CN104730019A - Method for in-vitro evaluation on safety of cigarettes and tobaccos - Google Patents
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Abstract
The invention discloses a method for in-vitro evaluation on safety of cigarettes and tobaccos. The method is characterized in that a biological reagent with TrxR (thioredoxin reductase) activity is adopted to detect TrxR activity inhibition ability of CSE (cigarette smoke extract) obtained after combustion of the cigarettes or the tobaccos so as to evaluate safety of the cigarettes and the tobaccos by taking an absorbance value of the CSE at 340nm as a quantification basis. By the method for in-vitro evaluation on safety of the cigarettes and the tobaccos, the problems of long evaluation period, complex evaluation mechanisms and the like in current cigarette harm evaluation are solved successfully to provide a reliable evaluation means for enterprises to improve tobacco quality and develop less harmful cigarettes.
Description
Technical field
The present invention relates to a kind of method of in-vitro evaluation cigarette and tobacco security.Specifically use containing the solution of thioredoxin reductase (TrxR) vigor, by detecting flue gas aqueous extract (CSE) that kinds of cigarettes and tobacco make to the rejection ability of TrxR vigor, with the absorbance OD of CSE at 340nm place
340nmas quantitative basis, the method for evaluating cigarette and tobacco security.
Background technology
Modern medicine study has enough evidences proof, and Smoking is harmful to your health, and flue gas aqueous extract CSE has also been widely used in research flue gas toxicological mechanism (see document 1-5).Containing 4800 kinds of materials determined such as tar, nicotine and aldehydes in CSE, wherein aldehydes comprises acetaldehyde, formaldehyde and unsaturated aldehydes, and the acryl aldehyde in unsaturated aldehydes and the rare aldehyde of fourth are one of maximum several materials of harmfulness.The unsaturated aldehydes such as acryl aldehyde is high active electrophilic compound, easy and nucleophile generation nucleophilic addition.Research shows that acryl aldehyde is that cigarette smoke causes Cytotoxic significant contributor (see document 6); In addition, acryl aldehyde can increase the risk (see document 7) of the Induced by Carcinogen cancer such as Benzpyrene in flue gas.
Thioredoxin reductase TrxR is a kind of dimer selenoenzyme, the selenocystein (Sec) of TrxR is most important for TrxR catalytic activity, it is the analog of halfcystine, its pKa (~ 5.2) is far below the pKa (~ 8.3) of halfcystine, therefore Sec is in the selenol salt state of aprotic when physiological pH, and this makes it have high nucleophilic vigor.TrxR plays key effect in the forming process of deoxynucleotide, be that DNA synthesizes and requisite material in proliferation process, TrxR has inhibited apoptosis, improves the multiple biological functions (see document 8) such as the activity of transcription factor simultaneously.TrxR vigor is suppressed causes the consequences such as tissue damage (see document 9 and 10).
Acryl aldehyde in flue gas, as active electrophilic compound, has also been proved and can be combined with the TrxR containing Sec, suppressed TrxR vigor (see document 11).
The current evaluation to cigarette harm mainly relies on constituent analysis and biological detection.Constituent analysis is widespread use in cigarette industry, but due to smoke components complexity, this disappears and other rises in the practice of reducing tar and reducing harm for different nuisance, and the evaluation depending on a few class material has certain limitation.Utilize cell and the animal experiment method evaluating cigarette security cycle long, in Less harmful cigarette R&D process, practicality is low.
Therefore, the means that Less harmful cigarette needs are a kind of immediately, rapid, high flux is evaluated cigarette and tobacco security are developed.
List of references:
1.Kosmider B,Messier EM,Chu HW,et.Epub 2011 Dec 7.Human alveolar epithelial cellinjury induced by cigarette smoke.PLoS One.2011；6(12).
2.Zhu L,Barrett EC,Xu Y,et.Regulation of Cigarette Smoke(CS)-Induced Autophagy byNrf2.PLoS One.2013 Apr 9；8(4).
3.Baglole CJ,Sime PJ,Phipps RP.Cigarette smoke-induced expression of heme oxygenase-1in human lung fibroblasts is regulated by intracellular glutathione.Am J Physiol Lung Cell MolPhysiol.2008Oct；295(4).
4.Yageta Y,Ishii Y,Morishima Y,et.Mar 2.Role of Nrf2 in host defense against influenzavirus in cigarette smoke-exposed mice.J Virol.2011 May；85(10).
5.Bertram KM,Baglole CJ,Phipps RP,rt.Molecular regulation of cigarette smokeinduced-oxidative stress in human retinal pigment epithelial cells:implications for age-relatedmacular degeneration.Am J Physiol Cell Physiol.2009 Nov；297(5).
6.Noya Y,Seki K,Asano H,et.Epub 2013 Aug 24.Identification of stable cytotoxic factors inthe gas phase extract of cigarette smoke and pharmacological characterization of their cytotoxicity.Toxicology.2013 Dec 6；314(1):1-10.
7.Feng Z,Hu W,Hu Y,Tang MS.Acrolein is a major cigarette-related lung cancer agent:Preferential binding at p53 mutational hotspots and inhibition of DNA repair.Proc Natl Acad SciUSA.2006,103:15404-15409
8.Holmgren A,Lu J.Thioredoxin and thioredoxin reductase:current research with specialreference to human disease.Biochem Biophys Res Commun.2010 May 21；396(1):120-4.
9.Wang X,Zhang J,Xu T.Thioredoxin reductase inactivation as a pivotal mechanism ofifosfamide in cancer therapy.Eur J Pharmacol.2008Jan 28；579(1-3):66-73.
10.Zhang J,Lu H.Ifosfamide induces acute renal failure via inhibition of the thioredoxinreductase activity.Free Radic Biol Med.2007 Dec 15；43(12):1574-83.
11.Zhang J1,Lu H.Randall MJ,Spiess PC,Hristova M.Acrolein-induced activation ofmitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase andthioredoxin 1.Redox Biol 2013,1:265-275.
12.A.D.Smith,O.A.Levander,High-throughput 96-well microplate assays for determiningspecific activities of glutathione peroxidase and thioredoxin reductase,Methods Enzymol.347(2002)113–121.
13.E.S.Arnér,A.Holmgren,Measurement of thioredoxin and thioredoxin reductase,in:M.Maines,L.Costa,D.Reed,S.Sassa(Eds.),Current Protocols in Toxicology,John Wiley & Sons,Inc.,New York,2000,pp.7.4.1–7.4.14.
Summary of the invention
The object of this invention is to provide a kind of can the method for instant, rapid, high-throughout in-vitro evaluation cigarette and tobacco security.
Technical solution problem of the present invention, adopts following technical scheme:
The method of in-vitro evaluation cigarette of the present invention and tobacco security, its feature is:
Step 1: each cigarette to be evaluated or tobacco are aspirated respectively, and be mixed with flue gas aqueous extract respectively, detects the absorbance OD of flue gas aqueous extract at 340nm place
340nm, with OD
340nmas the quantitative basis of evaluating cigarette or tobacco security;
Step 2: preparation, containing the biological reagent of thioredoxin reductase vigor, is used two (2-paranitrobenzoic acid) (DTNB) reducing process of 5,5 '-two sulphur to measure the energy value of thioredoxin reductase in described biological reagent, is designated as H0;
Step 3: carry out hatching of 20min by the flue gas aqueous extract adding different amount in the described biological reagent of same volume, flue gas aqueous extract is produced the thioredoxin reductase vigor in described biological reagent suppress, measure the energy value H1 suppressing thioredoxin reductase in artifact reagent, and calculate acquisition flue gas aqueous extract to inhibiting rate T=(H0-H1)/H0 × 100% of the thioredoxin reductase vigor in biological reagent, make the dose-effect curve of inhibiting rate, the suitable addition of addition as flue gas aqueous extract of any flue gas aqueous extract except zero point is selected in the addition of flue gas aqueous extract and the linear scope of inhibiting rate from the dose-effect curve of described inhibiting rate, be designated as m, unit is microlitre, determine that flue gas aqueous extract, to the inhibiting rate of the thioredoxin reductase vigor in biological reagent, is designated as T0 under described suitable addition,
Described suitable addition m is corrected to 10 microlitres, obtains the corrected value M=m/10 of suitable addition;
Step 4: determine the inhibiting rate T0 of the flue gas aqueous extract of each cigarette or tobacco to the thioredoxin reductase vigor in biological reagent and the corrected value M of suitable addition by the method for step 3; Kinds of cigarettes or its suitable addition of the made flue gas aqueous extract of tobacco different, therefore the suitable addition collective of all kinds of cigarettes or the made flue gas aqueous extract of tobacco is corrected to 10 microlitres, to evaluate the security of kinds of cigarettes or tobacco more accurately.
Step 5: calculate the safety evaluatio value S=T0/ (M × OD obtaining each cigarette or tobacco
340nm), the security of each cigarette or tobacco is compared with described safety evaluatio value S.
In-vitro evaluation cigarette of the present invention and tobacco security method menu, its feature is also: the aqueous extract of flue gas described in step 1 prepares as follows:
A cigarette to be evaluated or tobacco is aspirated at the standard conditions by vacuum pump, the main flume produced is collected by being respectively equipped with in the gas absorption bottle of 5mL supplementary set liquid of two series connection successively, after a cigarette to be evaluated or tobacco suction, suction 10s, supplementary set liquid is made fully to collect main flume, then will collect the supplementary set liquid of main flume through 0.22 μm of water film filtering, namely obtain flue gas aqueous extract; The phosphate buffer of described supplementary set liquid to be concentration be 0.1M, pH=7.0 is the disodium ethylene diamine tetraacetate (EDTA.Na of 10mM containing concentration in described phosphate buffer
2).
The homogenate of the homogenate of animal tissue, the homogenate of plant tissue, the homogenate of isolated cells or microorganism containing the biological reagent of thioredoxin reductase vigor described in step 2, or the pure enzyme preparation of thioredoxin reductase.
In step 2, the homogenate of animal tissue or the homogenate of plant tissue obtain as follows: take 0.15g at the animal tissue of-20 DEG C of low temperature cold storage or plant tissue, add 1.5mL tissue homogenate, then use the grinding of high-flux tissue mill, centrifugal 20min under 15000rcf, 4 DEG C of conditions, get supernatant, the homogenate of Ji get animal tissue or the homogenate of plant tissue; Described tissue homogenate is that concentration is 0.15M, pH=7.15 and contains the phosphate buffer that concentration is the disodium ethylene diamine tetraacetate of 1mM.
In step 2, the homogenate of isolated cells or the homogenate of microbial body obtain as follows: be suspended in cell pyrolysis liquid by isolated cells or microbial body with the concentration of 10-90 1,000,000/mL, then use ultrasonic cleaner at 4 DEG C, ultrasonic cell 20min under the condition of 40KHz, to reach broken object, then centrifugal 10min under 10000rpm, the condition of 4 DEG C, get supernatant, obtain the homogenate of isolated cells or the homogenate of microbial body; The solution of described cell pyrolysis liquid to be concentration the be trishydroxymethylaminomethane (Tris-HCl) of 0.1M, pH7.4, comprises the benzyl sulfonamide (PMSF) of dithiothreitol (DTT) (DTT) that concentration is 1mM and 1mM at the solution of described trishydroxymethylaminomethane.
Use two (2-paranitrobenzoic acid) (DTNB) reducing process of 5,5 '-two sulphur to measure the method for the energy value of thioredoxin reductase in described biological reagent see document 12 and 13 in the present invention, concrete steps are as follows:
First (phosphate buffer of described testing liquid to be concentration be 100mM, pH 7.0, comprises the EDTA-Na that concentration is 10mM in this testing liquid to prepare testing liquid
2, the DTNB of 5mM, NADPH and 0.2mg/mL of 240 μMs bovine serum albumin(BSA)), matching while using, 37 DEG C of temperature controls;
By biological reagent to be measured in killer tube and Anranofin by volume 9:1 mix, in non-killer tube biological reagent to be measured and 5% ethanol by volume 9:1 mix, at 37 DEG C, act on 20min respectively; Biological reagent in killer tube after process and non-killer tube is added in 96 orifice plates respectively, treats to add in gaging hole 250 microlitre testing liquid toward 96 orifice plates and start and react, and 96 orifice plates are placed in microplate reader detect immediately.Under 37 DEG C of conditions in microplate reader with 412nm wavelength detecting 6min, every 12s reads value once, read enzymatic kinetic curve, read the slope value (Vmax) of 120s ~ 300s time period, the Vmax in killer tube corresponding to sample is deducted with the Vmax in non-killer tube corresponding to sample, the energy value (TrxR energy value unit definition is the ability of 1mg albumen oxidation per minute μm ol NADPH) obtaining thioredoxin reductase in biological reagent is being calculated through specific formulation after obtaining this difference, or be directly reduced to this difference definition is that the energy value of thioredoxin reductase in biological reagent is to facilitate the calculating of data, namely this evaluation method utilizes this difference as the energy value of thioredoxin reductase in biological reagent.
Because CSE preparation there is no industry standard, and can not realize CSE quantification during preparation, different goods CSE needs to correct based on a kind of accessible index to TrxR vigor depression effect, thus realizes the comparison of different goods.The present invention adopts CSE at the absorbance OD at 340nm place
340nmcSE is corrected, with OD
340nmthe quantitative basis of TrxR vigor ability is suppressed, the quantitative basis of also i.e. evaluating cigarette or tobacco security as CSE.The quantitative basis of evaluating cigarette or tobacco security also can be tar content in flue gas aqueous extract or nicotine content.
Compared with the prior art, beneficial effect of the present invention is embodied in:
The invention provides a kind of immediately, the method for evaluating cigarette or tobacco security effectively, successfully solve present stage cigarette or the problem such as tobacco safety evaluatio mechanism is complicated, the cycle is long, running program is simple, evaluation result accurately and reliably, and there is high flux characteristic, for the research of tobacco enterprise development reducing tar and reducing harm technology and raising safety cigarette provides a reliable index.
Accompanying drawing explanation
Fig. 1 is CSE device for making figure;
Fig. 2 is that CSE is on main enzymatic activity impact several in mouse LH liquid;
Fig. 3 is the dose-effect curve of the inhibiting rate in the embodiment of the present invention 1;
Fig. 4 is the comparison diagram of the safety evaluatio value of 18 kinds of cigarette in the embodiment of the present invention 1;
Fig. 5 is the time effect curve of the inhibiting rate suppressing different biological reagent in the embodiment of the present invention 2 with CSE;
Fig. 6 is the dose-effect curve of the inhibiting rate suppressing different biological reagent in the embodiment of the present invention 3 with CSE;
Fig. 7 is the ratio versus figure of the safety evaluatio value of cigarette A and cigarette R under different biological reagent in the embodiment of the present invention 3.
Embodiment
Below in conjunction with accompanying drawing, by embodiment, the present invention is further described.
Embodiment 1
The present embodiment evaluates the security of 18 kinds of cigarette (naming from A to R with letter respectively) as follows:
Step 1: to be evaluated 18 kinds of cigarette are aspirated respectively, is then mixed with flue gas aqueous extract CSE respectively, and detect the absorbance OD of each CSE at 340nm place
340nm, with OD
340nmas the quantitative basis of evaluating cigarette or tobacco security;
As shown in Figure 1, CSE prepares as follows: by vacuum pump DP-01 at the standard conditions (vacuum pressure is 0.08Mpa) aspirate a cigarette to be evaluated, the main flume produced is collected by being respectively equipped with in the gas absorption bottle of 5mL supplementary set liquid of two series connection successively, after a cigarette to be evaluated or tobacco suction, suction 10s, supplementary set liquid is made fully to collect main flume, then by collecting the supplementary set liquid of main flume through 0.22 μm of water film filtering, namely CSE is obtained; The phosphate buffer of supplementary set liquid to be concentration be 0.1M, pH=7.0 is the disodium ethylene diamine tetraacetate (EDTA.Na of 10mM containing concentration in described phosphate buffer
2).
Step 2: preparation, containing the biological reagent of TrxR vigor, is used DTNB reducing process to measure the energy value of thioredoxin reductase in biological reagent, is designated as H0;
Compound method containing the biological reagent of TrxR vigor is: take the mouse liver of 0.15g-20 DEG C of low temperature cold storage, add 1.5mL tissue homogenate, then use the grinding of high-flux tissue mill, centrifugal 20min under 15000rcf, 4 DEG C of conditions, get supernatant, obtain mouse LH liquid, in this, as the biological reagent containing TrxR vigor; Tissue homogenate is that concentration is 0.15M, pH=7.15 and contains the phosphate buffer that concentration is the disodium ethylene diamine tetraacetate of 1mM.
For mouse LH liquid, several more common and there is bioactive enzyme (glutathione s-transferase by detecting when CSE highly suppresses TrxR vigor (80%) in mouse LH liquid in mouse LH liquid other, glutathione reductase, glutathione peroxidase, hydrogen peroxidase, superoxide dismutase) activity change (Fig. 2), find that the vigor of this several enzyme does not all change, illustrate in the biological reagent system containing TrxR vigor, CSE preferentially suppresses TrxR vigor, other has bioactive enzyme and suppresses the ability of TrxR all not affect on CSE, therefore TrxR can as the evaluation index of safety cigarette.
Step 3: undertaken hatching 20min by the flue gas aqueous extract of the cigarette A adding a series of difference amount in the biological reagent of same volume, flue gas aqueous extract is produced the thioredoxin reductase vigor in biological reagent suppress and measure the energy value H1 suppressing thioredoxin reductase in artifact reagent, calculate and obtain flue gas aqueous extract to inhibiting rate T=(H0-H1)/H0 × 100% of the thioredoxin reductase vigor in biological reagent, make the dose-effect curve of inhibiting rate, as shown in Figure 3, the suitable addition m of flue gas aqueous extract is determined from dose-effect curve, unit is microlitre (the present embodiment selects 20 μ L), suitable addition is the arbitrary value of the addition in the linear scope of the addition of flue gas aqueous extract in dose-effect curve and inhibiting rate, determine that flue gas aqueous extract, to the inhibiting rate of the thioredoxin reductase vigor in biological reagent, is designated as T0 under suitable addition, suitable addition m is corrected to 10 microlitres, obtains the corrected value M=m/10 of suitable addition,
Step 4: determine the inhibiting rate T0 of other flue gas aqueous extracts of 17 kinds to the thioredoxin reductase vigor in biological reagent and the corrected value M of suitable addition by the method for step 3;
Step 5: calculate the safety evaluatio value S=T0/ (M × OD obtaining each cigarette or tobacco
340nm), as shown in Figure 4, compare the security of each cigarette with safety evaluatio value S.As can be seen from the figure the S value of cigarette A is the highest, illustrate that the security of cigarette A is poor, and the S value of cigarette R is minimum, illustrates that the security of cigarette R is better; The gap of the S value of cigarette A and cigarette R reaches 2.59 times.
Embodiment 2
The present embodiment is for proving that in the inventive method step 3, incubation time selects the reason of 20min:
The CSE that 20 μ L are obtained by cigarette A is added in mouse LH liquid obtained in embodiment 1, and detect the inhibiting rate of different time points CSE to TrxR vigor in mouse LH liquid, draw the time effect curve of inhibiting rate, as shown in Figure 5 a, as can be seen from the figure when 20min, inhibiting rate tends towards stability, and the time of therefore selecting is 20min.
In addition, biological reagent is changed respectively into mouse lung homogenate liquid and the pure enzyme preparation of TrxR (production code member: T9698, sigma company produces), carry out same test, result as illustrated in figures 5 b and 5 c, as can be seen from the figure CSE is when suppressing different biological reagents, and 20min all can make inhibiting rate stablize.
Embodiment 3
The present embodiment is for proving the inventive method reliable results, repeatable strong.
Biological reagent in embodiment 1 is used respectively instead mouse lung homogenate liquid and the pure enzyme preparation of TrxR, carry out same test, the dose-effect curve of inhibiting rate obtained in step 3 respectively as shown in figure 6 a and 6b.
Three kinds of biopreparate evaluating cigarette security acquired results are comparatively used with the odds ratio of the S value of cigarette A and cigarette R, as shown in Figure 7, though can find out that gained ratio is not identical when selecting different biopreparate to evaluate, and has identical rule, it is all the S value that the S value of cigarette A is greater than cigarette R.Demonstrate the feasibility of the inventive method.
The foregoing is only embodiments of the invention, for a person skilled in the art, the present invention can have various modifications and variations, obtains better effect.Within the spirit and principles in the present invention all, any amendment done/equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. a method for in-vitro evaluation cigarette and tobacco security, is characterized in that:
Step 1: each cigarette to be evaluated or tobacco are aspirated respectively, and be mixed with flue gas aqueous extract respectively, detects the absorbance OD of flue gas aqueous extract at 340nm place
340nm, with OD
340nmas the quantitative basis of evaluating cigarette or tobacco security;
Step 2: preparation, containing the biological reagent of thioredoxin reductase vigor, is used two (2-paranitrobenzoic acid) reducing process of 5,5 '-two sulphur to measure the energy value of thioredoxin reductase in described biological reagent, is designated as H0;
Step 3: carry out hatching of 20min by the flue gas aqueous extract adding different amount in the described biological reagent of same volume, flue gas aqueous extract is produced the thioredoxin reductase vigor in described biological reagent suppress, measure the energy value H1 suppressing thioredoxin reductase in artifact reagent, and calculate acquisition flue gas aqueous extract to inhibiting rate T=(H0-H1)/H0 × 100% of the thioredoxin reductase vigor in biological reagent, make the dose-effect curve of inhibiting rate, the suitable addition of addition as flue gas aqueous extract of any flue gas aqueous extract except zero point is selected in the addition of flue gas aqueous extract and the linear scope of inhibiting rate from the dose-effect curve of described inhibiting rate, be designated as m, unit is microlitre, determine that flue gas aqueous extract, to the inhibiting rate of the thioredoxin reductase vigor in biological reagent, is designated as T0 under described suitable addition,
Described suitable addition m is corrected to 10 microlitres, obtains the corrected value M=m/10 of suitable addition;
Step 4: determine the inhibiting rate T0 of the flue gas aqueous extract of each cigarette or tobacco to the thioredoxin reductase vigor in biological reagent and the corrected value M of suitable addition by the method for step 3;
Step 5: calculate the safety evaluatio value S=T0/ (M × OD obtaining each cigarette or tobacco
340nm), the security of each cigarette or tobacco is compared with described safety evaluatio value S.
2. in-vitro evaluation cigarette according to claim 1 and tobacco security method menu, is characterized in that: the aqueous extract of flue gas described in step 1 prepares as follows:
A cigarette to be evaluated or tobacco is aspirated at the standard conditions by vacuum pump, the main flume produced is collected by being respectively equipped with in the gas absorption bottle of 5mL supplementary set liquid of two series connection successively, after a cigarette to be evaluated or tobacco suction, suction 10s, supplementary set liquid is made fully to collect main flume, then will collect the supplementary set liquid of main flume through 0.22 μm of water film filtering, namely obtain flue gas aqueous extract; The phosphate buffer of described supplementary set liquid to be concentration be 0.1M, pH=7.0 is the disodium ethylene diamine tetraacetate of 10mM containing concentration in described phosphate buffer.
3. in-vitro evaluation cigarette according to claim 1 and tobacco security method menu, it is characterized in that: the homogenate described in step 2 containing the biological reagent of thioredoxin reductase vigor being the homogenate of animal tissue, the homogenate of plant tissue, the homogenate of isolated cells or microorganism, or the pure enzyme preparation of thioredoxin reductase.
4. in-vitro evaluation cigarette according to claim 1 and tobacco security method menu, is characterized in that: in step 1, the quantitative basis of evaluating cigarette or tobacco security also can be the tar content in flue gas aqueous extract or nicotine content.
5. in-vitro evaluation cigarette according to claim 3 and tobacco security method menu, is characterized in that:
In step 2, the homogenate of animal tissue or the homogenate of plant tissue obtain as follows: take 0.15g at the animal tissue of-20 DEG C of low temperature cold storage or plant tissue, add 1.5mL tissue homogenate, then use the grinding of high-flux tissue mill, centrifugal 20min under 15000rcf, 4 DEG C of conditions, get supernatant, the homogenate of Ji get animal tissue or the homogenate of plant tissue; Described tissue homogenate is that concentration is 0.15M, pH=7.15 and contains the phosphate buffer that concentration is the disodium ethylene diamine tetraacetate of 1mM.
6. in-vitro evaluation cigarette according to claim 3 and tobacco security method menu, is characterized in that:
In step 2, the homogenate of isolated cells or the homogenate of microbial body obtain as follows: be suspended in cell pyrolysis liquid by isolated cells or microbial body with the concentration of 10-90 1,000,000/mL, use ultrasonic cleaner at 4 DEG C, ultrasonic 20min under the condition of 40KHz, to reach broken object, then centrifugal 10min under 10000rpm, the condition of 4 DEG C, get supernatant, obtain the homogenate of isolated cells or the homogenate of microbial body; The solution of described cell pyrolysis liquid to be concentration the be trishydroxymethylaminomethane of 0.1M, pH7.4, comprises the benzyl sulfonamide of dithiothreitol (DTT) that concentration is 1mM and 1mM at the solution of described trishydroxymethylaminomethane.
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