CN106434848B - A kind of active method of measurement ribosome inactivating protein - Google Patents
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Abstract
The present invention provides a kind of active methods of measurement ribosome inactivating protein.The present invention also provides active developing solutions of a kind of measurement ribosome inactivating protein and application thereof.Ribosome inactivating protein measuring method of the invention is easy to operate, at low cost, only need spectrophotometer that can complete quantitative determination process, can avoid interference of traditional RNA enzyme to reaction simultaneously has extensive actual application prospect in the research and development field of ribosome inactivating protein.
Description
Technical field
The invention belongs to biochemical analysis detection technique fields, and in particular to a kind of measurement ribosome inactivating protein activity
Method.
Background technique
Ribosome inactivating protein (ribosome-inactivating protein, RIP) is that one kind can make eukaryocyte core
Sugared body inactivates and inhibits the toxalbumin of protein synthesis, is widely present in angiosperm, bacterium, fungi and algae.RIP
With the multiple biological activities such as broad-spectrum anti-tumor, antiviral, immunological regulation, and to normal cell toxicity very little, recent two decades
Widely to be paid close attention to and be studied in the whole world.
But there is an obvious problem to the exploitation of RIP at this stage --- still without a kind of simple and convenient and reliable side
Method measures the activity of RIP.Existing method substantially has following several: first, increased by measurement RIP extracorporeal suppression tumor cell
It grows to demarcate its activity, this method needs many and diverse cell cultivation process, and minute is long.Second, there is researcher with same position
The method measurement RIP of element label inhibits the effect of external cell free system synthetic proteins matter.This method is equally complicated for operation, and
And need to detect radioactive isotope and guarantee to pollute without RNA enzyme, it is more demanding to laboratory condition.Third, by rRNA and RIP
It after reaction, is handled with acid aniline, carries out denaturation RNA electrophoresis detection.But this method is also required to guarantee to interfere without RNA enzyme, and
And can only qualitatively it measure, it is difficult to quantitative.4th, after RIP is reacted with RNA, free adenine is detected using RP-HPLC method
Burst size.This method can be quantitative determined accurately, but equally exclude the interference of RNA enzyme, and RP-HPLC is primary only
As soon as a sample can be measured, if there is a large amount of samples to seem inconvenient when needing to measure.
The patent of Publication No. 104498494A discloses a kind of utilization active method of plasmids detection RIP, but this method
Need to detect the activity of RIP by agarose gel electrophoresis, quantitative effect is bad.RIP can be quantitative determined by urgently finding one kind
Active effective and short-cut method.
Summary of the invention
The purpose of the present invention is to provide a kind of active developing solution of measurement ribosome inactivating protein and methods.
The present invention provides a kind of developing solution for ribosome inactivating protein determination of activity, the developing solution is with concentration
It is basic solution for 0.05~0.2M, pH the HEPES-NaOH buffer for being 6.5~7.3 or MES-NaOH buffer, and adds
The adenosine deaminase of final concentration of 5~9U/mL, the 4-AA of 4~6g/L, 30~50mM N- ethyl-N- (2-
Hydroxyl -3- sulfopropyl) -3- methylaniline sodium salt, 9~14U/mL peroxidase and 5~10U/mL xanthine oxidase.
0.05~0.2M: unit M, that is, mol/L.
4- hydroxyethyl piperazineethanesulfonic acid: HEPES；
2- (N- morpholine) ethanesulfonic acid: MES；
2- (N- morpholine) ethanesulfonic acid-NaOH buffer: MES-NaOH buffer；
N- ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt: TOOS.
Wherein, it for 0.1M, pH MES-NaOH buffer for being 7.0 is basic solution that the developing solution, which is with concentration, and is added
N- ethyl-N- (2- hydroxyl-the 3- of the adenosine deaminase of final concentration of 6U/mL, the 4-AA of 5g/L, 40mM are added
Sulfopropyl) -3- methylaniline sodium salt, 9U/mL peroxidase and 6U/mL xanthine oxidase；
Or, it for 0.1M, pH MES-NaOH buffer for being 7.0 is basic solution that the developing solution, which is with concentration, and add
The adenosine deaminase of final concentration of 5U/mL, the 4-AA of 4g/L, 35mM N- ethyl-N- (2- hydroxyl -3- sulphur
Propyl) -3- methylaniline sodium salt, 10U/mL peroxidase and 8U/mL xanthine oxidase；
Or, it for 0.1M, pH MES-NaOH buffer for being 7.0 is basic solution that the developing solution, which is with concentration, and add
The adenosine deaminase of final concentration of 8U/mL, the 4-AA of 5g/L, 50mM N- ethyl-N- (2- hydroxyl -3- sulphur
Propyl) -3- methylaniline sodium salt, 12U/mL peroxidase and 10U/mL xanthine oxidase；
Or, it for 0.2M, pH HEPES-NaOH buffer for being 7.3 is basic solution that the developing solution, which is with concentration, and add
N- ethyl-N- (2- hydroxyl-the 3- of the adenosine deaminase of final concentration of 9U/mL, the 4-AA of 6g/L, 40mM are added
Sulfopropyl) -3- methylaniline sodium salt, the peroxidase of 13U/mL, 8U/mL xanthine oxidase.
It wherein, further include the NaN of final concentration of 0.3~0.5g/L in the developing solution3With the Triton X- of 1~2mL/L
100；
It further, further include the NaN of final concentration of 0.3g/L in the developing solution3With the Triton X-100 of 2mL/L.
The present invention also provides purposes of the above-mentioned developing solution in ribosome inactivating protein determination of activity.
The present invention also provides a kind of active methods of measurement ribosome inactivating protein, it includes the following steps:
A. ribosome inactivating protein or the solution comprising ribosome inactivating protein are taken, as sample to be tested；
B. ribosome inactivating protein or the solution comprising ribosome inactivating protein are taken, 2~3min is boiled, as blank pair
According to；
C. sample to be tested, blank control are separately added into the substrate solution that isometric concentration is 40~80mM, mixed, 50
It is incubated for 20~35min under the conditions of~55 DEG C, then boils 3min, 5000~10000g is centrifuged 1~2min, takes supernatant；
Wherein, the substrate solution is formulated as follows: weighing rRNA analog, with concentration be 0.01~0.05M, pH is
2.0~3.0 buffer solution and dilution, the buffer is disodium hydrogen phosphate-citrate buffer solution, sodium hydroxide-lemon
Acid-hydrochloride buffer, glycine-HCI buffer or acetic acid-sodium acetate buffer solution；
The rRNA analog is that the compound for releasing adenine can be catalyzed by ribosome inactivating protein；
D. the above-mentioned developing solution of 3 times of volumes is added in the supernatant for taking step c, mixes, 35~45 DEG C of 25~40min of incubation；
E. with blank control zeroising, spectrophotometer method measures light of the sample to be tested under 500~600nm wavelength condition
Absorption value；
F. according to absorbance value, enzyme activity is calculated.
Solution comprising ribosome inactivating protein: it such as can be ribosome inactivating protein crude enzyme liquid.
The visible or ultraviolet-uisible spectrophotometer of absorbance value, common cuvette or microcolorimetric ware are measured, with blank
Pipe zeroising measures absorbance value.
Wherein, in step a and/or b, ribosome inactivating protein is α-Charantin, MAP30, trichosanthin.
Wherein, in step a, ribosome inactivating protein or crude enzyme liquid are diluted, keep sample to be tested anti-according to step a-e
Ying Hou, the absorbance value under 500~600nm wavelength condition are controlled 0.1~0.4；
Further, retarder thinner is disodium hydrogen phosphate-citric acid that concentration is 0.01~0.05M, pH is 2.0~3.0
Buffer, sodium hydroxide-citrate-hcl buffer or acetic acid-sodium acetate buffer solution.
Wherein, in step c, before sample to be tested or blank control are mixed with substrate solution, sample to be tested, blank control, bottom
Object solution is respectively at 50~55 DEG C, 2~5min of lower incubation；
And/or in step c, sample to be tested, blank control are separately added into substrate solution, mix, incubation time be 25~
35min；Then 3min is boiled, centrifugal condition is that 8000g/min is centrifuged 1min；
And/or in step c, the rRNA analog is AMP, NAD+Or NADH.
Wherein, in step d, incubation temperature is 40 DEG C, and incubation time is 25~30min；
In step e, the wavelength is 555nm.
Ribosome inactivating protein active unit is defined as: under this experiment condition, catalysis substrate per minute discharges 1 μm of ol gland
The enzyme amount of purine is 1U.
Wherein, in step f, the method for calculating enzyme activity is as follows:
The relative activity of analyte sample fluid (ribosome inactivating protein) is calculated according to following formula:
Δ A: sample absorbance value variable quantity；0.2142: under 555nm wavelength, the corresponding adenine concentration (mM) of 1A；
Vt: reaction total volume (mL)；103: 1mM=103μM；df: enzyme solution extension rate；T: the accurate response time of enzyme and substrate
(min)；Ve: enzyme solution volume (mL)；D: cuvette optical path (1cm)；Vr: the volume (mL) to react with conjugate enzyme developing solution；C:
Protein concentration (mg/mL).
Each buffer according to the present invention the preparation method is as follows:
1, the MES-NaOH buffer that 0.05~0.2M, pH are 6.5~7.3
The MES solution of 0.3M is prepared, then pH to 7.0-7.3 is adjusted with sodium hydroxide, uses ddH2O is diluted to corresponding dense
Degree.
2, the HEPES-NaOH buffer that 0.05~0.2M, pH are 6.5~7.3
Compound concentration is the HEPES solution of 0.3M, then adjusts pH to 6.5-7.3 with sodium hydroxide, uses ddH2O is diluted to
Corresponding concentration.
3, disodium hydrogen phosphate-citrate buffer solution that concentration is 0.01~0.05M, pH is 2.0~3.0
The sodium dihydrogen phosphate and citric acid solution for preparing 0.01~0.05M respectively survey pH up to 2.0 after being mixed in a certain proportion
~3.0.
4, the sodium hydroxide that concentration is 0.01~0.05M, pH is 2.0~3.0-citrate-hcl buffer citric acid
Add ddH after 21g, NaOH8.4g and concentrated hydrochloric acid 16mL mixing2O is dissolved to 1000ml.
5, the acetic acid-sodium acetate buffer solution that concentration is 0.01~0.05M, pH is 2.0~3.0
The acetic acid and sodium acetate solution for preparing 0.01M~0.05M respectively, take appropriate acetic acid solution, are gradually added sodium acetate
Solution measures with pH meter and controls mixed liquor pH value, stops that sodium acetate solution is added as pH=2, only when for 0.01~
The acetic acid-sodium acetate buffer solution of 0.05mol/L, pH 2.0.
Developing solution provided by the invention can be used for the Activity determination of ribosome inactivating protein, and the method for the present invention can be effective
The activity for measuring ribosome inactivating protein, is quick on the draw, accurately and reliably, and can avoid interference of traditional RNA enzyme to reaction；Separately
Outer operation of the present invention is simple, low in cost, only needs spectrophotometer that can complete quantitative determination process.
It can be used to analyze using the method for the present invention to compare the active size of separate sources RIP；It separates and purified in RIP
The whereabouts of Cheng Zhong, detection or tracking different phase destination protein or active recovering state；Detect ribosomes in plant, microorganism etc.
The expression situation of inactivating protein gene；Evaluate the work before and after RIP structure of modification (such as modification of polyethylene glycol chemistry, rite-directed mutagenesis)
Property variation etc., in the research and development field of RIP, there is extensive actual application prospect.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 is the silica gel thin sheet chromatography that three kinds of ribosome inactivating proteins substrate reactions similar with five kinds of rRNA generate adenine
Map figure；
A is standard items, and each road substrate is respectively 1.AMP；2.ATP；3.NAD+；4.NADH；5.NADP+；6. adenine；B is
MAP30 is first boiled inactivation by control group, then with each substrate reactions.Each road substrate is respectively 1.AMP；2.ATP；3.NAD+；
4.NADH；5.NADP+；C be reaction group, by MAP30 respectively with various substrates, in 55 DEG C of reaction 2h.Each road substrate is respectively
1.AMP；2.ATP；3.NAD+；4.NADH；5.NADP+；6 be the adenine of release.
Fig. 2 is standard items NAD+With chromatography map of the standard items adenine on RP-HPLC；
A is NAD+Standard items；B is adenine standard items.
Fig. 3 is under the conditions of pH2.0,4.0 and 6.0, MAP30 and NAD+The RP-HPLC testing result figure of reaction product；
A:pH2.0；B:pH 4.0；C:pH 6.0.
Fig. 4 is under the conditions of pH2.0,4.0 and 6.0, α-MMC and NAD+The RP-HPLC testing result figure of reaction product；
A:pH2.0；B:pH 4.0；C:pH 6.0.
Fig. 5 is the working curve diagram that high concentration adenine is reacted with conjugate enzyme developing solution.
Fig. 6 is the working curve diagram that low concentration adenine is reacted with conjugate enzyme developing solution.
Fig. 7 is pH to the active influence diagram of ribosome inactivating protein；
A: α-MMC；B:MAP30；C:TCS.
Fig. 8 is ribosome inactivating protein concentration to catalyzing hydrolysis NAD+Discharge adenine influence diagram；
A: α-MMC；B:MAP30；C:TCS.
Fig. 9 is to be catalyzed NAD to RIP in the reaction time+Discharge the influence diagram of adenine.
Figure 10 is NAD+Concentration is to the active influence diagram of ribosome inactivating protein.
Figure 11 is that temperature is catalyzed NAD to RIP+Discharge the influence diagram of adenine；
A: α-MMC；B:MAP30；C:TCS.
Specific embodiment
It is described further below with embodiment, but the present invention is not limited to these embodiments.
Major experimental reagent and instrument used in the present invention are as follows:
Reagent: 2- (N- morpholine) ethanesulfonic acid or 4- hydroxyethyl piperazineethanesulfonic acid, N- ethyl-N- (2- hydroxyl -3- sulphur third
Base) -3- methylaniline sodium salt (TOOS), AMP, ATP, NAD+、NADH、NADP+For Sigma reagent, 4-AA is wheat
Crin reagent；Xanthine oxidase, adenosine deaminase or adenine deaminase, peroxidase are that upper sea blue garden bioengineering has
Limit Products；Other reagents are A.R grades domestic (analysis is pure).
Instrument: UV-2102C/PC/PCS type spectrophotometric is calculated as UNICO's product.
The preparation of the developing solution of the present invention of embodiment 1
Developing solution used in the present invention is the coupling enzymatic reagent for detecting adenine content, and developing solution is using preceding fresh super
Pure water is prepared, and is in colourless transparent solution, 4 DEG C of preservations, if containing at least three moon stabilizer validity period.If discovery is empty in measurement
When the absorbance value of white pipe is greater than 0.01, then it should prepare again.
Conjugate enzyme developing solution is formulated as follows:
The MES-NaOH buffer for taking 0.1M, pH7.0, sequentially add TOOS, 4-AA, peroxidase,
Adenosine deaminase, xanthine oxidase are eventually adding MES-NaOH buffer constant volume, so that containing final concentration of in developing solution
The 4-AA of TOOS, 5g/L of 40mM, the peroxidase of 9U/mL, the adenosine deaminase of 6U/mL, 6U/mL Huang are fast
Purine oxidizing ferment.Developing solution as of the invention, places it in brown bottle, saves backup in 4 DEG C.
The preparation of the developing solution of the present invention of embodiment 2
The MES-NaOH buffer for taking 0.1M, pH7.0, sequentially add TOOS, 4-AA, peroxidase,
Adenosine deaminase, xanthine oxidase are eventually adding MES-NaOH buffer constant volume, so that containing final concentration of in developing solution
The 4-AA of TOOS, 4g/L of 35mM, the peroxidase of 10U/mL, the adenosine deaminase of 5U/mL, 8U/mL are yellow
Purine oxidase.Developing solution as of the invention, places it in brown bottle, saves backup in 4 DEG C.
The preparation of the developing solution of the present invention of embodiment 3
The HEPES-NaOH buffer for taking 0.2M, pH7.3, sequentially adds TOOS, 4-AA, peroxide
Enzyme, adenosine deaminase, xanthine oxidase are eventually adding HEPES-NaOH buffer constant volume, so that containing dense eventually in developing solution
Spend adenosine deaminase, the 8U/ of the 4-AA of TOOS, 6g/L for being 40mM, the peroxidase of 13U/mL, 9U/mL
ML xanthine oxidase.Developing solution as of the invention, places it in brown bottle, saves backup in 4 DEG C.
The preparation of the developing solution of the present invention of embodiment 4
The MES-NaOH buffer for taking 0.1M, pH7.0, sequentially add TOOS, 4-AA, peroxidase,
Adenosine deaminase, xanthine oxidase, NaN3With Triton X-100, it is eventually adding MES-NaOH buffer constant volume, so that aobvious
The gland of the 4-AA of TOOS, 5g/L, the peroxidase of 9U/mL, 6U/mL in color liquid containing final concentration of 40mM
Guanosine deaminase, 6U/mL xanthine oxidase, 0.3g/L NaN3, 2mL/L Triton X-100.Colour developing as of the invention
Liquid places it in brown bottle, saves backup in 4 DEG C.
5 ribosome inactivating protein determination of activity-α of embodiment-Charantin (α-MMC)
The working principle of the method for the present invention (the active method of conjugate enzyme determination of color ribosome inactivating protein) are as follows: according to
Ribosome inactivating protein (RIP) is catalyzed AMP, NAD+The characteristics of being broken with N- glycosidic bond in NADH and releasing product-adenine,
Again through adenosine deaminase, xanthine oxidase and peroxidase and auxiliary reagent: N- ethyl-N- (2- hydroxyl -3- sulphur third
Base) compositions such as -3- methylaniline sodium salt (TOOS), 4-AA (4-AA) chromogenic reagent, measure color product
Absorbance value, can reflect the bioactivity of ribosome inactivating protein indirectly.Shown in the principle is as follows:
Operating method is as follows:
A. the preparation of substrate: suitable NAD is weighed+Powder is acetic acid-acetate buffer of 0.04M, pH2.5 with concentration
Liquid dissolves and concentration is made to reach 40mM.
B. the dilution of sample to be tested: taking testing protein sample α-Charantin (α-MMC), is control with bovine serum albumin(BSA),
Measurement protein content is 0.5mg/mL.
C. test sample: by sample to be tested and substrate solution, 2min is preheated respectively in 55 DEG C of water-baths, then respectively takes 100 μ
L mixing, is immediately placed on 55 DEG C of accurate response 30min, boils 3min immediately and terminates reaction.Reaction solution 8000g is centrifuged 1min, is received
Collect supernatant.
Blank tube: first enzyme is inactivated in 100 DEG C of processing 2min, then plus substrate.Other same sample cells of process.
D. process color: taking 100 μ L of supernatant and 300 μ L developing solutions, (developing solution is prepared: the MES-NaOH of 0.1M, pH7.0
Buffer, adenosine deaminase 5U/mL, 4-AA 4g/L, TOOS35mM, peroxidase 10U/mL and xanthine oxidase
Change enzyme 8U/mL).30min is kept the temperature in 37 DEG C of water-baths.
E. continuous mode:
Optical path is 1cm cuvette, and with blank tube zeroising, measuring absorption value of the sample cell under 555nm wavelength is 0.24.
The enzymatic activity of analyte sample fluid: U/mL=34U, U/mg=70U is calculated according to following formula
Δ A: sample absorbance value variable quantity；0.2142: under 555nm wavelength, the corresponding adenine concentration (mM) of 1A；
Vt: reaction total volume (mL)；103: 1mM=103μM；df: enzyme solution extension rate；T: the accurate response time of enzyme and substrate
(min)；Ve: enzyme solution volume (mL)；D: cuvette optical path (1cm)；Vr: the volume (mL) to react with conjugate enzyme developing solution；C:
Protein concentration (mg/mL).
Ribosome inactivating protein α-MMC (α-Charantin) used in the present embodiment can be obtained by purchase commercial product,
Present literature procedure preparation can be used, the present invention is prepared with the following method:
α-MMC is extracted from the white seed of bitter gourd of Lanshan County head, method are as follows: seed of bitter gourd is peeled off and takes out bitter melon seed, by powder
Broken, extracting, acidification, neutralization, ammonium sulfate precipitation, dialysis, cation-exchange chromatography, sieve chromatography combine Blue-
Sepharose affinity chromatography obtains.Obtained α-MMC borrows SDS-PAGE, Acid PAGE, IEF-PAGE and MALDI-TOF-
TOF and N- end sequencing is identified.Protein content is measured using bovine serum albumin(BSA) as standard using Lowry method.
6 ribosome inactivating protein determination of activity-MAP30 of embodiment
Operating method is as follows:
A. the preparation of substrate: appropriate NAD is weighed+Powder is disodium hydrogen phosphate-citric acid of 0.05M, pH3.0 with concentration
Buffer solution simultaneously makes NAD+Concentration reaches 50mM.
B. the dilution of sample to be tested: taking testing protein sample MAP30, and being diluted to concentration with distilled water is 0.1mg/mL.
C. 100 μ L samples to be tested and 100 μ L substrates reaction process: are preheated into 3min respectively at 52 DEG C of incubations.Then it respectively takes
100 μ L mixing, is immediately placed on 52 DEG C of accurate response 30min, boils 3min immediately and terminates reaction.Blank tube: by enzyme at 100 DEG C
Manage 2min inactivation, then plus substrate.Other same sample cells of process.
D. process color: being centrifuged 1min for the reaction solution 5000g of previous step, and 50 μ L of supernatant and 150 μ L developing solutions is taken to mix
It closes, 35 DEG C of water-baths keep the temperature 35min；
The conjugate enzyme developing solution is grouped as by the group of following concentration: MES-NaOH buffer, the 4- amino of 0.1MpH7.0
Antipyrine 5g/L, peroxidase 12U/mL, adenosine deaminase 8U/mL, xanthine oxidase 10U/mL and TOOS 0.05M.
E. continuous mode: optical path is 1cm cuvette, with blank tube zeroising, measures suction of the sample cell under 550nm wavelength
Receipts value is 0.21.
The enzymatic activity of analyte sample fluid: U/mL=30U, U/mg=310U is calculated according to following formula
Δ A: sample absorbance value variable quantity；0.2142: under 555nm wavelength, the corresponding adenine concentration (mM) of 1A；
Vt: reaction total volume (mL)；103: 1mM=103μM；df: enzyme solution extension rate；T: the accurate response time of enzyme and substrate
(min)；Ve: enzyme solution volume (mL)；D: cuvette optical path (1cm)；Vr: the volume (mL) to react with conjugate enzyme developing solution；C:
Protein concentration (mg/mL).
Ribosome inactivating protein MAP30 used in the present embodiment (Momordica Antiviral Protein30kD, it is bitter
Melon anti-AIDS toxalbumin) it can be obtained by purchase commercial product, present literature procedure preparation can also be used, the present invention adopts
It prepares with the following method:
MAP30 is extracted from commercially available seed of bitter gourd, method are as follows: seed of bitter gourd is peeled off and takes out bitter melon seed, by crushing, taking out
It mentions, be acidified, neutralizing, ammonium sulfate precipitation, dialysis, cation-exchange chromatography, sieve chromatography, the last desalination of cation chromatography
And degerming is dispensed, obtained MAP30 reflects by the methods of SDS-PAGE, Acid PAGE, IEF-PAGE, MALDI-TOF-TOF
It is fixed.Protein content is measured with Lowry method, using bovine serum albumin(BSA) as standard, makes standard protein curve.
7 ribosome inactivating protein determination of activity of embodiment-trichosanthin (TCS)
Operating method is as follows:
A. the preparation of substrate: suitable NAD is weighed+Powder is acetic acid-acetate buffer of 0.02M, pH3.0 with concentration
Liquid dissolves and concentration is made to reach 45mM.
B. the dilution of sample to be tested: trichosanthin to be measured (TCS) is taken, is diluted to 0.5mg/mL with distilled water.
C. 100 μ L samples to be tested and 100 μ L substrates reaction process: are preheated into 3min respectively at 55 DEG C of incubations.Then it respectively takes
100 μ L mixing, is immediately placed on 55 DEG C of accurate response 30min, boils 3min immediately and terminates reaction.Blank tube: by enzyme at 100 DEG C
Manage 2min inactivation, then plus substrate.Other same sample cells of process.
D. process color: the reaction solution 6000g of previous step is centrifuged 1min, takes the developing solution of supernatant 100 μ L and 300 μ L
37 DEG C of water-baths keep the temperature 35min；
The conjugate enzyme developing solution is grouped as by the group of following concentration: HEPES-NaOH buffer, the gland of 0.2M, pH7.3
Guanosine deaminase 9U/mL, 4-AA 6g/L, TOOS 40mM, peroxidase 13U/mL, xanthine oxidase 8U/
mL、NaN30.3g/L and Triton X-100 2mL/L.
E. continuous mode: optical path is 1cm cuvette, with blank tube zeroising, measures suction of the sample cell under 550nm wavelength
Receipts value is 0.22.
It is U/mL=34U, U/mg=65U according to the enzymatic activity that analyte sample fluid is calculated in following formula.
Δ A: sample absorbance value variable quantity；0.2142: under 555nm wavelength, the corresponding adenine concentration (mM) of 1A；
Vt: reaction total volume (mL)；103: 1mM=103μM；df: enzyme solution extension rate；T: the accurate response time of enzyme and substrate
(min)；Ve: enzyme solution volume (mL)；D: cuvette optical path (1cm)；Vr: the volume (mL) to react with conjugate enzyme developing solution；C:
Protein concentration (mg/mL).
Ribosome inactivating protein trichosanthin (TCS) used in the present embodiment can be obtained by purchase commercial product,
Present literature procedure preparation can be used, the present invention is prepared with the following method:
TCS is isolated and purified from Snakegourd Fruit stem tuber, and following steps are passed through in purifying:
(1) it crushes: the Snakegourd Fruit stem tuber dried being first ground into fritter with mortar, is then broken into powder, mistake with pulverizer
80 mesh steel sieve, collects powder.
(2) extract: the phosphate buffer that addition is equivalent to 5~6 times of volumes of powder quality (is containing 0.15MNaCl, concentration
The phosphate buffer of 50mM, pH6.0), it is stirred overnight, the filtering of nylon cloth.
(3) ammonium sulfate fractionation separates: 12000g is centrifuged 10min, collects supernatant.Magnetic agitation is slowly added to sulfuric acid at 4 DEG C
Ammonium powder makes ammonium sulfate saturation degree up to 80% in supernatant, and after standing 2h, 12000g is centrifuged 20min, discards supernatant and recycles
Precipitating.
(4) dialyse: the concentration by precipitating minimum volume is that 10mM, pH6.0 phosphate buffer sufficiently dissolve, and is packed into retention
In the bag filter of molecular weight 10000, ammonium sulfate is removed to the phosphate buffer dialysis that concentration is 10mM, pH6.0.
(5) ion-exchange chromatography: removing undissolved impurity for dialysis sample 12000g centrifugation 5min, then will be on sample
SP-Sepharose F.F. chromatographic column uses the same buffer elution of 0.1MNaCl instead with the abundant column scrubber of same buffer,
Collect protein peak.
(6) affinity chromatography: slow with pH6.0 30mM phosphoric acid by Blue-sepharose F.F. chromatographic column on upper step sample
Then the abundant column scrubber of fliud flushing uses NaCl containing 0.5M, the elution of concentration 50mM, pH6.0 phosphate buffer instead, collects protein peak.
Obtained TCS is identified by SDS-PAGE, Acid PAGE and MALDI-TOF-TOF and Lowry method is used to measure albumen
Content.
Illustrate beneficial effects of the present invention with the mode of test example below:
Screening test of 1 ribosome inactivating protein of test example to rRNA analog substrate
One, test material
Testing protein sample α-MMC, MAP30 and TCS used in Example 5-7 respectively.
Two, silica gel thin sheet chromatographic assays screen rRNA analog substrate
1. the activation of silica gel thin sheet
Long 20cm silica gel plate is placed in 110 DEG C of baking 2h in baking box, is put into drier after its cooling stand-by.
2. the preparation of ribosome inactivating protein and rRNA analog reaction product
NAD is weighed respectively+、NADH、NADP+, AMP, ATP powder, with disodium hydrogen phosphate-citric acid of 0.1M, pH3.0
Buffer solution makes the concentration of every kind of substrate be 40mM.
α-the MMC of preparation, MAP30 and TCS solution are diluted to 2mg/mL, 0.4mg/mL and 2mg/ with distilled water respectively
mL.It takes 50 μ L RIP solution to mix with 50 μ L substrate solutions, is placed in insulation reaction 2h in 55 DEG C of water-baths.
Blank control group is to take corresponding RIP solution 50 μ L, 100 DEG C of processing 3min simultaneously, adds 50 μ L substrates, 55 DEG C
Keep the temperature 2h.
3. silica gel thin sheet chromatographs
Standard items, blank control and each 10 μ L of reaction mixture are taken with microsyringe, is put respectively from silica gel thin-layer
At plate lower edge 0.6cm, point of sample spacing distance is 1.0cm.Silica gel plate inclination is put into chromatography cylinder, an end margin of point sample
Immerse ascending development in solvent.The composition of solvent is n-butanol: water: acetic acid=5:2:3 (v/v).Solvent ascending development
Silica gel plate is taken out from chromatography cylinder when to apart from silica gel plate top edge 0.5cm, and is dried up with hair dryer cold wind, in ultra-violet analysis
It is observed under instrument, records speckle displacement, make schematic diagram.As a result as shown in figure 1 and table 1.
Table 1: thin plate chromatography detects three kinds of ribosome inactivating proteins and is catalyzed five kinds of rRNA similar to substrate generation adenine ability
Note: "+" indicates adenine generation；"-" indicates that no adenine generates.How much expressions of "+" observe that gland is fast
The power of purine spot.
By table 1 and Fig. 1 it is found that three kinds of ribosome inactivating proteins are equal after the small molecule substrates reaction similar with five kinds of rRNA
Energy and NAD+, NADH and AMP react and hydrolyze the N- glycosidic bond in substrate and release adenine, qualitatively observe three kinds of enzymes
Albumen and NAD+The maximum amount of adenine can be released when reaction, NADH takes second place, and AMP is minimum；And three kinds of ribosome inactivating proteins
With ATP and NADP+The generation of adenine is not detected in reaction product,
Illustrate NAD+, NADH and AMP can be used as the rRNA analog substrate of ribosome inactivating protein zymetology reaction,
In, especially NAD+Catalytic effect when for substrate is best.
Three, RP-HPLC (reversed-phase high performance liquid chromatography) method detects
1. the preparation of ribosome inactivating protein substrate reactions product similar with rRNA
Weigh NAD+Powder makes its concentration 40mM with different pH0.1M buffer solution powder.Different pH buffers point
Not are as follows: pH2.0 glycine-HCI buffer；PH4.0 acetic acid-sodium acetate buffer solution；PH6.0 disodium hydrogen phosphate-sodium dihydrogen phosphate
Buffer.
α-MMC and MAP30 sample, respectively 1mg/mL and 0.2mg/mL are diluted with distilled water.Take two kinds of albumen of 0.1mL molten
The liquid NAD with 0.1mL difference pH buffer system respectively+Solution mixing, is incubated in 55 DEG C immediately, after accurate response 1h, boils
3min terminates reaction, and subsequent 8000g is centrifuged 1min.The cold dehydrated alcohol of 0.5ml is added in reaction solution, mixes well, is placed in 4 DEG C
30min is stood in refrigerator, 12000g is centrifuged 5min, and it is spare that supernatant is sucked out.
2.RP-HPLC detection
Mobile phase is pH7.5 40mM borax-phosphate sodium dihydrogen buffer solution containing 32% (v/v) methanol, and with 0.45 μm
Film filtering and ultrasonic wave degassing.Reverse-phase chromatographic column is Grace Smart C18 column (150mm × 46mm).First with flowing before sample introduction
Chromatographic column is mutually rinsed until baseline is steady, flow velocity 0.5mL/min, sampling volume is 20 μ L, and on-line checking device Detection wavelength is set
It is set to 260nm.Standard items are 10mM NAD+With 10mM adenine solution (solvent: acetic acid-acetate buffer of pH2.0,0.1M
Liquid), other processing are same as above.Tomographic results are as shown in Figure 2, Figure 3, Figure 4.
As shown in Figure 2, NAD+The retention time of standard items is 3.2min, and the retention time of adenine standard items is
5.7min.MAP30 and α-MMC is reflected in Fig. 3 and Fig. 4 respectively and is catalyzed NAD under condition of different pH+Discharge the detection of adenine
As a result.As can be seen that α-MMC and MAP30 can be catalyzed NAD+Adenine is released, retention time and the gland for discharging adenine are fast
Purine standard items are consistent；As pH value in reaction increases, it is catalyzed the adenine activity released and gradually decreases.
To sum up, ribosome inactivating protein can be catalyzed NAD+, NADH and AMP the enzyme hydrolysis of N- glucosides and release free
Adenine, NAD+, NADH and AMP can be used as the rRNA analog substrate of ribosome inactivating protein zymetology reaction, wherein it is outstanding
Its NAD+Catalytic effect when for substrate is best.
The sensitivity technique of the developing solution of the present invention of test example 2
(1) conjugate enzyme developing solution
The MES-NaOH buffer of 10mL 0.2M, pH7.0
160μL TOOS
160 μ L 4-AA, 160 μ L peroxidase (1U/ μ L)
100 μ L adenosine deaminases (1U/ μ L)
100 μ L xanthine oxidases (1U/ μ L)
It is sequentially added by said sequence, is settled to 15mL with MES-NaOH buffer.TOOS is final concentration of in developing solution
40mM, the final concentration of 5g/L of 4-AA.
Developing solution is placed in brown bottle, 4 DEG C of preservations.
(2) standard adenine
First weigh suitable adenine powder, dissolved with distilled water and be allowed to concentration and reach 0.5mM, then according to table 2 into
Row dilution obtains the adenine aqueous solution of various concentration.
The process for preparation of 2 various concentration adenine aqueous solution of table
(3) reaction process and detection
The adenine of 50 μ L various concentrations is taken to mix with 150 μ L conjugate enzyme developing solutions, 37 DEG C of water-baths keep the temperature 20min.With point
Its absorbance value at 550nm of light photometric determination.With adenine concentration to A550nmMapping, as adenine working curve.
As a result as shown in Fig. 5 (low concentration), Fig. 6 (high concentration), it will thus be seen that no matter adenine is in low concentration, or
High concentration, R2Value can reach > 0.99.Show to show good linear relationship；For adenine in low concentration, minimum detection is dense
Degree is 0.025mM, and at high concentrations, maximum detectable concentration is 0.2mM.Illustrate that developing solution of the invention can convert completely RIP and urge
Change the adenine of hydrolysis rRNA substrate analogue release, the high sensitivity of developing solution of the present invention.
The Accuracy Verification of 3 the method for the present invention of test example
1, pH is on the active influence of ribosome inactivating protein
(1) NAD is weighed+Powder makes its concentration 40mM with the buffer solution powder of suitable difference pH.Different pH,
The buffer of 0.01M is respectively as follows: pH2.0 to pH7.0 disodium hydrogen phosphate-citrate buffer solution；PH8.0~pH9.0 is barbital-
Hydrochloride buffer.
(2) mother liquor for taking suitable α-MMC, MAP30 and TCS, is diluted with distilled water respectively, makes to measure after end reaction
OD555nmReach 0.1-0.5.50 μ L protein solutions are added in the different pH solution of 50 μ L difference substrates, and are placed in 55 DEG C at once
It is kept the temperature in water-bath, accurate response 30min, boils 3min at once and terminate reaction.
Blank control group is that protein solution is first boiled 2min to be allowed to inactivate, and other operating process are same as above.
(3) the reaction solution 6000g for boiling termination is centrifuged 1min, draws the coupling of 50 μ L reaction solutions and 150 μ L embodiments 1
The mixing of enzyme developing solution, keeps the temperature 25min in 37 DEG C of water-baths.It is to use in the micro quartz colorimetric utensil of 1cm that reaction solution, which is transferred to optical path,
Ultra-violet and visible spectrophotometer measures its absorbance value at 555nm, with the relative activity mapping under different pH.
As a result as shown in Figure 7.
As seen from Figure 7, three kinds of ribosome inactivating proteins and NAD+It is reacted under condition of different pH, they can be catalyzed
NAD+Adenine is discharged, activity is reduced with the raising of pH value.Therefore, selected pH for the slow of 2-3 in the method for the present invention
Fliud flushing, ribosome inactivating protein activity is best at this time.
2, ribosome inactivating protein concentration is to catalysis NAD+Discharge the influence of adenine
A. preparation of reagents
Various concentration RIP: α-MMC and TCS mother liquor are diluted to distilled water respectively 0.05mg/mL, 0.1mg/mL,
0.2mg/mL, 0.5mg/mL, 0.7mg/mL and 1mg/mL；By MAP30 mother liquor be diluted to 0.01mg/mL, 0.03mg/mL,
0.05mg/mL, 0.1mg/mL, 0.15mg/mL and 0.2mg/mL.
The preparation of substrate: disodium hydrogen phosphate-citrate buffer solution of 0.01M, pH3.0 are solvent, make NAD+ 40mM.
B. reaction process
The RIP solution of 100 μ L various concentrations is taken to mix with 100 μ L substrate solutions, immediately in 55 DEG C of accurate response 30min,
3min is boiled immediately and terminates reaction, and reaction solution 8000g is centrifuged 1min, collects supernatant.
C. process color
100 μ L supernatants and the developing solution of 300 μ L embodiments 1 are taken to mix, 37 DEG C of water-baths keep the temperature 25min.
D. continuous mode
With spectrophotometer, optical path is the micro quartz colorimetric utensil of 1cm, measures the absorption value under 500nm wavelength.With A500
It maps to various concentration RIP, as a result as shown in Figure 8.
According to enzyme kinetics principle, enzyme concentration is one of principal element for influencing enzymatic reaction speed and enzyme
Promote one of the notable feature of kinetics.I.e. under given conditions, increase as enzyme concentration increases reaction speed, that is,
It says, enzyme concentration is proportional to reaction speed.
As seen from Figure 8, the ribosome inactivating protein catalyzing hydrolysis NAD of various concentration+Line is presented in the adenine amount released
Property proportional relation, result meet enzymatic reaction move educational level feature.
Therefore, the method for the present invention can measure the activity of various concentration ribosome inactivating protein, as a result accurately and reliably.
3, the reaction time is catalyzed NAD to RIP+Discharge the influence of adenine
Dilute α-MMC, MAP30 and TCS respectively with distilled water to 1mg/mL, 0.2mg/mL and 1mg/mL, it is other to operate
Journey is the same as above-mentioned 2.
Three kinds of ribosome inactivating proteins and NAD+Reacted, and in differential responses time sampling, then with coupling enzymatic reagent
After reaction, the burst size of adenine is detected.As a result as shown in Figure 9.
As seen from Figure 9, at the RIP of same concentrations, with the growth in reaction time, the free adenine amount that releases
Increasing.When being reacted to after a certain period of time, reaction speed is in stable state.Speed of the reaction time in 60min, i.e. generation
The initial velocity of reaction of three kinds of ribosome inactivating proteins of table.
Therefore, the reaction time is catalyzed NAD to RIP+Discharge the influence of adenine as a result, it to follow and meet enzymatic reaction dynamic
The rule of mechanics.
4, influence of the concentration of substrate to enzymatic reaction speed
Reaction total volume is 0.3mL, by the NAD of final concentration of 1mM~30mM of 0.15mL+(solvent is pH3.0 0.02M
Disodium hydrogen phosphate-citrate buffer solution) and the water-reducible MAP30 composition of 0.15mL.
In 55 DEG C of accurate response 50min, then boils 3min and terminate reaction, 10000g is centrifuged 2min, takes supernatant 0.1mL
With coupling enzymatic reagent 0.3mL, 37 DEG C of reactions keep the temperature 0.5h.555nm absorbance value is measured, it is dense to substrate with 555nm absorbance value
Degree mapping.The result is shown in Figure 10.
In biochemical reaction, if the concentration of enzyme is definite value, when the initial concentration of substrate is lower, enzymatic reaction speed and substrate
Concentration is directly proportional, i.e., increases with the increase of concentration of substrate.After all enzyme-to-substrates, which combine, generates intermediate product, even if
Increase concentration of substrate, concentration of intermediate products will not increase, and enzymatic reaction speed does not also increase.As seen from Figure 10, MAP30 is catalyzed
In the reaction for discharging adenine, when concentration is lower, with NAD+The increase of concentration, the adenine amount and NAD released+Concentration
It is directly proportional.Work as NAD+When concentration reaches higher, the adenine amount released is not also further added by.
Therefore, MAP30 is catalyzed NAD+Discharge the reaction of adenine, it then follows enzyme kinetics-concentration of substrate is to reaction
The Hyperbolic Feature that speed influences.
α-MMC and TCS also can get the result similar with MAP30.
5, influence of the temperature to enzymatic reaction speed
Take 100 μ L various concentrations RIP (α-MMC 1mg/mL, MAP30 0.2mg/mL and TCS1.5mg/mL, water be it is molten
Agent) respectively with 100 μ L 40mM NAD+(disodium hydrogen phosphate-citrate buffer solution of solvent 0.1M, pH3.0), 40~80
DEG C accurate response 30min, 100 DEG C of heating 3min terminate reaction.Reaction solution is centrifuged 3min in 10000g, takes supernatant 0.1mL
It is mixed with 0.3mL coupling enzymatic reagent, is placed in 37 DEG C of incubation 30min.Then 555nm absorbance value is measured, with 555nm absorbance value
It maps to different temperatures, the result is shown in Figure 11.
As seen from Figure 11, the optimum temperature of α-MMC, MAP30 and TCS are 50~55 DEG C, when being lower than this temperature, activity
It is increased with the raising of temperature；When higher than this temperature, the raising of their activity with temperature is gradually decreased, and shows rapid deactivation
Feature.Therefore, the present invention selects reaction temperature for 50~55 DEG C.
This test example is by verifying pH on the active influence of ribosome inactivating protein, ribosome inactivating protein concentration, reaction
Time is to catalysis NAD+Discharge the influence of adenine, the influence of concentration of substrate, temperature to enzymatic reaction speed, it is determined that the present invention
Enzymatic reaction is consistent with theory, illustrates the active standard with the method for the present invention the method for the present invention detection ribosome inactivating protein
True property.
To sum up, developing solution of the invention can be used for the Activity determination of ribosome inactivating protein, and ribosomes of the invention loses
Living protein measuring method accuracy is high, is quick on the draw, easy to operate, and can avoid interference of traditional RNA enzyme to reaction, application
It has good prospects.
Claims (11)
1. a kind of developing solution for ribosome inactivating protein determination of activity, it is characterised in that: the developing solution is to be with concentration
The HEPES-NaOH buffer or MES-NaOH buffer that 0.05~0.2M, pH are 6.5~7.3 are basic solution, and are added to
The adenosine deaminase of final concentration of 5~9U/mL, the 4-AA of 4~6g/L, 30~50mM N- ethyl-N- (2- hydroxyl
Base -3- sulfopropyl) -3- methylaniline sodium salt, 9~14U/mL peroxidase and 5~10U/mL xanthine oxidase.
2. developing solution according to claim 1, it is characterised in that:
It for 0.1M, pH MES-NaOH buffer for being 7.0 is basic solution that the developing solution, which is with concentration, and is added to final concentration
For the adenosine deaminase of 6U/mL, the 4-AA of 5g/L, 40mM N- ethyl-N- (2- hydroxyl -3- sulfopropyl) -3-
Methylaniline sodium salt, the peroxidase of 9U/mL and 6U/mL xanthine oxidase；
Or, it for 0.1M, pH MES-NaOH buffer for being 7.0 is basic solution that the developing solution, which is with concentration, and it is added to end
Concentration is the N- ethyl-N- (2- hydroxyl -3- sulphur third of the adenosine deaminase of 5U/mL, the 4-AA of 4g/L, 35mM
Base) -3- methylaniline sodium salt, 10U/mL peroxidase and 8U/mL xanthine oxidase；
Or, it for 0.1M, pH MES-NaOH buffer for being 7.0 is basic solution that the developing solution, which is with concentration, and it is added to end
Concentration is the N- ethyl-N- (2- hydroxyl -3- sulphur third of the adenosine deaminase of 8U/mL, the 4-AA of 5g/L, 50mM
Base) -3- methylaniline sodium salt, 12U/mL peroxidase and 10U/mL xanthine oxidase；
Or, it for 0.2M, pH HEPES-NaOH buffer for being 7.3 is basic solution that the developing solution, which is with concentration, and it is added to
The adenosine deaminase of final concentration of 9U/mL, the 4-AA of 6g/L, 40mM N- ethyl-N- (2- hydroxyl -3- sulphur third
Base) -3- methylaniline sodium salt, the peroxidase of 13U/mL, 8U/mL xanthine oxidase.
3. developing solution according to claim 1 or 2, it is characterised in that: further include final concentration of 0.3 in the developing solution~
The NaN of 0.5g/L3With the Triton X-100 of 1~2mL/L.
4. developing solution according to claim 1 or 2, it is characterised in that: further include final concentration of 0.3g/ in the developing solution
The NaN of L3With the Triton X-100 of 2mL/L.
5. purposes of the developing solution in ribosome inactivating protein determination of activity described in claim 1-4 any one.
6. a kind of active method of measurement ribosome inactivating protein, it is characterised in that: it includes the following steps:
A. ribosome inactivating protein or the solution comprising ribosome inactivating protein are taken, as sample to be tested；
B. ribosome inactivating protein or the solution comprising ribosome inactivating protein are taken, 2~3min is boiled, as blank control；
C. sample to be tested, blank control are separately added into the substrate solution that isometric concentration is 40~80mM, mixed, 50~55
It is incubated for 20~35min under the conditions of DEG C, then boils 3min, 5000~10000g is centrifuged 1~2min, takes supernatant；
Wherein, the substrate solution is formulated as follows: weigh rRNA analog, with concentration be 0.01~0.05M, pH be 2.0~
3.0 buffer solution and dilution, the buffer is disodium hydrogen phosphate-citrate buffer solution, sodium hydroxide-citric acid-salt
Acid buffer, glycine-HCI buffer or acetic acid-sodium acetate buffer solution；
The rRNA analog is that the compound for releasing adenine can be catalyzed by ribosome inactivating protein；
D. the developing solution of 3 times of volumes is added in the supernatant for taking step c, mixes, 35~45 DEG C of 25~40min of incubation；
The developing solution is developing solution described in claim 1-4 any one；
E. with blank control zeroising, spectrophotometer method measures light absorption of the sample to be tested under 500~600nm wavelength condition
Value；
F. according to absorbance value, enzyme activity is calculated.
7. according to the method described in claim 6, it is characterized by: ribosome inactivating protein is α-balsam pear in step a and/or b
Element, MAP30, trichosanthin.
8. according to the method described in claim 6, it is characterized by: being carried out in step a to ribosome inactivating protein or crude enzyme liquid
Dilution, make sample to be tested according to step a-e reaction after, the control of absorbance value under 500~600nm wavelength condition 0.1~
0.4；
Further, retarder thinner is disodium hydrogen phosphate-lemon acid buffering that concentration is 0.01~0.05M, pH is 2.0~3.0
Liquid, sodium hydroxide-citrate-hcl buffer or acetic acid-sodium acetate buffer solution.
9. according to the method described in claim 6, it is characterized by: in step c, sample to be tested or blank control and substrate solution
Before mixing, sample to be tested, blank control, substrate solution are respectively at 50~55 DEG C, 2~5min of lower incubation；
And/or in step c, sample to be tested, blank control are separately added into substrate solution, mix, and incubation time is 25~35min；
Then 3min is boiled, centrifugal condition is that 8000g/min is centrifuged 1min；
And/or in step c, the rRNA analog is AMP, NAD+Or NADH.
10. according to the method described in claim 6, it is characterized by:
In step d, incubation temperature is 40 DEG C, and incubation time is 25~30min；
In step e, the wavelength is 555nm.
11. according to the method described in claim 6, it is characterized by: the method for calculating enzyme activity is as follows in step f:
The relative activity of analyte sample fluid (ribosome inactivating protein) is calculated according to following formula:
Δ A: sample absorbance value variable quantity；0.2142: under 555nm wavelength, the corresponding adenine concentration (mM) of 1A；Vt: reaction
Total volume (mL)；103: 1mM=103μM；df: enzyme solution extension rate；T: the accurate response time (min) of enzyme and substrate；Ve: enzyme
Liquid product (mL)；D: cuvette optical path (1cm)；Vr: the volume (mL) to react with conjugate enzyme developing solution；C: protein concentration
(mg/mL)。
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| CN1763087A (en) * | 2005-11-08 | 2006-04-26 | 中国人民解放军军事医学科学院野战输血研究所 | Sponge gourd seed ribosome deactivated protein and encoding gene and application |
| CN101386644A (en) * | 2007-01-04 | 2009-03-18 | 中国科学院福建物质结构研究所 | Pumpkin protein 2 and preparation method and application thereof |
| CN101738480A (en) * | 2009-09-18 | 2010-06-16 | 中国计量科学研究院 | Nanoparticle probes for detecting ribosome inactivating protein, manufacturing method thereof and use thereof |
| CN104498494A (en) * | 2015-01-06 | 2015-04-08 | 山西大学 | Plasmid for detecting activity of ribosome inactivating protein as well as construction method and application of plasmid |
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| CN1763087A (en) * | 2005-11-08 | 2006-04-26 | 中国人民解放军军事医学科学院野战输血研究所 | Sponge gourd seed ribosome deactivated protein and encoding gene and application |
| CN101386644A (en) * | 2007-01-04 | 2009-03-18 | 中国科学院福建物质结构研究所 | Pumpkin protein 2 and preparation method and application thereof |
| CN101738480A (en) * | 2009-09-18 | 2010-06-16 | 中国计量科学研究院 | Nanoparticle probes for detecting ribosome inactivating protein, manufacturing method thereof and use thereof |
| CN104498494A (en) * | 2015-01-06 | 2015-04-08 | 山西大学 | Plasmid for detecting activity of ribosome inactivating protein as well as construction method and application of plasmid |
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