CN104865376A - Method for observing natural rubber particles and application of method in strain screening of rubber plants - Google Patents
Method for observing natural rubber particles and application of method in strain screening of rubber plants Download PDFInfo
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Abstract
The invention relates to a method for observing natural rubber particles and an application of the method in the strain screening of rubber plants with high latex yield, especially koksaphyz with high latex yield. The method is characterized in that a mark immunological recognition technology is used for observation. The method of the invention can be used for the qualitative and quantitative observation of natural rubber particles, so that the rubber plant with high latex yield, especially koksaphyz with high latex yield, can be screened quickly and effectively.
Description
[technical field]
The present invention relates to the method for the examination rubber plant strain utilizing labelled immune recognition technology, particularly utilize the method for the rubber plant strain (particularly Russian dandelion strain) such as labelled immune electron microscopy examination dandelion, thorn lettuce, fig, guayule rubber.
[background technology]
Comprise rubber grain in the rubber latex of rubber plant, namely natural rubber molecule synthesizes on the surface of rubber grain.Small rubber granule protein (SRPP) is that a kind of of rubber particle surface synthesizes with natural rubber the memebrane protein be closely related, and it becomes positive correlation (see Oh, S.K. with the product glue ability of rubber plant; Kang, H.; Shin, D.H.; Yang, J.; Chow, K.S.; Yeang, H.Y.; Wagner, B.; Breiteneder, H.; Han, K.H.J.Biol.Chem.1999,274,17132 – 17138.).Therefore by the SRPP in a kind of rubber plant emulsion of observation and the product glue ability of measurable this kind of rubber plant.
Russian dandelion is per nnial herb, comprises composite family dandelion etc.Russian dandelion contains nature rubber latex, can manufacture natural rubber.China's area is vast, all can estate rubber grass from temperate zone to subtropical species.Therefore, Chinese research mechanism has begun one's study from Russian dandelion and has gathered Heveatex, to prepare natural rubber.But gel content is general lower in Russian dandelion.Need from wild Russian dandelion, filter out the high Russian dandelion strain of natural emulsion content and strain, carry out artificial culture, plant with large-scale promotion.Therefore find a kind of examination of Russian dandelion strain or the method for screening, no matter be significant from economic implications or the angle of the rubber safety of China.
Common electron microscope can realize the particle morphological analysis of high power and high-res, such as, use scanning electron microscope and transmission electron microscope, clearly can characterize the size of nanoparticles, form and distribution characteristics thereof, thus establish methodology basis for the important research of association area and application.
Common electron microscopy carries out follow-up electron microscopic observation by rubber plant samples of latex being made metal spraying or fix process on wire netting, and under good condition of work, common electron microscopy can obtain particle electron microscopic picture comparatively clearly.
In prior art, apply common electron microscopy and can observe directly particle in rubber plant emulsion.But, owing to may contain multiclass albumen, resin, rubber molecule and other particles in rubber plant emulsion simultaneously, under ordinary electronic microscope, these particles demonstrate similar image, so ordinary electronic microscope cannot distinguish the rubber grain in rubber plant emulsion, namely common electron microscopy does not have specificity, and whether cannot confirm viewed particle is natural rubber particle.
Immuno-labelling technique uses the mark substances such as radioactive nuclide, fluorescein, enzyme, nanogold particle, quantum dot, the antigen-antibody reaction of binding specificity, and the specific molecular means of identification carried out.
In immuno-labelling technique, labelled immune electron microscopy is combined with the electron microscopy of high magnification and high-res by Ag-Ab specific molecular recognition reaction, labelled immune electron microscope technique has a very wide range of applications in fields such as clinical diagnosis, disease treatment and physiological and pathological researchs, in recent years, along with the progress of labelling technique and the continuous renewal of electron microscopic relevant device, the research field of labelled immune electron microscopy is constantly expanded, and application is also increasingly extensive.
[summary of the invention]
[inventing problem to be solved]
Problem to be solved by this invention is the rubber grain that common electron microscope cannot distinguish in rubber plant emulsion, namely common electron microscopy does not have specificity, whether be desired target natural rubber particle, cannot judge the product glue ability of rubber plant if cannot confirm viewed particle.
[solving the means of problem]
The present invention is based on the small rubber granule protein of rubber particle surface, utilize labelled immune recognition technology, identify the rubber grain in rubber plant emulsion specifically, judge the rubber grain content height in rubber plant emulsion, and by observing the Density Distribution of hevein label, fast prediction and the product glue ability height judging rubber plant, solve the crucial technical problem producing the examination of glue Russian dandelion strain.
Specifically, to solve the means of problem as follows in the present invention:
1. observe a method for rubber grain, it is characterized in that, described method utilizes labelled immune recognition technology to carry out observing and identifying.
2. the method as described in project 1, is characterized in that, described rubber grain is from rubber plant emulsion.
3, the method as described in project 1 or 2, is characterized in that, described rubber plant is rubber tree or Russian dandelion, such as, be selected from the group be made up of Para rubber tree, dandelion, thorn lettuce, fig and guayule rubber any one.
4. the method according to any one of project 1 to 3, it is characterized in that, described labelled immune recognition technology is immunoelectronmicroscopy, enzyme linked immunosorbent assay, fluorescence microscopy, radioimmunoassay labelling technique, albumen or gene chips or the test strips method based on sidestream immune principle.
5. the method according to any one of project 1 to 4, is characterized in that, described immunology recognition technology is immunoelectronmicroscopy.
6, the method according to any one of project 1 to 5, is characterized in that, described method comprises the steps:
(1) to take from the rubber latex of rubber plant as testing sample, small rubber granule protein (SRPP) wherein is fully contacted with first antibody, make both combine;
(2) second antibody making label labeled fully contacts with the testing sample marking first antibody, makes both combine, obtains antibody labeling sample;
(3) antagonist mark sample carries out observing and identifying.
7, the method according to any one of project 1 to 6, is characterized in that, described first antibody is the antibody stemming from small rubber particle albumen, and be preferably mouse source small rubber particle protein antibodies, described second antibody is the antibody of first antibody.
8, the method according to any one of project 1 to 7, is characterized in that, described second antibody is golden labelled antibody.
9, the method according to any one of project 1 to 8, is characterized in that, described method comprises the steps:
(1) osmium tetroxide is made under room temperature, to fix 1 hour from the rubber latex of rubber plant with collection, obtain through the fixing rubber latex of osmium tetroxide, it is air-dry to the nickel screen of polyvinyl formal by the rubber latex of fixing through osmium tetroxide described in this then to drip a droplet;
(2) before sample is combined with first antibody, will be immersed in deionized water, PBS damping fluid and the PBS damping fluid containing bovine serum albumin (BSA), Triton100 successively through air-dry nickel screen;
(3) described nickel screen and first antibody is made at 37 DEG C fully in conjunction with 120 minutes;
(4) take out described nickel screen, drip PBS damping fluid, PBS damping fluid containing skimmed milk power and Tween 20 successively;
(5) described nickel screen and second antibody is made at room temperature fully in conjunction with 2 hours;
(6) nickel screen is washed with TBS-Tween 20, TBS successively, finally spend deionized water 4 times;
(7) air-dry for nickel screen rear use transmission electron microscope is observed.
10, the application in the strain examination that the method according to any one of project 1 to 9 is carried out for Rubber Yield on rubber plant especially Russian dandelion.
11, the application described in project 10, wherein, described strain examination is qualitatively screening and/or quantitatively examination.
[effect of invention]
The present invention utilizes labelled immune recognition technology, identify the rubber grain in rubber plant emulsion specifically, and by observing the Density Distribution of hevein label, achieve fast quantification and judge that the rubber grain content in rubber plant emulsion just and fast judges that rubber plant produces glue ability height, solves the crucial technical problem producing the examination of glue Russian dandelion strain.
[accompanying drawing explanation]
Fig. 1 is the transmission electron microscope photo of embodiment 1 (having first antibody).
Fig. 2 is the enlarged drawing of Fig. 1.
Fig. 3 is the transmission electron microscope photo of comparative example 1 (without first antibody).
Fig. 4 is the transmission electron microscope photo of embodiment 2 (having first antibody).
Fig. 5 is the transmission electron microscope photo of comparative example 2 (without first antibody).
Fig. 6 is colloidal goldlabeling electron microscopy principle schematic.
[embodiment]
The method of the rubber grain utilizing immuno-labelling technique to identify specifically in rubber plant emulsion of the present invention goes for rubber tree, dandelion, thorn lettuce, fig or guayule rubber.Method of the present invention is more suitable for Russian dandelion, is specially adapted to dandelion.
Method of the present invention comprises the steps:
(1) using the rubber latex extracted as testing sample, SRPP wherein is fully contacted with first antibody, make both combine;
(2) second antibody label being marked fully contacts with testing sample, makes both combine;
(3) use recognition technology to observe, identify natural rubber particle.
More particularly, method of the present invention comprises the steps:
(1) will the above-mentioned rubber plant emulsion crossing embrane method collection and purifying be utilized to use 1 quality % osmium tetroxide solution (osmium tetroxide is dissolved in 50mM sodium cacodylate buffer liquid), be placed in room temperature to fix for lower 1 hour, drip on a droplet osmium tetroxide solution to the nickel screen of polyvinyl formal air-dry;
(2) first antibody combine before (namely, before first antibody and sample (nickel screen with rubber grain) combine), nickel screen is immersed in successively in deionized water, PBS damping fluid and the PBS damping fluid containing 1 quality % bovine serum albumin (BSA), 0.5 volume %Triton100 (soaking 5min respectively in each liquid)
(3) SRPP antibody (mouse source) (first antibody) 1:200 is diluted, obtain dilution, nickel screen is fully contacted dilution described in 120min at 37 DEG C, the sample be loaded on nickel screen is combined with first antibody;
(4) nickel screen being loaded with sample is taken out, PBS damping fluid (50 μ L), the PBS damping fluid (50 μ L) containing 2 quality % skimmed milk powers and 0.5 volume %Tween 20 is dripped successively with pipettor, nickel screen is porous web, the nearly 4-5mm of diameter, after dripping, sample covers the whole surface of nickel screen.
(5) second antibody (taking first antibody as the sheep source antibody that the antigen obtains) 1:20 (volume ratio) that gold marks is diluted, obtain dilution, the nickel screen being loaded with sample is at room temperature contacted 2 hours with the described dilution containing second antibody, the first antibody be combined on sample is combined with second antibody;
(6) nickel screen being adsorbed with sample that (5) step obtains is used successively TBS-Tween 20 (consumption 50 μ L), TBS (consumption 50 μ L) washs, and finally spends deionized water (consumption 50 μ L) 4 times;
(7) by air-dry for the sample on nickel screen, nickel screen is loaded onto specimen holder and use transmission electron microscope (TEM) to observe rubber grain under the accelerating potential of 60/80kV.
Described first antibody can be, such as, and anti-SRPP antibody (small rubber particle protein antibodies, mouse source).The antibody of described second antibody to be second antibody be first antibody such as, take first antibody as the sheep source anti-mouse antibody that antigen obtains.Described label can be, such as, and nm of gold or other labels.
Described recognition technology can be various direct or indirect image recognition technologys, such as, uses various electron microscope (transmission electron microscope, scanning electron microscope etc.).Described labelled immune recognition technology is preferably immunoelectronmicroscopy.Described Electronic Speculum is preferably transmission electron microscope.
Described rubber plant is rubber tree or Russian dandelion, such as, be selected from the group be made up of Para rubber tree, dandelion, thorn lettuce, fig and guayule rubber any one.
Embrane method process can be crossed to vegetable rubber emulsion, such as, use 0.22 μm of filter membrane to process, to remove the impurity in vegetable rubber emulsion.Cross embrane method and use needle tubing and filter membrane.Cross embrane method can such as carry out in the following way: natural rubber latex is extracted into needle tubing (such as plastics needle tubing), take off syringe needle (such as metal needle), connect at the syringe needle place of needle tubing an aperture be micron-sized filter membrane (such as, 0.22um aperture), then rubber latex is pushed out, can filter effect be reached.Cross embrane method and there is the advantage that step is simple, amount of samples is few.
Crossing embrane method film used can be the polymeric membrane of inertia to solution and antibody, such as, and poly tetrafluoroethylene, polyvinylidene fluoride film, poly (ether sulfone) film etc.The aperture of described film can be less than 0.45 μm, preferably about 0.22 μm.
The principle of colloidal goldlabeling electron microscopy as shown in Figure 6.Specifically, natural rubber molecule synthesizes on the surface of natural rubber particle, and SRPP, on the surface of natural rubber particle, is closely connected with the synthesis of natural rubber.Utilize SRPP monoclonal antibody (first antibody) and SRPP specific bond, anti-and the first antibody specific bond of recycling gold mark two, the distribution of golden nanometer particle and density is observed to realize characterizing SRPP eventually through TEM, simultaneously characterization natural rubber grain specifically.
[embodiment]
Below with Taraxacum rubber emulsion for example, illustrate method of the present invention.
[material and reagent]
SRPP monoclonal antibody reagent (trade name Mouse Monoclonal Antibody) is purchased from IcosagenAS (Tartu, Estonia) company.Gold mark second antibody available from Sigma.All solution all also uses the following membrane filtration in 0.22 μm, aperture before use with ultrapure water preparation.
Filter membrane is the water system film that Tian Jinjin rises company's production, and material is PES (polyethersulfone), and starting material film is German MEMBRANA company film imported, and aperture is 0.22um.
Phosphate (PBS) damping fluid, osmium tetroxide, BSA, Triton 100, skimmed milk power, Tris, HCl, D-sorbite, NaCl, MgCl
2, DTT available from Sigma.
The nickel screen of polyvinyl formal is purchased from the emerging Bry Technology Co., Ltd. in Beijing.
Tem observation JEOL JEM-1230.
Taraxacum sp.1, Taraxacum sp.2 two kinds of Taraxacum rubber grass are all city of Beijing suburb field-collected strains.
Other reagent available from Sigma.
[collection of vegetable rubber emulsion and purifying]
0.22 μm is carried out to vegetable rubber emulsion to be measured and crosses film process, to remove the impurity in vegetable rubber emulsion.Cross embrane method and use membrane filtration vegetable rubber emulsion.Cross embrane method and use needle tubing and filter membrane.Cross embrane method can such as carry out in the following way: natural rubber latex is extracted into plastics needle tubing, take off metal needle, connecting an aperture at the syringe needle place of needle tubing is micron-sized filter membrane (0.22um aperture), is then pushed out by rubber latex, can reach filter effect.Cross embrane method and there is the advantage that step is simple, amount of samples is few.
Crossing embrane method film used can be the polyvinylidene fluoride film of inertia to solution and antibody.
[embodiment 1]
(1) will the above-mentioned Taraxacum sp.1 Taraxacum rubber emulsion crossing embrane method collection and purifying be utilized to use 1 quality % osmium tetroxide solution (osmium tetroxide is dissolved in 50mM sodium cacodylate buffer liquid), be placed in room temperature to fix for lower 1 hour, drip on a droplet osmium tetroxide solution to the nickel screen of polyvinyl formal air-dry;
(2) first antibody combine before (namely, before first antibody and sample (nickel screen with rubber grain) combine), nickel screen is immersed in successively in deionized water, PBS damping fluid and the PBS damping fluid containing 1 quality % bovine serum albumin (BSA), 0.5 volume %Triton100 (soaking 5min respectively in each liquid)
(3) SRPP antibody (mouse source) (first antibody) 1:200 is diluted, obtain dilution, nickel screen is fully contacted dilution described in 120min at 37 DEG C, the sample be loaded on nickel screen is combined with first antibody;
(4) nickel screen being loaded with sample is taken out, PBS damping fluid (50 μ L), the PBS damping fluid (50 μ L) containing 2 quality % skimmed milk powers and 0.5 volume %Tween 20 is dripped successively with pipettor, nickel screen is porous web, the nearly 4-5mm of diameter, after dripping, sample covers the whole surface of nickel screen.
(5) second antibody (taking first antibody as the sheep source antibody that the antigen obtains) 1:20 (volume ratio) that gold marks is diluted, obtain dilution, the nickel screen being loaded with sample is at room temperature contacted 2 hours with the described dilution containing second antibody, the first antibody be combined on sample is combined with second antibody;
(6) nickel screen being adsorbed with sample that (5) step obtains is used successively TBS-Tween 20 (consumption 50 μ L), TBS (consumption 50 μ L) washs, and finally spends deionized water (consumption 50 μ L) 4 times;
(7) by air-dry for the sample on nickel screen, nickel screen is loaded onto specimen holder and use transmission electron microscope (TEM) to observe rubber grain under the accelerating potential of 60/80kV.
The rubber grain with nano Au particle can be observed in embodiment 1 specifically clearly.
[comparative example 1]
Except (3) step saving embodiment 1, other steps are identical with embodiment 1.
Cannot observe in comparative example 1 specifically with the rubber grain of nano Au particle.
[embodiment 2]
Except rubber latex is changed to Taraxacum sp.2 Taraxacum rubber emulsion, the step identical with above-described embodiment 1 is adopted to carry out observing, identifying.
The rubber grain with nano Au particle can be observed in example 2 specifically.
[comparative example 2]
Except rubber latex is changed to Taraxacum sp.2 Taraxacum rubber emulsion, the step identical with above-mentioned comparative example 1 is adopted to carry out observing, identifying.
Cannot observe in comparative example 2 specifically with the rubber grain of nano Au particle.
Through the contrast of above-mentioned two kinds of embodiments and comparative example, method of the present invention can be observed target rubber grain specifically, qualitative, quantitative examination, and judges the product glue ability height of target rubber plant fast by the Density Distribution of protein marker on rubber grain.
In addition, the rubber grain in rubber plant (such as Para rubber tree) emulsion of known product glue ability can be observed by method of the present invention, and then compare with the rubber grain Density Distribution in target rubber plant emulsion, the product glue ability of quantitative forecast target rubber plant.
Claims (11)
1. observe a method for rubber grain, it is characterized in that, described method utilizes labelled immune recognition technology to carry out observing and identifying.
2. the method for claim 1, is characterized in that, described rubber grain is from rubber plant emulsion.
3. method as claimed in claim 1 or 2, it is characterized in that, described rubber plant is rubber tree or Russian dandelion, such as, be selected from group be made up of Para rubber tree, dandelion, thorn lettuce, fig and guayule rubber any one, preferred rubber is careless.
4. method as claimed any one in claims 1 to 3, it is characterized in that, described labelled immune recognition technology is immunoelectronmicroscopy, enzyme linked immunosorbent assay, fluorescence microscopy, radioimmunoassay labelling technique, albumen or gene chips or the test strips method based on sidestream immune principle.
5. the method according to any one of Claims 1-4, is characterized in that, described immunology recognition technology is immunoelectronmicroscopy.
6. the method according to any one of claim 1 to 5, is characterized in that, described method comprises the steps:
(1) to take from the rubber latex of rubber plant as testing sample, small rubber granule protein (SRPP) wherein is fully contacted with first antibody, make both combine;
(2) second antibody making label labeled fully contacts with the testing sample marking first antibody, makes both combine, obtains antibody labeling sample;
(3) antagonist mark sample carries out observing and identifying.
7. the method according to any one of claim 1 to 6, is characterized in that, described first antibody is the antibody stemming from small rubber particle albumen, and be preferably mouse source small rubber particle protein antibodies, described second antibody is the antibody of first antibody.
8. the method according to any one of claim 1 to 7, is characterized in that, described second antibody is golden labelled antibody.
9. the method according to any one of claim 1 to 8, is characterized in that, described method comprises the steps:
(1) osmium tetroxide is made under room temperature, to fix 1 hour with the rubber latex gathered from rubber plant (preferred rubber grass), obtain through the fixing rubber latex of osmium tetroxide, it is air-dry to the nickel screen of polyvinyl formal by the rubber latex of fixing through osmium tetroxide described in this then to drip a droplet;
(2) before sample is combined with first antibody, will be immersed in deionized water, PBS damping fluid and the PBS damping fluid containing bovine serum albumin (BSA), Triton100 successively through air-dry nickel screen;
(3) described nickel screen and first antibody is made at 37 DEG C fully in conjunction with 120 minutes;
(4) take out described nickel screen, drip PBS damping fluid, PBS damping fluid containing skimmed milk power and Tween 20 successively;
(5) described nickel screen and second antibody is made at room temperature fully in conjunction with 2 hours;
(6) nickel screen is washed with TBS-Tween 20, TBS successively, finally spend deionized water 4 times;
(7) air-dry for nickel screen rear use transmission electron microscope is observed.
10. the application as claimed in any one of claims 1-9 wherein in the strain examination carried out for Rubber Yield on rubber plant preferred rubber grass of method.
11. application according to claim 10, wherein, described strain examination is qualitatively screening and/or quantitatively examination.
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Cited By (1)
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| CN115558041A (en) * | 2022-02-28 | 2023-01-03 | 中国热带农业科学院橡胶研究所 | Method for improving molecular weight of natural rubber and product |
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| CN115558041A (en) * | 2022-02-28 | 2023-01-03 | 中国热带农业科学院橡胶研究所 | Method for improving molecular weight of natural rubber and product |
| CN115558041B (en) * | 2022-02-28 | 2023-12-22 | 中国热带农业科学院橡胶研究所 | Method for improving molecular weight of natural rubber and product |
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