CN106053795B - A kind of methyl transferase activity real-time assay and kit - Google Patents
A kind of methyl transferase activity real-time assay and kit Download PDFInfo
- Publication number
- CN106053795B CN106053795B CN201610549785.7A CN201610549785A CN106053795B CN 106053795 B CN106053795 B CN 106053795B CN 201610549785 A CN201610549785 A CN 201610549785A CN 106053795 B CN106053795 B CN 106053795B
- Authority
- CN
- China
- Prior art keywords
- sah
- methyl
- concentration
- transmethylase
- added
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
Abstract
The invention discloses a kind of methyl transferase activity real-time assay and kits.Including two reactions carried out simultaneously in a reaction system: (1) guaranteeing in the buffer system that s-adenosylmethionine has biological activity as the transmethylase of methyl donor, sample, methyl donor and corresponding substrate containing transmethylase (MT) is added or addition s-adenosylmethionine methyl donor and corresponding substrate react to generate AdoHcy (SAH) product directly in the liquid sample containing transmethylase, substrate is the receptive material of methyl；(2) using the content of immunological method detection SAH, to determine the activity of transmethylase in reaction system.The immune response of the biochemical reaction that the present invention is catalyzed MT and measurement product SAH carries out simultaneously, the two processes are organically combined, are especially of great significance for accurately measuring the extremely unstable SAH of molecularity.
Description
Technical field
The present invention relates to transmethylase detection technique fields, and in particular to one kind is using s-adenosylmethionine as methyl donor
Transmethylase biological activity real-time assay and detection kit.
Background technique
Transmethylase (Methyltransferase, MT) is the enzyme of a kind of catalytic methylation reaction, generally with S- adenosine
Methionine (S-Adenosyl Methionine, SAM) is used as methyl donor.Methylation reaction is widely present in organism, he
Substrate specificity range almost enumerate intracellular all bioactive molecules, including nucleic acid, protein, polysaccharide, lipid with
And many small molecules, therefore the inhibition or closing, the reparation of DNA damage of much important physiology link such as gene expressions, and
The synthesis of physiology course intermediate product and degradation reaction etc. are directed to the regulating and controlling effect of MT in microorganism, animal and plant body.
Two classes: inhereditary material transmethylase and non-inhereditary material methyl can be divided into according to current research transmethylase
Transferase.The methylation of inhereditary material methyl transferase catalytic inhereditary material cytimidine physiological and the work as caused by mutagen matter
For the episomal methylation of inhereditary material.The MT of catalysis cytosine methylation has had found 6 kinds, wherein dnmt rna
(DNA Methyltransferase, Dnmt) Dnmt1, Dnmt2, Dnmt3 are the dnmt rna of mammal.MT1,
MT3 is then the homogeneity transmethylase existed in plant, furthermore chromatin transmethylase (Chromomethylase,
It CMT) is a kind of MT specific to plant.The transmethylase type for being catalyzed non-inhereditary material is very more, from participation chromosome
Formed the synthesis of hormone all with its have it is certain be associated with, the substrate of effect is very extensive, including steroids, protein is residual
Base, flavones, chlorophyll, inorganic arsenic, phosphoglycerol esters etc. list in table 1 and study the non-inhereditary material of more catalysis at present
Transmethylase.
Table 1
MT is catalyzed SAM and substrate in vivo, methyl is transferred to substrate and generate SAH (AdoHcy) and
Methide.By taking homocysteine methyltransgerase (Homocysteine Methyltransferase, HMT) as an example, enzyme
It is as follows to promote reaction:
SAM is the most common mesostate in organism.In methionine circulation it may first have to activate Met, be formed
Methyl is supplied to important substance such as DNA in life process by SAM, SAM, RNA, lipoprotein, hormone, becomes S- after neurotransmitter etc.
Adenosyl homocysteine (SAH), SAH generate homocysteine (Hcy) after sloughing adenosine, provide finally by tetrahydrofolic acid
Methyl, and become Met.In liver, methionine circulation has an additional function, mainly drinks in homomethionin or high protein
After food, to remove methionine excessively high in blood rapidly, finally by homocysteine, cysteine, cystathionie, paddy Guang
Sweet peptide is transported to other organs.
SAM be in nature only several functions extremely multiplicity and the bioactive substance of important sulfur-bearing, be egg ammonia
Key substance in acid circulation is unique donor of methyl in the vital methylation modification of the mankind.In transmethylase, turn
Sulphur turns have consequence in aminopropyl reaction.For the relevant cell function of methylating, polyamines synthesis, adjust methionine with
The ratio etc. of homocysteine has direct influence.Have in the Proliferation, Differentiation etc. of various life-critical metabolic responses and cell
Significance.Research shows that certain density SAM is maintained to play a crucial role the performance of liver function in liver.
About 85% methylation reaction and about 50% Methionine metabolism are carried out in liver, and liver is not only prompted to adjust blood
Liquid Methionine Levels have an important role, and prompt SAM regulation liver cell regeneration, differentiation and to various factors (alcohol,
Chemical substance, radiation, pathogenic microorganism, virus, helminth etc.) caused by hepatocellular injury sensibility important meaning.?
In the mouse of Glycine N-methyltransferase (GNMT) missing, SAM level rises, and under both of these case, liver cancer occurs in mouse
Probability obviously increases.
Coding accounts for the 1% of the gene number of human genome dependent on the gene of the transmethylase of SAM.Many research tables
Bright MT and its methyl substrate such as embryo's generation, development, growth, break up, health, disease in the different phase of human life activity
In play a crucial role, exactly because SAM methionine circulation and one carbon metabolism in have very important special role,
It is directly related with human metabolism's situation.Intracorporal SAM content can be because of age, sex, race, weight, diet, medication feelings
Condition, health and disease condition and fluctuated.In view of this characteristic, we should be from two sides of synthesis and catabolism of SAM
Reason is investigated in face.Therefore, understand MT bioactivity in varied situations height we are studied it is many vital
Metabolism has very big practical significance with health problem.
There are three types of the activity of method measurement MT at present: method one: tagging microanalysis.With isotope labelling
SAM is as methyl donor, using another substrate of target enzyme as acceptors, under rigid condition, turns in the analytical unit time
The isotope liquid moved on on another substrate, which dodges every point of count value, indicates the relative activity of MT；Method two: high pressure liquid phase (HPLC) or
Mass spectrum (LC-MS/MS) method measurement MT catalyzes and synthesizes the amount of SAH or methylate；Method three: multienzyme round-robin method.MT enzyme with
Through SAH hydrolase etc., further catalysis finally synthesizes photochemistry side to the SAH that SAM and its corresponding acceptors substance reaction generate
The detectable NAD of method+。
Wherein, it although tracer method has many advantages, such as that high sensitivity, positioning and quantitative are accurate, is lacked there is also some
It falling into, this method needs cumbersome substrate and the subsequent chromatographic isolation of product, and it is time-consuming and laborious, and poor repeatability, in most cases
It can only accomplish semi-quantitative analysis, be engaged in radioisotopic staff and have to pass through specialized training, while also needing to have
Certain safety prevention measure.As there are chemical property between the radioactive isotope and corresponding common element of tracer element
Fine difference so as to cause individual qualitative significant differences, for light element, this so-called isotope
Benefit Transfer is serious.In addition, this method is it is also possible to cause radioactive pollution.
There is also certain limitations for the method for high pressure liquid phase (HPLC) method measurement SAH or methylate.In fact without
It is all very inaccurate for assessment MT activity by being which kind of method measurement SAH of HPLC or LC-MS/MS or methylate content
's.The sample of HPLC and LC-MS/MS, needs specially treated, and SAH or methylate must after the long period, compared with multi-step
So there is loss, furthermore the product of synthetic reaction is possible to unstable, or even degrades in a short time, if detection will cause not in time
Detected value deviation.All of these factors taken together all can cause surveyed value very inaccurate, needless to say attempt to reach reflection enzyme kinetics
The purpose of reaction.
As for multienzyme round-robin method, often it is related to two kinds of even two or more enzyme reactions, process is complicated, and influence factor is very
It is more, and detection method needs the expensive instrument using this kind of large size of biochemical instruments.Therefore, it is accurate, quick, direct to develop a kind of energy
Measurement is very important by the detection method of the transmethylase biological activity of methyl donor of SAM.
Summary of the invention
It accurate, quickly, in real time, directly can be measured using s-adenosylmethionine as methyl the purpose of the present invention is to provide a kind of
The method of the transmethylase biological activity of donor and kit matched with this method.The present invention is based on method simultaneously is direct
Measure the AdoHcy product assay of its synthesis.As long as MT to be detected can be supplied by methyl of s-adenosylmethionine
The methyl of SAM is transferred to its corresponding another substrate by body, MT, and SAM is converted into SAH, and no matter internal or in-vitro method is surveyed
Determine MT enzymatic activity, the present invention most directly can rapidly measure the content of SAH at the first time, to calculate the active height of MT
It is low.
Innovation of the invention is that the biochemical reaction for allowing MT to be catalyzed and the immune response of measurement SAH carry out simultaneously,
The two processes organically combine, and anti-AdoHcy specific antibody quickly, is easily measured with S- gland
Glycosides methionine is a kind of transmethylase biological activity of methyl donor, and is explored by a large number of experiments and a set of allow the two
Reaction while the optimal reaction system and condition for carrying out and obtaining optimum detection effect.For accurately measuring molecularity extremely
Unstable SAH is especially of great significance.
Methyl transferase activity real-time assay of the present invention including two in a reaction system while carrying out anti-
It answers: (1) guaranteeing to be added in the buffer system that s-adenosylmethionine has biological activity as the transmethylase of methyl donor
S- is directly added in sample, methyl donor and substrate containing transmethylase in the liquid sample containing transmethylase
Ademetionine methyl donor and substrate react to generate AdoHcy product, and substrate is methyl
Receptive material；(2) using the content of immunological method detection AdoHcy, to determine, methyl turns in reaction system
Move the activity of enzyme.
The methyl transferase activity real-time assay utilizes envelope antigen method and coated antibody method two ways
The content of competition law measurement AdoHcy；
1) envelope antigen method: in advance by the SAH antigen coat of coupling in solid phase elisa plate, while antigen to be checked is added
With the anti-SAH antibody of tracer-labelling, antigen to be checked is to react the SAH product generated, and antigen to be checked and envelope antigen are jointly competing
The anti-SAH antibody of tracer-labelling is striven, the anti-SAH antibody of board-washing after reaction, the tracer-labelling in conjunction with envelope antigen retains,
Then be washed off with the antibody of the tracer-labelling of antigen binding to be checked, be eventually adding substrate colour developing, it is final develop the color result with it is to be checked
Amount of antigen is inversely proportional, and in continuous mode, uses a series of SAH of gradient known quantities of preparation as standard items, it is bent to make standard
Line；
2) coated antibody method: first anti-SAH antibody incubation, Huo Zhezhi will be added in sheep or the coated microwell plate of rabbit anti-mouse igg
Connect the anti-SAH antibody of coating, board-washing；The SAH antigen of tracer-labelling is added, adds antigen to be checked, antigen to be checked, which reacts, to be produced
Raw SAH product, the SAH of antigen and tracer-labelling to be checked compete the anti-SAH specific antibody being coated on elisa plate, are incubated for
Board-washing；Substrate colour developing is added, in continuous mode, uses a series of SAH of gradient known quantities of preparation as standard items, production mark
Directrix curve.
The methyl transferase activity real-time assay is carried out even using the analog substitution SAH antigen of SAH antigen
Connection or tracer-labelling.
The methyl transferase activity real-time assay, tracer include enzyme, fluorescein, colloidal gold, chemiluminescence
Substance, biotin, digoxin, radioactive marker substance and various types of latex beads.
The methyl transferase activity real-time assay, AdoHcy acid antigen are that poly relies ammonia
Acid, bovine serum albumin(BSA) or other carrier protein couplets.
The methyl transferase activity real-time assay, using envelope antigen method, specific step is as follows:
(1) preparation of various concentration standard items: taking AdoHcy standard items, with buffer at difference
The standard curve sample of concentration；
(2) in the microwell plate for being coated with albumen-SAH conjugate or polymer-SAH conjugate, horseradish peroxidating is added
The anti-AdoHcy monoclonal antibody of object enzyme or alkali phosphatase enzyme mark, contains s-adenosylmethionine, first to be measured
The buffer solution system of the sample of based transferase and acceptors substance corresponding with transmethylase to be measured；If it is liquid-like
Product, then be directly added into s-adenosylmethionine, the sample of transmethylase to be measured and methyl corresponding with transmethylase to be measured by
Body substance；The SAH standard items of prepared various concentration gradient are added in standard curve hole；It is reacted after mixing, board-washing；
(3) substrate of horseradish peroxidase or alkaline phosphatase is added, colour developing is added terminate liquid and terminates reaction, in enzyme
Appropriate wavelength is selected to read the absorbance reading OD450 in every hole on mark instrument；
(4) the SAH standard items that a series of known quantities prepared are used in same elisa plate, according to its OD450 and known standard
Product concentration obtains curvilinear equation, then sample OD450 substitution equation is acquired to the production quantity of its corresponding reaction product SAH；
(5) it is catalyzed the concentration of generation product SAH within the unit time according to the sample containing transmethylase to calculate first
The activity of based transferase.
The methyl transferase activity real-time assay,
Buffer is to include potassium concentration range in 25-200mM, and BSA addition concentration is in 0.1%-0.9%, pH range
For Tris the or PB buffer system of 7.0-8.5；Preferably including potassium concentration is 100mM, and BSA adds concentration 0.1%, and pH is
7.8 PB buffer system.
The methyl transferase activity real-time assay,
The standard concentration of the various concentration for making standard curve of buffer is respectively 0,0.3125,
0.625、1.25、2.5、5、10μM。
The methyl transferase activity real-time assay,
Concentration range after methyl donor is added in liquid sample or buffer is 10-30 μM, dense after substrate addition
Spending range is 10-60 μM, the antibody dilution 1:3000-1:10000 of tracer-labelling, and coated antigen concentration is 0.2-1 μ g/
ml；It is preferred that 20 μM of concentration after methyl donor is added in liquid sample or buffer, 20 μM of concentration after substrate addition, tracer
The dilution 1:10000 that the antibody of substance markers is added, coated antigen concentration are 1ug/ml；
The volume of buffer solution system or liquid sample that each micropore is added is 50 μ l, the tracer-labelling after dilution
The volume of antibody is 50 μ l.
The methyl transferase activity real-time assay, when measuring HMT, reaction time and temperature are respectively 37 DEG C
With 20 minutes.
The methyl transferase activity real-time assay, using envelope antigen method: BSA being coupled SAH antigen in advance
Be coated in solid phase elisa plate, peridium concentration is after 1ug/ml closes with BSA, while be added antigen to be checked (sample containing SAH) and
The anti-SAH antibody of HRP enzyme mark.Antigen to be checked and envelope antigen compete enzyme labelled antibody, board-washing after reaction, in conjunction with envelope antigen jointly
Enzyme labelled antibody retain, with antigen to be checked (free antigen) combine enzyme labelled antibody be then washed off.It is eventually adding substrate colour developing, most
Colour developing result is inversely proportional with amount of antigen to be checked eventually, i.e. OD450 value is higher, and SAH content is lower in sample to be tested.In experimentation
In, use a series of SAH for the gradient known quantities prepared as standard items in same elisa plate, as standard curve.According to it
OD450 reading obtains curvilinear equation with known standard concentration, then sample OD450 substitution equation is acquired its corresponding concentration value.
The methyl transferase activity real-time assay, the sample source containing transmethylase is in genetic engineering table
Transmethylase in the sample that is purified out in the product that reaches, biological tissue cell, histocyte, biological fluid or tissue are thin
Born of the same parents' culture solution, biological fluid are blood, blood plasma, serum, saliva, urine, cerebrospinal fluid, abdominal cavity or pleural effusion, tissue fluid.
There is provided kits matched with the above method for another object of the present invention, comprising: is coated with anti-S- adenyhomotype
The microwell plate of cysteine antibody or AdoHcy antigen；The anti-AdoHcy of tracer-labelling
The AdoHcy antigen of antibody or tracer-labelling；AdoHcy standard items；Guarantee with S- gland
Glycosides methionine is that the transmethylase of methyl donor has the buffer of biological activity, s-adenosylmethionine, methyl to be measured transfer
The corresponding acceptors substance of enzyme；Positive quality control product transmethylase；Tracer detection system.
Compared with prior art, the beneficial effects of the present invention are:
1, the content of product SAH can be measured directly to directly reflect the ability of the synthesis SAH of MT；
2, SAH is generated in the present invention and measurement is completed at the same time in the same system, there is no lag, it is as a result accurate and
When；
3, not only special but also very sensitive with determination of immunological methods SAH, not by SAH existence form be mating type
With the influence of sequestered；
4, compared with prior art, the present invention does not need specific apparatus, merely with the microplate reader in Routine Test Lab
It is detected；
Therefore, make MT determination of activity more accurate, reliable, direct, simple and quick using kit of the invention, it can
MT activity and SAH content in sample are obtained simultaneously.
Compared with prior art, key point of the invention is:
1. simultaneously method, i.e., MT enzymatic chemically react and generate key product dosing process put together and meanwhile into
Row, for the very low sample of MT activity, especially then it is necessary to be re-introduced into the detection of the quantitative amount of product of immunological response progress for this.
And during immune response, the chemical reaction of MT catalysis is still carrying out, i.e., constantly has new product SAH to generate.This hair
Bright key point is combined into one catalysis reaction and immune response to reach optimum detection sensitivity.
2. most accurately, without any delay ground reflection active dynamic change of MT, since product SAH is unstable, if
It cannot be measured in first time, can not effectively, reliably and correctly calculate MT enzymatic activity.It can using this method
In the shortest possible time, while MT activity and SAH content in sample being measured.
3. substrate specificity corresponding with MT to be measured need to be provided, the activity of MT in sample can be measured using this method.
4. the combination of immunological method and biochemical reaction enables the substance in biochemical reaction to be had
Effect quickly, specifically, is sensitively measured.
5. enabling this, method sensitively reflects the reaction formula of liquid of MT activity change simultaneously, has both guaranteed the life of zymetology catalysis
Object chemical reaction can be normally carried out, and immune detection step is effectively implemented.
The present invention also has made intensive studies and is inquired into from the following using HMT as major experimental enzyme:
First: determine anti-SAH antibody with s-adenosylmethionine and another substrate whether there is or not intersecting, to avoid cross reaction to competing
The interference of ELISA is striven, if it exists cross reaction, then needs to set control removal cross reaction bring background.
Second: groping condition of different pH reaction buffer and potassium ion, BSA etc. and SAH's is generated to methyl transferase catalytic
It influences.
Third: grope the optium concentration of MT concentration and substrate.
4th: the true MT suitable reactions time, and suitable periods are set and survey SAH reaction production quantity.
5th: testing the optium concentration for optimizing each component repeatedly, reaction time, buffer formulation and pH value, standard
Curve and standard items range etc..
Therefore, the present invention is groped by a series of conditions, finds a set of biochemical reaction for being suitable for MT catalysis and measurement SAH
Immune response carry out simultaneously, and obtain the optimal reaction system and condition of optimum detection effect.
Above-mentioned technology path has obtained abundant detailed confirmation in the following example.It is not difficult to see from it that the present invention relates to
And the superiority of the method arrived includes but is not limited to following: (1) the directly important products SAH of measurement MT rather than secondary species because
But most directly reflect the SAH synthesis capability of MT；(2) SAH generates and measures a step in the same system and is completed at the same time, energy
It is enough to capture the active variation of MT under different situations well, the enzyme reaction time is preferably controlled, thus result can be very accurate
And it is timely；(3) specificity, sensitivity are used, all very high anti-SAH antibody of affinity quantifies SAH product in time, not only specifically, and
And it is very sensitive, it is not influenced by SAH existence form (mating type and sequestered)；(4) convenient, fast, therefore it is very beneficial for
Various factors is studied on the active influence of MT in sample, even if influence is very delicate, can also determine and.
Important meaning of the invention is, provides new MT measuring method and product, enable researcher at any time
It is convenient and accurately measure MT enzymatic activity, help to understand in depth to the various relevant research-and-development activities of MT enzyme, for
Methionine metabolism is answered, internal methylation status (methylation procedure) is the discussion of the methylation mechanism in various situations, apparently
The further investigation of science of heredity, metabolic disease, the generation of tumour, development, prognosis, nutrition, the research and monitoring of disease and health
There is great meaning.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below to embodiment or existing
Attached drawing needed in technical description is briefly described.
Fig. 1: direct competitive ELISA step and component part schematic diagram.Show that coating to what is combined on plate is anti-in figure
The former and example of non-antibody.
Fig. 2: SAH standard curve schematic diagram.It is bottom that wherein horizontal axis, which is with 10, the logarithm of SAH standard concentration, and the longitudinal axis is
OD450/OD450Zero competitionWith 1-OD450/OD450Zero competitionRatio natural logrithm value, or be LOGIT.
Specific embodiment
It further elaborates to the present invention below with reference to experimental result and data.The raw material that do not mention is customary commercial
Change reagent, is available on the market.
The anti-SAH antibody of embodiment 1 and homocysteine methyltransgerase substrate SAM and Hcy cross reaction
Experimental material
The anti-SAH antibody of HRP enzyme mark: Arthus Biosystems, MAH00301；Bovine serum albumin(BSA) BSA-SAH:
Arthus Biosystems,ACT00301；AdoHcy (SAH) standard items: Arthus Biosystems,
AST00301；
S-adenosylmethionine: Sigma, A2408,；Homocysteine methyltransgerase (HMT): Beijing love must believe biology
Technology Co., Ltd. Abt-P-005；BSA, Tris-Cl, NaCl, NaH2PO4, Na2HPO4,Na2HPO4.12H2O: Beijing is magnificent to be added
At Bioisystech Co., Ltd；KCl: Tianjin great Mao chemical reagent factory；Homocysteine (Hcy), ProClin 300:
Sigma；TMB developing solution: Huzhou Ying Chuan Bioisystech Co., Ltd；Sulfuric acid: Hu'nan Kangdou Pharmaceutical Co., Ltd.；96 holes are enzyme-linked
Plate: U.S. Corning high adsorbs elisa plate item.
Preparation of reagents:
According to initial experiment, BSA-SAH peridium concentration used in experimental system is 0.2ug/ml, the detection model of SAH
It encloses for 0-1uM, and enzyme reaction SAH generated has exceeded the detection range, therefore the peridium concentration of SAH antigen is adjusted again
After whole, the detection range of SAH is expanded as into 0-10uM, the peridium concentration of BSA-SAH is 1ug/ml, facilitates the various conditions in later period
Investigation.
Enzyme reaction buffer solution: 100mM PB, 100mM KCl, BSA 0.1% adjusts pH7.80, pH8.0, pH8.5；SAH standard
Product (SAHNa): 10uM, 5uM, 2.5uM, 1.25uM, 0.625uM, 0.3125uM, 4uM, 0uM, buffer are that 7.80 enzyme of pH is anti-
Answer buffer；SAH quality-control product (SAHNa): 4uM, buffer are pH7.80 enzyme reaction buffer solution.
Take coating BSA-SAH antigen ELSIA lath, SAH standard curve with 0,0.3125,0.625,1.25,2.5,5,
10uM standard items, SAM and Hcy intersect object concentration 0,10,50,100,500uM.The anti-SAH enzyme labelled antibody 1:10000 HRP of HRP-
Diluted.Every hole adds 50ul after standard items and the anti-SAM antibody dilution of the intersection every hole 50ul of analog Hcy and SAM, HRP-.
37 DEG C of reaction 1h, 100ul TMB37 DEG C develops the color 15min after board-washing, and terminate liquid is read after adding 50ul.As a result as follows:
The reactivity (A/A0) of the anti-SAH monoclonal antibody of table 2 and SAM and Hcy
| SAH uM | SAH mark is bent | SAM | Hcy | uM | Crossing-over rate |
| 0 | 1 | 1 | 1 | 0 | |
| 0.625 | 1.040477 | 1.032789 | 1.070128 | 10 | 50% |
| 1.25 | 0.939679 | 0.9205 | 1.024436 | 50 | 10% |
| 2.5 | 0.821427 | 0.920895 | 0.988759 | 100 | 5% |
| 5 | 0.678864 | 0.433362 | 0.69237 | 500 | 1% |
| 10 | 0.524789 | ||||
| Crossing-over rate | 1%~5% | < 1% |
A/A0；Reactivity (OD450 is read when A0 is 0nM, and A is the OD450 reading in free small molecule competition hole)
Crossing-over rate=(50% inhibition of C free antigen/C intersects object 50% and inhibits) * 100%
From the point of view of result, the cross reaction of Hcy and anti-SAH monoclonal antibody is much smaller than 1%, and SAM and anti-SAH Dan Ke
Intersect although grand antibody exists, intersect substantially in 1%~5% range, therefore, experiment can add only SAM's when implementing
Experimental group is to remove background caused by cross reaction.
Standard curve example according to Fig.2, it is bottom that horizontal axis, which is with 10, the logarithm of SAH standard concentration, and the longitudinal axis is
OD450/OD450Zero competitionWith 1-OD450/OD450Zero competitionNatural logrithm value.Sample OD450 is substituted into equation after the curve of fitting
It can be acquired and correspond to SAH concentration value.
Embodiment 2: external test HMT activity optimum buffer system
The elisa plate item for taking coating BSA-SAH antigen, respectively with the 20mM of pH7.79, pH8.04, pH8.23, pH8.39
100mM PB buffer SAM containing 20uM, the 20uM Hcy of Tris buffer and pH7.0, pH7.4, pH7.8, pH8.0 and
The HMT system reaction solution of 1ug/ml HMT, the every hole 50ul of antibody of reaction solution every hole 50ul, HRP label.37 DEG C of reaction 20min,
100ul TMB is added in every hole after board-washing, reads after terminate liquid 50ul is added after 37 DEG C of colour developing 15min.Wherein SAH standard curve is used
0,0.3125,0.625,1.25,2.5,5,10uM standard items, and pH7.8PB buffer and pH 8.25Tris buffer are used respectively
It prepares.Detected mark song is fitted, and calculates separately the production quantity of each experimental group SAH.As a result as shown in the table:
HMT is catalyzed SAH and generates comparison under 3 difference pH of table, buffer conditions
The production quantity of SAH is indicated by detected value removal background value.As can be known from the above results: (1) under condition of different pH,
When substrate, enzyme amount are constant, the SAH production quantity in the Tris buffer of pH8.38 and the PB buffer of pH7.8 be respectively Tris and
Highest in PB buffer；(2) with the raising of pH, the production quantity of SAH is improved to some extent, but as pH rises to centainly
After range, the raising of SAH production quantity is not very significant；(3) Tris and PB buffer is compared, the SAH in PB buffer is generated
It measures slightly higher.
To investigate influence of the ion addition to HMT system, tentatively with the PB buffer K of pH7.8+, BSA investigate.As a result such as
Shown in following table:
4 various concentration K of table+, HMT catalysis SAH generates comparison (PB buffer) under the conditions of BSA
From the above results: (1) K+Addition remarkable result is had no to the generation for promoting SAH, but in contrast,
The K of 100mM+To a small amount of facilitation of the generation of SAH, and the K of slightly higher concentration+As 200mM will cause the production quantity of SAH to reduce；
(2) within the scope of a certain concentration production quantity of SAH can improve for the addition of BSA, but with the increase of BSA concentration, this promotion
Effect will gradually weaken.
5 various concentration K of table+、Mg2+Under the conditions of HMT catalysis SAH generate comparison (Tris buffer)
Due to Mg2+Stronger hydrolysis will occur in PB solution, therefore be not Mg in PB buffer2+Investigation, and
K is added in Tris+、Mg2+Result also indicate that: (1) Mg2+Concentration increases to 200mM from 25mM, under the production quantity of SAH will continue
Drop, illustrates Mg2+Methyl transferase catalytic can not be promoted to synthesize SAH.Therefore the enzyme reaction used in the experiment in later period is slow
Fliud flushing does not add Mg2+；(2)K+Influence, the K similar to the trend in PB buffer that additive amount generates SAH+Concentration be
When 100mM, the generation of SAH is higher, but on the whole the production quantity of SAH lower than not adding K+Experimental group；(3) in Tris
Either add K+Or Mg2+, the production quantity of SAH is lower in the production quantity ratio PB buffer of SAH.
In summary experimental result, later experiments will select 0.1%BSA, 100mM K+, the PB buffer conduct of pH7.80
Enzyme reaction buffer solution.
Embodiment 3: the enzyme concentration and concentration of substrate of external test HMT
Take coating BSA-SAH antigen ELSIA lath, with enzyme reaction buffer solution pH7.8 prepare SAH standard items 0,0.3125,
0.625,1.25,2.5,5,10uM and 4uM Quality Control.And prepare SAM, Hcy and HMT enzyme formula of various concentration.Standard items and enzyme
The every hole 50ul of antibody of reactant every hole 50ul, HRP label.37 DEG C of reaction durations take 20min, 30min, 40min, after board-washing
It is read after 100ul TMB 37 DEG C of colour developings 15min, terminate liquid 50ul.
HMT catalysis SAH generates comparison under the different concentration of substrate of table 6, HMT concentration conditions
According to upper table as a result, the increase of SAM concentration will cause the increase of background value, this is by SAM and anti-SAH monoclonal
Caused by the cross reaction of antibody.Meanwhile the increase of SAM concentration will remarkably promote the increase of the production quantity of SAH, but in enzyme concentration
And Hcy concentration it is certain under conditions of, SAM's continues to increase, and the conversion ratio for being converted into SAH decreases, thus from
From the aspect of background value and cost two, the concentration of SAM is unsuitable excessively high.Opposite SAM, the promotion that the increase of Hcy concentration generates SAH
It acts on weaker.And the raising of HMT enzyme concentration is overall, and the amount of SAH can be made slightly to improve, but raising amount is not shown
It writes.
Further to investigate the influence that Hcy concentration and enzyme concentration generate SAH, the concentration of SAM is set to 20uM, is prepared not
With the reaction solution of the Hcy and HMT enzyme of concentration.
The generation comparison of SAH under 7 difference Hcy concentration of table, HMT concentration conditions
When the concentration of HMT enzyme is 0.3ug/ml, Hcy concentration is improved, and the production quantity of SAH increased, especially when Hcy's
When concentration is increased to 60uM from 40uM, the production quantity of SAH is dramatically increased, and when the concentration of Hcy continues to be increased to 80uM,
The production quantity of SAH is begun to decline, and the concentration of HMT be 0.5ug/ml and 1ug/ml when trend be also such.Therefore Hcy is most
It is good that 60uM is not to be exceeded using concentration.And for the use concentration of HMT, under conditions of SAM and Hcy are 20uM, from
The generation of 0.3ug/ml to 0.5ug/ml, SAH dramatically increase, when HMT enzyme being further increased to 1ug/ml, the generation of SAH
It still improves to some extent, but compares the experiment of previous step, SAM and Hcy are 20uM, and HMT is in 2ug/ml, the generation of SAH
Amount is not much different with 1ug/ml experimental group.
Embodiment 4: external test HMT activity optimum reaction period determines
Take coating BSA-SAH antigen ELSIA lath, with enzyme reaction buffer solution pH7.8 prepare SAH standard items 0,0.3125,
0.625,1.25,2.5,5,10uM and 4uM Quality Control.Prepare SAM and Hcy 20uM, HMT enzyme 1ug/ml, SAM and Hcy 20uM,
HMT enzyme 0.7ug/ml, SAM and Hcy 20uM, HMT enzyme 0.5ug/ml, (being final concentration) is as generation SAH substrate and enzyme amount.
The every hole 50ul of antibody of standard items and enzyme reaction object every hole 50ul, HRP label.37 DEG C of reaction durations take 20min, 40min,
60min is read after 100ul TMB 37 DEG C of colour developings 15min, terminate liquid 50ul after board-washing.
The 8 differential responses time of table catalyzes and synthesizes the influence of the amount of SAH to HMT
It shows from the above, the results show that HMT enzyme generates SAH amount in 20-60min, SAH amount is without significant change, base
This is in plateau, illustrate after 20min may concentration of substrate it is inadequate or SAH generated produces product to HMT enzyme
Inhibit, causes SAH production quantity without increase.
Embodiment 5: the MT activity of normal liver cell system L02
Experimental material:
Sterile PBS, 0.25% pancreatin (Aladdin), 1640 culture mediums (Gibco) of 10%FBS (first Heng Shengma), 75cm2
Square vase (Corning), phosphate buffer (PBS): NaCl 8g, Na2HPO4.12H2O 2.885g, KCl 0.2g, KH2PO4
0.2g, ultrapure water 1000ml；Pancreatin (0.25% trypsase): 200 μ l, 1 × PBS of trypsase 0.1g, 4%EDTA solution are molten
Liquid 39.8ml；Trypsase uses 0.2 μm of aseptic filtration head (Pall) filtering after completely dissolution.(4% bent sharp benzene is blue for trypan blue solution
Solution, Aladdin): bent benefit benzene indigo plant 1.6g, ultrapure water 40ml are filtered with filter paper.
Experimental procedure:
1. observation square vase inner cell is in 80% or so convergence degree, after 1640 culture mediums of 10% fetal calf serum are removed
It is washed one time with PBS；
2. the pancreatin of appropriate volume is added, guarantee that pancreatin covers entire square vase bottom；
3. square vase is placed in 37 DEG C, 5%CO22-3min in incubator is added 5ml and contains when cell starts fall in flakes
1640 culture mediums of 10%FBS terminate.
4. being resuspended after centrifugation with 1ml or so PBS, trypan blue is counted；
5. taking the ultrasonication in ice bath of 1ml group of cells suspension, the centrifuging and taking supernatant at 15000g.
6. taking coating BSA-SAH antigen ELSIA lath, the peridium concentration of BSA-SAH is 1ug/ml, with enzyme reaction buffer solution
PH7.8 prepares SAH standard items 0,0.3125,0.625,1.25,2.5,5,10uM and 4uM Quality Control.
7. preparing SAM 5uM and 20uM and SAM 5uM&Hcy20uM, SAM respectively using clasmatosis liquid as enzyme reaction solution
The reaction solution of 20uM&Hcy 20uM, standard items and the anti-SAH enzyme labelled antibody 1:10000 HRP of enzyme reaction object every hole 50ul, HRP-
Diluted, every hole 50ul, 37 DEG C of reaction 20min.After board-washing after 100ul TMB 37 DEG C of colour developings 15min, terminate liquid 50ul
Reading.In conjunction with trypan blue counting as a result, the cell concentration of L02 is adjusted to 108Cells, and correspondingly by SAH
The reaction production quantity being adjusted under the density.
The SAH that the MT of 9. normal liver cell system L02 of table is catalyzed and synthesized
| SAH(uM) | |
| SAM 5uM | 1.618283 |
| SAM 20uM | 2.718398 |
| SAM 5uM+Hcy 20uM | 2.694052 |
| SAM 20uM+Hcy 20uM | 3.724836 |
As seen from the above table, SAM is added, there is SAH generation, illustrates that the MT in cell pyrolysis liquid has been catalyzed the methyl transfer of SAM,
To generate SAH, the concentration of SAM increases, and the generation of SAH also has a degree of increase.And add again on the basis of adding SAM
Add Hcy, the production quantity of SAH also has increase, this illustrates that there are a certain amount of HMT in the cell pyrolysis liquid, but also there are other
The MT of type.It suffices to show that and is measured with this method using SAM as the exploitativeness of a kind of methyl transferase activity of methyl donor.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (6)
1. a kind of methyl transferase activity real-time assay, which is characterized in that including two simultaneously in a reaction system
The reaction of progress: (1) in the buffering for guaranteeing to have biological activity using s-adenosylmethionine as the transmethylase of methyl donor
In system, the sample containing transmethylase, methyl donor and corresponding substrate is added or is directly containing transmethylase
Liquid sample in SAM methyl donor and corresponding substrate is added, react to generate AdoHcy and produce
Object, substrate are the acceptor substance of methyl；(2) using the content of immunological method detection SAH, to determine first in reaction system
The activity of based transferase；
Utilize the content of the competition law of envelope antigen method and coated antibody method two ways measurement AdoHcy；
1) using envelope antigen method, specific step is as follows:
(1) preparation of various concentration standard items: taking AdoHcy standard items, with buffer at different dense
The standard curve sample of degree；
(2) in the microwell plate for being coated with albumen-SAH conjugate or polymer-SAH conjugate, horseradish peroxidase is added
The anti-AdoHcy monoclonal antibody of enzyme or alkali phosphatase enzyme mark, contains s-adenosylmethionine, methyl to be measured
The buffer solution system of the sample of transferase and Methyl acceptors substance corresponding with transmethylase to be measured；If it is liquid-like
Product are then directly added into s-adenosylmethionine, the sample of transmethylase to be measured and methyl corresponding with transmethylase to be measured and connect
Receptive material；The SAH standard items of prepared various concentration gradient are added in standard curve hole；It is reacted after mixing, board-washing；
(3) substrate of horseradish peroxidase or alkaline phosphatase is added, colour developing is added terminate liquid and terminates reaction, in enzyme mark
Appropriate wavelength is selected to read the absorbance reading OD value in every hole on instrument；
(4) the SAH standard items that a series of known quantities prepared are used in same elisa plate, according to its OD value and known standard
Product concentration obtains curvilinear equation, then sample OD value substitution equation is acquired to the production quantity of its corresponding reaction product SAH；
(5) it is catalyzed the concentration of generation product SAH within the unit time according to the sample containing transmethylase to calculate methyl
The activity of transferase；
Buffer is to include potassium concentration range in 25-200mM, and BSA addition concentration is 7 in 0 .1%-0 .9%, pH range
.0-8 Tris the or PB buffer system of .5；
The standard concentration of the various concentration for making standard curve of buffer be respectively 0,0 .3125,0 .625,
1 .25,2 .5,5,10μM；
Concentration range after methyl donor is added in liquid sample or buffer is 10-30 μM, the concentration model after substrate addition
Enclose is 10-60 μM；The volume of buffer solution system or liquid sample that each micropore is added is 50 μ l, the tracer-labelling after dilution
The volume of the antibody of note is 50 μ l；
Reaction time and temperature are respectively 37 DEG C and 20 minutes；
2) coated antibody method: anti-SAH antibody incubation will first be added in sheep or the coated microwell plate of rabbit anti-mouse igg, or directly wrap
By anti-SAH antibody, board-washing；The SAH antigen of tracer-labelling is added, adds antigen to be checked, antigen to be checked, that is, biochemical reaction produces
Raw product SAH, the SAH of antigen and tracer-labelling to be checked compete the anti-SAH specific antibody being coated on elisa plate, are incubated for
Board-washing；Substrate colour developing is added, in continuous mode, uses a series of SAH of gradient known quantities of preparation as standard items, production mark
Directrix curve.
2. methyl transferase activity real-time assay according to claim 1, which is characterized in that using SAH antigen
Analog substitution SAH antigen carries out coupling or tracer-labelling.
3. methyl transferase activity real-time assay according to claim 1, which is characterized in that buffer be include potassium
Ion concentration is 100mM, and BSA adds 0 .1% of concentration, and pH is the PB buffer system of 7 .8.
4. methyl transferase activity real-time assay according to claim 1, which is characterized in that liquid sample is slow
20 μM of concentration after methyl donor is added in fliud flushing, 20 μM of concentration after substrate addition.
5. methyl transferase activity real-time assay according to claim 1, which is characterized in that contain transmethylase
Sample source in the sample being purified out in the product, biological tissue cell of gene engineering expression, the methyl in histocyte
Transferase, biological fluid or histocyte culture solution；Biological fluid is blood, blood plasma, serum, saliva, urine, cerebrospinal fluid, abdomen
Chamber or pleural effusion, tissue fluid.
6. the matched kit of the described in any item methyl transferase activity real-time assays of claim 1-5, feature exist
In, comprising: it is coated with the microwell plate of anti-AdoHcy antibody or AdoHcy antigen；Tracer
The anti-AdoHcy antibody of label or the AdoHcy antigen of tracer-labelling；S- adenyhomotype
Cysteine standard product；Guarantee using s-adenosylmethionine as the transmethylase of methyl donor have biological activity buffer,
The corresponding acceptors substance of s-adenosylmethionine, transmethylase to be measured；Positive quality control product transmethylase；Tracer analyte detection
System.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610549785.7A CN106053795B (en) | 2016-07-13 | 2016-07-13 | A kind of methyl transferase activity real-time assay and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610549785.7A CN106053795B (en) | 2016-07-13 | 2016-07-13 | A kind of methyl transferase activity real-time assay and kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106053795A CN106053795A (en) | 2016-10-26 |
| CN106053795B true CN106053795B (en) | 2019-02-12 |
Family
ID=57185745
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610549785.7A Active CN106053795B (en) | 2016-07-13 | 2016-07-13 | A kind of methyl transferase activity real-time assay and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106053795B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825551B (en) * | 2019-02-21 | 2022-08-02 | 深圳大学 | Method for evaluating histone lysine demethylase activity |
| CN112415196A (en) * | 2020-11-25 | 2021-02-26 | 刘世杰 | System and application for analyzing molecular activity |
| CN113533745A (en) * | 2021-07-21 | 2021-10-22 | 苏州立禾生物医学工程有限公司 | Homocysteine detection kit and detection method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103954774A (en) * | 2014-05-09 | 2014-07-30 | 山东博科生物产业有限公司 | Stable homocysteine detection kit |
| CN104046682A (en) * | 2013-03-13 | 2014-09-17 | 苏州博泰安生物科技有限公司 | Homocysteine (HCY) detection method and diagnostic kit thereof |
| CN104479022A (en) * | 2014-10-23 | 2015-04-01 | 湖南天合生物技术有限公司 | Anti-S-adenosyl-L-homocysteine monoclonal antibody 301, hybridomas thereof, composition, colloidal gold test paper, kit and uses |
| CN105296596A (en) * | 2015-11-17 | 2016-02-03 | 山东博科生物产业有限公司 | Enzymatic homocysteine detection kit with strong stability |
| CN105445464A (en) * | 2015-11-07 | 2016-03-30 | 湖南天合生物技术有限公司 | Method for measuring biological activity of methionine adenosyltransferase and kit |
-
2016
- 2016-07-13 CN CN201610549785.7A patent/CN106053795B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046682A (en) * | 2013-03-13 | 2014-09-17 | 苏州博泰安生物科技有限公司 | Homocysteine (HCY) detection method and diagnostic kit thereof |
| CN103954774A (en) * | 2014-05-09 | 2014-07-30 | 山东博科生物产业有限公司 | Stable homocysteine detection kit |
| CN104479022A (en) * | 2014-10-23 | 2015-04-01 | 湖南天合生物技术有限公司 | Anti-S-adenosyl-L-homocysteine monoclonal antibody 301, hybridomas thereof, composition, colloidal gold test paper, kit and uses |
| CN105445464A (en) * | 2015-11-07 | 2016-03-30 | 湖南天合生物技术有限公司 | Method for measuring biological activity of methionine adenosyltransferase and kit |
| CN105296596A (en) * | 2015-11-17 | 2016-02-03 | 山东博科生物产业有限公司 | Enzymatic homocysteine detection kit with strong stability |
Non-Patent Citations (1)
| Title |
|---|
| "银杏类黄酮O甲基转移酶活性的高效液相色谱分析;仲月明等;《食品工业科技》;20120831;第33卷(第8期);第95-98,116页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106053795A (en) | 2016-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200333327A1 (en) | Method and system for determining integrated metabolic baseline and potential of living cells | |
| Allen et al. | A semisynthetic epitope for kinase substrates | |
| Matschinsky et al. | Quantitative histochemical analysis of glycolytic intermediates and cofactors with an oil well technique | |
| Llenado et al. | Surfactants | |
| CN105445464B (en) | A kind of methionine adenosyltransferase Determination of biological activity method and kit | |
| CN106053795B (en) | A kind of methyl transferase activity real-time assay and kit | |
| CN104630324B (en) | Improved homocysteine detection reagent and method | |
| US20180364249A1 (en) | Method of quantitative determination of sarcosine in a biological sample | |
| SK281517B6 (en) | Method for assaying homocysteine and homocysteine assay | |
| US20100151481A1 (en) | Cell-signaling assays | |
| Gercel-Taylor | Diphenylamine assay of DNA fragmentation for chemosensitivity testing | |
| Kricka | Chemiluminescence and bioluminescence | |
| Dresler et al. | 2', 3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: evidence for involvement of DNA polymerase. delta. | |
| Goud et al. | Direct real-time measurement of intra-oocyte nitric oxide concentration in vivo | |
| EP1592813A2 (en) | Assay method for group transfer reactions | |
| CN105603049A (en) | Compound stabilizer and kit for in vitro diagnosis reagents | |
| Wang et al. | A new assay of phosphorylase based on the filter paper technique | |
| CN103575914B (en) | The enzyme linked immunological kit of detection vitamin B12 and application thereof | |
| CN113549673B (en) | Methods involving testing for lysosomal storage disorders | |
| Blake | Target validation in drug discovery | |
| CA2468275C (en) | Assaying apparatus, kit, and method for lipids and associated enzymes | |
| EA012438B1 (en) | A method and kit for determination of thymidine kinase activity and use thereof | |
| CN106680512B (en) | A kind of quick detection kit of NMP-22 and its application | |
| WO2009069980A2 (en) | Protein chip for determining kinase or phosphatase activity | |
| EP0106615B1 (en) | Assay for the free portion of substances in biological fluids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210623 Address after: Room 2006, building a, data building, 799 Yaocheng Avenue, Taizhou City, Jiangsu Province, 225300 Patentee after: Taizhou Huifeng Hetai Biotechnology Co.,Ltd. Address before: 410000 Hunan Province Economic and Technological Development Zone Changsha Panpan Road No. 9 Patentee before: Hunan Skyworld Biotechnologies Co. |
|
| CI03 | Correction of invention patent | ||
| CI03 | Correction of invention patent |
Correction item: Patentee|Address Correct: Taizhou Huifeng Hetai Biotechnology Co.,Ltd.|Room 2006, building a, data building, 799 Yaocheng Avenue, Taizhou City, Jiangsu Province, 225300 False: Taizhou Huifeng Hetai Biotechnology Co.,Ltd.|Room 2006, building a, data building, 799 Yaocheng Avenue, Taizhou City, Jiangsu Province, 225300 Number: 28-01 Volume: 37 |