CN100425688C - Stabilization technology for intact phycobilisomes of red algae and blue algae - Google Patents
Stabilization technology for intact phycobilisomes of red algae and blue algae Download PDFInfo
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- CN100425688C CN100425688C CNB2005100420263A CN200510042026A CN100425688C CN 100425688 C CN100425688 C CN 100425688C CN B2005100420263 A CNB2005100420263 A CN B2005100420263A CN 200510042026 A CN200510042026 A CN 200510042026A CN 100425688 C CN100425688 C CN 100425688C
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Abstract
The present invention relates to a technology for stabilizing the intact phycobilisome of blue algae and red algae, which belongs to the technical field of marine biotechnology. In the present invention, red algae and blue algae are used as materials; cells are broken by ultrasonic waves, the phycobilisome is dissociated from a thylakoid membrane by using a detergent TritonX-100, the TritonX-100, chlorophyll and cell debris are conventionally removed centrifugally, and then, the intact phycobilisome is prepared by sucrose density gradient ultracentrifugation; subsequently, a polysaccharide glucan non-covalent bond coating method and a formaldehyde covalence crosslinking method are used for protecting the integrity of the phycobilisome. The integrity of the phycobilisome can still be kept without dissociation when the phycobilisome is preserved for 30 days at room temperature. Thus, the present invention provides the basis for causing the phycobilisome to be used for super-sensitive biomedical detection.
Description
(1) technical field
The present invention relates to the stabilization technology of blue-green algae and red algae intact phycobilisome, belong to the marine biotechnology field.
(2) background technology
Phycobiliprotein is that a class photosynthesis is caught photopigment-protein complex, mainly is present in blue-green algae, red algae, latent algae and the minority dinoflagellate.In photosynthesis, play a part to catch and transmit luminous energy, have intensive fluorescence.In blue-green algae and red algae, form the supramolecular structure phycobilisome by 2-3 kind phycobiliprotein, form transmission ofenergy sequence efficiently.At the mid-80, the scholar of American Studies algae photosynthesis proposition as fluorescent marker, is used for diagnostic reagent with phycobiliprotein.Compare with fluorescent marker commonly used, phycobiliprotein has following advantage: production process safety, nontoxic, and luminous energy absorbs strong, the fluorescent yield height surpasses 90%, and bias light interference and false positive rate are low, stable in the scope of pH4-11, can make double-colored, three looks and four color markers.So the range of application of this class reagent constantly enlarges.Because its unique advantage, the diagnostic reagent of phycobiliprotein and phycobiliprotein mark enters the world market in the early 1990s.But,, be not applied to popular reagent for clinical diagnosis as yet owing to cost an arm and a leg.The whole imports of China also only limit to the diagnosis of carrying out with cell streaming instrument.
Phycobiliprotein has its special advantages as fluorescent probe, but one phycobiliprotein molecule, because its molecule is less, the pigment group that contains in the molecule is less, therefore, the brightness that produces fluorescence is not enough, makes in high-resolution detection medium sensitivity not high.The Mo Siman of the U.S. (J.P.Morseman) proposes, and applying immobilized intact phycobilisome can be used for multiple biomedical the detection as fluorescent probe.Because contain more pigment group in the phycobilisome, therefore, its brightness is higher, and excitation wavelength is the absorbing wavelength of phycoerythrin (PE) or Phycocyanins, C-(PC), and fluorescent emission is from APC, therefore, excitation wavelength and emission wavelength are avoided the phase mutual interference at a distance of farther.But phycobilisome is to be connected under the proteic effect by phycobiliprotein, the supramolecular complex that self-assembly forms under non covalent bond effects such as hydrophobic, electric charge, it is stable that phycobilisome only keeps in than the buffering system of high ionic strength, than under the low ionic strength, very easily dissociate, even under higher ionic strength, intact phycobilisome also is difficult to long-time the preservation, thereby has seriously limited phycobilisome as the application of fluorescent probe in biomedicine detects.Therefore, the stabilization of intact phycobilisome is used for biomedical the detection for the preservation of phycobilisome with as fluorescent probe, seems particularly important.
(3) summary of the invention
The present invention is directed to phycobilisome and be used for biomedical the detection as fluorescent probe as fluorescent marker and need the key technical problem that solves, a kind of stabilization technology of intact phycobilisome is provided, thereby lays the foundation for intact phycobilisome is used as fluorescent probe.
The stabilization technology of blue-green algae of the present invention and red algae intact phycobilisome, method is as follows:
1, the fragmentation of raw material: with fresh blue-green algae or red algae is raw material, is dissolved in the Sodium phosphate dibasic-potassium phosphate buffer of pH=6.8～7.0, adopts the ultrasonic disruption cell.
Above-mentioned blue-green algae is selected from spirulina plalensis or anabena; Above-mentioned red algae is selected from unicellular red algae Porphyridium cruentum, large-scale red algae multitube algae or Ceramium kondoi.
2, phycobilisome dissociates: add stain remover tween X-100 (TritonX-100) in the frond of above-mentioned fragmentation, phycobilisome is disintegrated down from thylakoid membrane.Centrifugal, remove stain remover, chlorophyll and broken cell, get the crude extract of phycobilisome.
3, the extraction of intact phycobilisome: extract the intact phycobilisome of above-mentioned blue-green algae of preparation or red algae with the ultracentrifugal method of sucrose density gradient.
4, the stability protection of intact phycobilisome adopts one of following method:
(1) adopt dextran, non-covalent crosslinked to phycobilisome, accurately the weighing relative molecular mass is 20000 dextran, joins in the above-mentioned steps 3 intact phycobilisome solution, and dissolving makes final concentration reach 9～10% (W/V).
(2) adopt the formaldehyde crosslinking technology, the phycobilisome intramolecularly is carried out covalent cross-linking, get above-mentioned steps 3 intact phycobilisome solution, Sodium phosphate dibasic-potassium phosphate buffer (pH=6.98) with 0.75mol/L is adjusted protein concentration to 0.25～0.30mg/mL, then, get this phycobilisome solution 1.8mL, slowly drip 3～5% (W/V) formaldehyde strong solution 0.2mL of Sodium phosphate dibasic-potassium phosphate buffer preparation with 0.75mol/L while vibrating, until concentration of formaldehyde is 0.3～0.5% (W/V), 20 ℃ of insulation 12-13h.
The fragmentation of above-mentioned steps 1 raw material is specially: fresh frond 0.75～1.0g is dissolved in 15～20mL 1molL
-1Sodium phosphate dibasic-potassium phosphate buffer (pH=6.8～7.0) in, ultrasonic disruption, broken power are 12～15W, broken 5～6 times, each 1.5～2min, 2～3min at interval.
Above-mentioned steps 2 phycobilisome dissociate and are specially: add concentration of volume percent 1.9～2.1% stain remover tween X-100 in the frond solution of fragmentation, under the room temperature, slowly stirred 1 hour～1.5 hours, centrifugal 1～2 time of 13000rpm～15000rpm, remove the cell debris of stain remover tween X-100, chlorophyll and the bottom on top layer, collect out the solution at centrifuge tube middle part, be the phycobilisome crude extract.
Add-on the present invention of above-mentioned stain remover is not particularly limited, and is by state of the art, suitable so that phycobilisome is dissociated into from thylakoid membrane.
The method that above-mentioned steps 3 is extracted the sucrose density gradient centrifugation of phycobilisome can adopt prior art, the invention provides following concrete grammar:
Get the phycobilisome crude extract 3～4mL of step 2 preparation, the upper strata of the sucrose density gradient centrifugation that is added to pipe, volumetric molar concentration from bottom to upper strata sucrose and volume: 2molL
-14mL, 1molL
-13mL, 0.5molL
-15mL, 0.25molL
-15mL; Centrifugal 3～the 4h of 40000rpm～45000rpm gets 1molL
-1The blue liquid of layer is to 1molL
-1Sodium phosphate dibasic-potassium phosphate buffer dialysed overnight remove sucrose, use 1molL at last
-1The dilution of Sodium phosphate dibasic-potassium phosphate buffer, to phycobiliprotein concentration be 0.25～0.30mg/mL, measure the fluorescence spectrum of intact phycobilisome.(as shown in Figure 1)
Above-mentioned steps 4 intact phycobilisomes stability is measured:
1, will reduce to 0.6mol/L with the ionic strength of the Sodium phosphate dibasic-potassium primary phosphate of dextran stability protection method (1) gained phycobilisome solution, 20 ℃ of preservations were preserved 30 days.In contrast be to reduce to the phycobilisome that does not add dextran that 1.0mol/L preserves (in the Sodium phosphate dibasic-potassium phosphate buffer of phycobilisome without stability protection at 0.75～1.0mol/L in the ionic strength of Sodium phosphate dibasic-potassium primary phosphate; can be kept perfectly; and in Sodium phosphate dibasic-potassium phosphate buffer of 0.5mol/L; dissociate easily, can't be kept perfectly).Measure the fluorescence spectrum of phycobilisome, phycobilisome still is kept perfectly (as shown in Figures 2 and 3).
2, formaldehyde crosslinking technical stability guard method (2) gained phycobilisome solution; be diluted to low ion concns 0.1mol/L with deionized water, 20 ℃ of incubations 30 days are measured its fluorescence spectrum; the result shows under the 0.1mol/L low ionic strength and to preserve 30 days that the phycobilisome of stabilization still is kept perfectly.(as shown in Figure 4)
The present invention is a material with red algae and blue-green algae; using ultrasound ripple smudge cells, stain remover TritonX-100 prepare intact phycobilisome from dissociate phycobilisome, conventional centrifugal removal TritonX-100, chlorophyll and cell debris, application sucrose density gradient ultracentrifugation of thylakoid membrane; use one of dextran non covalent bond bag quilt and two kinds of methods of formaldehyde covalent cross-linking then; stability to phycobilisome is protected; room temperature preservation 30 days; phycobilisome still is kept perfectly, and does not dissociate.Thereby lay a good foundation for phycobilisome being applied to overdelicate biomedical the detection, have good application prospects.
(4) description of drawings
Fig. 1 is the room temperature fluorescence spectrum of the spirulina plalensis phycobilisome of embodiment 1 sucrose density gradient centrifugation extraction.Curve a is the room temperature fluorescence emmission spectrum of spirulina plalensis phycobilisome, and its emission peak is positioned at the 680nm place.Curve b is the excitation spectrum of phycobilisome, and there are two places at the fluorescence excitation peak of 680nm, and wherein the 622nm place is the maximum excitation peak, and the 650nm place is an acromion.
Fig. 2 is that embodiment 1 adds after the dextran phycobilisome V value with the variation of shelf time.
Fig. 3 is that embodiment 1 adds dextran (a) and do not add 30 days room temperature fluorescence emmission spectrum of dextran contrast (b) phycobilisome preservation.
Fig. 4 is the room temperature fluorescence spectrum of the spirulina plalensis phycobilisome of embodiment 2 stabilizations, wherein, and a fluorescence excitation spectrum, Em=665nm; The b fluorescence emission spectrum, Ex=580nm.
(5) embodiment
The stabilization technology of embodiment 1, blue-green algae intact phycobilisome, method is as follows:
1, the fragmentation of raw material: fresh spirulina plalensis 0.8g is dissolved in 16mL 1molL
-1Sodium phosphate dibasic-potassium phosphate buffer (pH=6.9) in, adopt the ultrasonic disruption cell, broken power is 12～15W, broken 5 times, each 1.5min, 2min at interval.
2, phycobilisome dissociates: add concentration of volume percent 2.0% stain remover tween X-100 in the frond of above-mentioned fragmentation, under the room temperature, slowly stirred 1 hour～1.5 hours, phycobilisome is disintegrated down from thylakoid membrane, centrifugal 1～2 time of 13000rpm～15000rpm, remove the cell debris of stain remover tween X-100, chlorophyll and the bottom on top layer, collect out the solution at centrifuge tube middle part, be the phycobilisome crude extract.
3, the extraction of intact phycobilisome: extract intact phycobilisome with the ultracentrifugal method of sucrose density gradient.Get the phycobilisome crude extract 3mL of step 2 preparation, the upper strata of the sucrose density gradient centrifugation that carefully is added to pipe, volumetric molar concentration from bottom to upper strata sucrose and volume: 2molL
-14mL, 1molL
-13mL, 0.5molL
-15mL, 0.25molL
-15mL; Centrifugal 3～the 4h of 40000rpm～45000rpm gets 1molL
-1The blue liquid of layer is to 1molL
-1Sodium phosphate dibasic-potassium phosphate buffer dialysed overnight remove sucrose, use 1molL at last
-1The dilution of Sodium phosphate dibasic-potassium phosphate buffer, to phycobiliprotein concentration is 0.25～0.30mg/mL.
Measure the fluorescence spectrum of intact phycobilisome, as shown in Figure 1, curve a is the room temperature fluorescence emmission spectrum of spirulina plalensis phycobilisome, its emission peak is positioned at the 680nm place, this peak value is the fluorescence emission peak of AP-B, does not have C-algae basket albumen (C-PC) (648nm) and different algae basket albumen (APC) fluorescence emission peak (660nm), illustrates in the phycobilisome of purifying, the luminous energy that C-PC and APC absorb all can pass to the terminal acceptor AP-B of energy efficiently, thereby the phycobilisome of proof gained is complete.Curve b is the excitation spectrum of phycobilisome, there are two places at the fluorescence excitation peak of 680nm, wherein the 622nm place is the maximum excitation peak, come from C-PC, the energy of this explanation phycobilisome 680nm fluorescence emission peak is mainly derived from C-PC, and the 650nm place is an acromion, comes from different algae basket albumen (APC), and it is complete further specifying phycobilisome.
4, the stability protection of intact phycobilisome: adopt dextran; non-covalent crosslinked to phycobilisome; accurately the weighing relative molecular mass is 20000 dextran; join in the above-mentioned steps 3 intact phycobilisome solution; dissolving makes final concentration reach 9～10% (W/V), and the ionic strength of the Sodium phosphate dibasic-potassium primary phosphate of this solution is reduced to 0.6mol/L; 20 ℃ of preservations were preserved 30 days.
In contrast be to reduce to the phycobilisome that does not add dextran that 1.0mol/L preserves in the ionic strength of Sodium phosphate dibasic-potassium primary phosphate, measure the fluorescence spectrum of phycobilisome, phycobilisome still is kept perfectly, as shown in Figures 2 and 3.
V=F among Fig. 2
676nm/ F
650nm, the dissociation degree of reflection phycobilisome, the V value is big more, and phycobilisome is complete more, and the V value is more little, and the dissociation degree of phycobilisome is big more.The adding of dextran makes the V value of spirulina plalensis phycobilisome (0.6mg/mL) increase, and illustrates that dextran makes phycobilisome more tight in conjunction with getting.
Fig. 3 showed in examination phase (in the 1mol/L damping fluid, 7,20 ℃ of preservations of pH) at 30 days, compared with the contrast phycobilisome, and the V value of phycobilisome that adds dextran is all the time greater than the corresponding V value of control group, and the reduction amount of V value is less in the preservation process.Preserve after 30 days, the fluorescence emission peak change in location of the phycobilisome of adding dextran is little, is blue shifted to 671nm by 676nm, and acromion does not occur about 650nm, and the acromion about tangible 650nm has appearred in contrast phycobilisome, and the fluorescent emission main peak also is blue shifted to 668nm.See in appearance, preserve after 30 days that the phycobilisome that adds dextran forms the blue test tube bottom that is deposited in, the supernatant liquor clarification, and more white flocks has appearred in control group phycobilisome blue solution, and existing a small amount of phycobiliprotein sex change is described.These presentation of results dextran have significant protective effect to the stability of phycobilisome.
Embodiment 2, as described in the embodiment 1, different is:
The stability protection of step 4 intact phycobilisome: adopt the formaldehyde crosslinking technology; the phycobilisome intramolecularly is carried out covalent cross-linking; get above-mentioned steps 3 intact phycobilisome solution; adjust protein concentration to 0.28mg/mL with Sodium phosphate dibasic-potassium phosphate buffer (pH=6.98) of 0.75mol/L; then; get this phycobilisome solution 1.8mL; slowly drip 4% (W/V) formaldehyde strong solution 0.2mL of Sodium phosphate dibasic-potassium phosphate buffer preparation with 0.75mol/L while vibrating; until concentration of formaldehyde is 0.4% (W/V), 20 ℃ of insulation 12h.Gained phycobilisome solution is diluted to low ion concns 0.1mol/L with deionized water, and 20 ℃ of incubations 30 days are measured its fluorescence spectrum, and the result shows under the 0.1mol/L low ionic strength and preserved 30 days that the phycobilisome of stabilization still is kept perfectly.As shown in Figure 4.
Fig. 4 is the room temperature fluorescence spectrum of the spirulina plalensis phycobilisome of stabilization, wherein, and a fluorescence excitation spectrum, Em=665nm; The b fluorescence emission spectrum, Ex=580nm.Its fluorescence emission peak is the fluorescence emission peak of Allophyxoxyanin (APC) at 665nm, the fluorescence emission peak of 650nm C-Phycocyanins, C-do not occur, illustrates that energy can be delivered to Allophyxoxyanin from the C-Phycocyanins, C-efficiently; The maximum fluorescence excitation peak of stabilization phycobilisome contrasts (622nm) little to some extent moving of phycobilisome at 626nm, and the stabilization phycobilisome also has one to excite acromion about 650nm.The stabilization treatment of these presentation of results spirulina plalensis phycobilisome does not change the original energy transfer properties of phycobilisome.
The stabilization technology of embodiment 3, red algae intact phycobilisome, method is as follows:
As described in embodiment 1, different is:
The raw material of step 1 is unicellular red algae Porphyridium cruentum 0.9g, is dissolved in 18mL 1molL
-1Sodium phosphate dibasic-potassium phosphate buffer (pH=6.8) in, ultrasonic disruption cell, broken power are 12～15W, broken 6 times, each 2min, 3min at interval.
Embodiment 4, as described in the embodiment 2, different is:
The raw material of step 1 is large-scale red algae multitube algae 1.0g, is dissolved in 20mL 1molL
-1Sodium phosphate dibasic-potassium phosphate buffer (pH=7.0) in, ultrasonic disruption cell, broken power are 12～15W, broken 6 times, each 2min, 3min at interval.
Step 3 is extracted the method for the sucrose density gradient centrifugation of phycobilisome: get the phycobilisome crude extract 4mL of step 2 preparation, the upper strata of the sucrose density gradient centrifugation that carefully is added to pipe, volumetric molar concentration from bottom to upper strata sucrose and volume: 2molL
-14mL, 1molL
-13mL, 0.5molL
-15mL, 0.25molL
-15mL; Centrifugal 3～the 4h of 40000rpm～45000rpm gets 1molL
-1The blue liquid of layer is to 1molL
-1Sodium phosphate dibasic-potassium phosphate buffer dialysed overnight remove sucrose, use 1molL at last
-1The dilution of Sodium phosphate dibasic-potassium phosphate buffer, to phycobiliprotein concentration is 0.30mg/mL.
Claims (5)
1. the stabilization method of blue-green algae and red algae intact phycobilisome, step is as follows:
(1) fragmentation of raw material: with fresh blue-green algae or red algae is raw material, is dissolved in the Sodium phosphate dibasic-potassium phosphate buffer of pH=6.8～7.0, adopts the ultrasonic disruption cell;
(2) phycobilisome dissociates: in the frond of above-mentioned fragmentation, add stain remover tween X-100, phycobilisome is disintegrated down from thylakoid membrane, and centrifugal, remove stain remover, chlorophyll and broken cell, get the crude extract of phycobilisome;
(3) extraction of intact phycobilisome: extract the intact phycobilisome of above-mentioned blue-green algae of preparation or red algae with the ultracentrifugal method of sucrose density gradient;
(4) stability protection of intact phycobilisome adopts following method:
Adopt the formaldehyde crosslinking technology, the phycobilisome intramolecularly is carried out covalent cross-linking, get above-mentioned steps (3) intact phycobilisome solution, Sodium phosphate dibasic one potassium phosphate buffer pH=6.98 with 0.75mol/L, adjust protein concentration to 0.25～0.30mg/mL, then, get this phycobilisome solution 1.8mL, slowly drip the 3～5%W/V formaldehyde strong solution 0.2mL for preparing with Sodium phosphate dibasic one potassium phosphate buffer of 0.75mol/L while vibrating, until concentration of formaldehyde is 0.3～0.5%W/V, 20 ℃ of insulation 12-13h.
2. the stabilization method of blue-green algae as claimed in claim 1 and red algae intact phycobilisome is characterized in that blue-green algae is selected from spirulina plalensis or anabena, and red algae is selected from unicellular red algae Porphyridium cruentum, large-scale red algae multitube algae or Ceramium kondoi.
3. the stabilization method of blue-green algae as claimed in claim 1 and red algae intact phycobilisome is characterized in that the fresh frond 0.75～1.0g of step (1), is dissolved in 15～20mL 1molL
-1Sodium phosphate dibasic one potassium phosphate buffer in, ultrasonic disruption, broken power are 12～15W, broken 5～6 times, each 1.5～2min, 2～3min at interval.
4. the stabilization method of blue-green algae as claimed in claim 1 and red algae intact phycobilisome, the concentration of volume percent that it is characterized in that step (2) adding stain remover tween X-100 is 1.9～2.1%, under the room temperature, slowly stirred 1 hour～1.5 hours, centrifugal 1～2 time of 13000rpm～15000rpm collects out the solution at centrifuge tube middle part.
5. the stabilization method of blue-green algae as claimed in claim 1 and red algae intact phycobilisome, it is characterized in that step (3) is specially: the phycobilisome crude extract 3～4mL that gets step (2) preparation, the upper strata of the sucrose density gradient centrifugation that is added to pipe, volumetric molar concentration from bottom to upper strata sucrose and volume: 2molL
-14mL, 1molL
-13mL, 0.5molL
-15mL, 0.25molL
-15mL; Centrifugal 3～the 4h of 40000rpm～45000rpm gets 1molL
-1The blue liquid of layer is to 1molL
-1Sodium phosphate dibasic one potassium phosphate buffer dialysed overnight remove sucrose, use 1molL at last
-1The dilution of Sodium phosphate dibasic-potassium phosphate buffer, to phycobiliprotein concentration be 0.25～0.30mg/mL.
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| US20030134325A1 (en) * | 1995-04-10 | 2003-07-17 | Cubicciotti Roger S. | Modified phycobilisomes and uses therefore |
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| US20030134325A1 (en) * | 1995-04-10 | 2003-07-17 | Cubicciotti Roger S. | Modified phycobilisomes and uses therefore |
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| 藻胆体稳定性的研究（V）－pH对藻胆体稳定性的影响. 杨忠慧等.首都师范大学学报（自然科学版），第15卷第4期. 1994 |
| 藻胆体稳定性的研究（V）－pH对藻胆体稳定性的影响. 杨忠慧等.首都师范大学学报（自然科学版），第15卷第4期. 1994 * |
| 钝顶螺旋藻藻胆体的稳定性研究. 张熙颖.海洋科学，第28卷第8期. 2004 |
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