Aminosaeuren to binding crystal conservative in CBM fragment is very crucial.
Embodiment 4, CBM fragment are as the application identifying the cellulosic probe of plant-sourced
Since CBM fragment can specific recognition in conjunction with the crystallization Mierocrystalline cellulose of various plant origin, so CBM fragment can as the cellulosic probe of identification plant-sourced crystallization.For the using value of checking CBM fragment, the method confirmation that the present invention dye by immunochemistry, CBM fragment can specific combination crystallization Mierocrystalline cellulose, thus identification CBM fragment is applied to, the cellulosic probe of plant-sourced crystallization.
One, the structure of recombinant plasmid
1, extract japonica rice variety " gold is fine " total serum IgE and reverse transcription is cDNA.
2, the cDNA extracted with step 1, for template, carries out pcr amplification with the primer of F3 and R3 composition, reclaims pcr amplification product (target sequence is as shown in the sequence 11 of sequence table).
F3：5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGGATAACAGCTTTGTGGAT-3'；
R3：5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTATCAGAGCTTATCCATGTCGGC-3’。
3, PCR primer step 2 obtained is carried out agarose gel electrophoresis and is reclaimed the fragment (sequence 11) of about 1.9kb, recovery product and carrier pDONR207 is carried out BP reaction, obtains recombinant plasmid pDONR207-IIP4.
4, recombinant plasmid pDONR207-IIP4 and carrier pDEST17 is carried out LR reaction, obtain recombinant plasmid pDEST17-IIP4.IIP4 fragment.IIP4 is ILA1 (increased leaf angle1 (ILA1)) interactingprotein4, the albumen done mutually with ILA1 is a nucleoprotein, can not identify the crystallization Mierocrystalline cellulose of plant-sourced in theory, be a negative control fragment.
Two, the preparation of IIP4 fragment
1, recombinant plasmid pDEST17-IIP4 is imported intestinal bacteria Rosetta (DE3), obtain recombinant bacterium.
2, the recombinant bacterium that step 1 obtains is seeded to the LB liquid nutrient medium containing 50 μ g/mL penbritins, 50 μ g/mL paraxin and 5g/100mL glucose, 37 DEG C, 200rpm shaking culture is to OD
600nmadd IPTG when=0.8 and make its final concentration be 0.8mM, 28 DEG C, 200rpm shaking culture 3 hours, then 4 DEG C, the centrifugal 10min of 4000rpm, collecting bacterial sediment.
3, the precipitation that step 2 obtains is got, with binding buffer, (solvent is water, containing 20mM sodium phosphate, 0.5M sodium-chlor and 20mM imidazoles, pH7.4) suspend, put ultrasonication on ice (70w, ultrasonic 90 times, each 10s, 15s interval) centrifugal 30 minutes of 10000rpm afterwards, collects supernatant liquor.
4, the supernatant liquor that step 3 obtains is splined on affinity column (the Glutathione Ni Sepharose6Fast Flow4B of GE company) and carries out affinity chromatography.Wash-out is carried out successively with the binding buffer containing 200mM imidazoles of the bindingbuffer containing 80mM imidazoles of the binding buffer containing 50mM imidazoles of the binding buffer containing 35mM imidazoles of 5 column volumes, 5 column volumes, 5 column volumes, binding buffer and 5 column volume containing 100mM imidazoles of 5 column volumes, collection, containing solution after the post excessively of the binding buffer of 80mM imidazoles, is the IIP4 fragment solution that purifying obtains.
Three, Immunochemical staining assay
The crystallization cellulose suspension in the paddy rice source of preparing 1 of the step 2 of 100 μ g embodiments 3, in 100 μ lPBS damping fluids, drips on slide glass, 45 DEG C of roasting sheet platforms toasts and spends the night.Above-mentioned slide glass is put into PBS damping fluid to be washed once, blockades 1 hour with the PBS damping fluid containing 1%BSA.Then, IIP4 fragment solution (containing 1 μ g albumen) incubated at room 2h prepared by the fusion rotein His-CBM solution (containing 1 μ g albumen) prepared with 10 microliter Examples 2 or step one, washes 3 times, each 10 minutes with the PBS damping fluid containing 0.1%BSA.Add Anti-His primary antibodie, incubated at room 2h, wash 3 times, each 10 minutes with the PBS damping fluid containing 0.1%BSA.Add marked by fluorescein isothiocyanate two resist, incubated at room 2 hours, mounting after washing 3 times with PBS damping fluid.Last at ZeissImager D2 fluorescent microscope photographs picture.Result as shown in Figure 7, can be seen very strong fluorescent signal with the slide glass that CBM fragment temperature is hatched, and with the fluorescent signal that the slide glass that IIP4 fragment temperature is hatched can be can't see, illustrate that CBM probe can apply specific recognition plant origin crystallization Mierocrystalline cellulose.
Claims (10)
1.CBM fragment or there is the fusion rotein of described CBM fragment; Described CBM fragment is following (a) or (b) or (c):
A protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 3;
B protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 5;
C protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 6.
2. there is the fusion rotein of described CBM fragment as claimed in claim 1, it is characterized in that: described fusion rotein is following (f) or (g) or (h):
F protein that () is made up of the aminoacid sequence shown in sequence in sequence table 3;
G protein that () is made up of the aminoacid sequence shown in sequence in sequence table 5;
H protein that () is made up of the aminoacid sequence shown in sequence in sequence table 6.
3. the gene of CBM fragment described in claim 1 of encoding or the gene of fusion rotein described in claim 1 of encoding.
4. gene as claimed in claim 3, is characterized in that: described gene is the DNA molecular described in following (1) or (2) or (3):
(1) sequence 7 of sequence table is from the DNA molecular shown in 5 ' end 109-669 position Nucleotide;
(2) sequence 9 of sequence table is from the DNA molecular shown in 5 ' end 109-669 position Nucleotide;
(3) sequence 10 of sequence table is from the DNA molecular shown in 5 ' end 109-669 position Nucleotide.
5. gene as claimed in claim 3, is characterized in that: described gene is the DNA molecular described in following (6) or (7) or (8):
(6) DNA molecular shown in sequence 7 of sequence table;
(7) DNA molecular shown in sequence 9 of sequence table;
(8) DNA molecular shown in sequence 10 of sequence table.
6. the expression cassette containing gene described in claim 3 or 4 or 5, recombinant vectors, transgenic cell line or recombinant bacterium.
7.CBM fragment or there is the application of fusion rotein in the product for the preparation of binding crystal cellulose of described CBM fragment;
Described CBM fragment is following (a) or (b) or (c):
A protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 3;
B protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 5;
C protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 6.
8., for a product for binding crystal cellulose, its activeconstituents is CBM fragment or the fusion rotein with described CBM fragment;
Described CBM fragment is following (a) or (b) or (c):
A protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 3;
B protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 5;
C protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 6.
9.CBM fragment or the fusion rotein with described CBM fragment are for the preparation of the application identified in the cellulosic product of crystallization;
Described CBM fragment is following (a) or (b) or (c):
A protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 3;
B protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 5;
C protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 6.
10., for identifying the cellulosic product of crystallization, its activeconstituents is CBM fragment or the fusion rotein with described CBM fragment;
Described CBM fragment is following (a) or (b) or (c):
A protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 3;
B protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 5;
C protein that () is made up of from N-terminal the 37 to 223 amino acids residue sequence in sequence table 6.
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| CN1875098A (en) * | 2003-10-30 | 2006-12-06 | 诺和酶股份有限公司 | Carbohydrate-binding modules of a new family |
| CN102174532A (en) * | 2011-01-19 | 2011-09-07 | 中国科学技术大学 | Application of family 3 cellulose binding domain serving as affinity tag for expression and purification of recombinant protein in eukaryote |
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| CN102174532A (en) * | 2011-01-19 | 2011-09-07 | 中国科学技术大学 | Application of family 3 cellulose binding domain serving as affinity tag for expression and purification of recombinant protein in eukaryote |
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