CN107219201B - Utilize the immune rear bullfrog antibody of fluorescence microscope citrinin and affine method - Google Patents
Utilize the immune rear bullfrog antibody of fluorescence microscope citrinin and affine method Download PDFInfo
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- CN107219201B CN107219201B CN201710259433.2A CN201710259433A CN107219201B CN 107219201 B CN107219201 B CN 107219201B CN 201710259433 A CN201710259433 A CN 201710259433A CN 107219201 B CN107219201 B CN 107219201B
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- citrinin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
Abstract
The present invention relates to food safety detection technologies, it is desirable to provide a method of the rabphilin Rab being immunized using fluorescence microscope citrinin.It include: the protein extract fetched from citrinin antibody mediated immunity group bullfrog and control group bullfrog, it is each that citrinin standard items are added, it is stood in refrigerator after mixing；Then bag filter is respectively charged into dialyse；The reservation drop in bag filter is taken to be placed in fluorescence microscope in glass slide center；Filter plate, the size of analysis of fluorescence spot are excited using blue green light.The present invention identifies citrinin binding protein by measurement fluorescence spot area in the form of the bullfrog haemocyanin and muscle protein after fluorescence microscopy observation citrinin is immune.Have the characteristics that relative to traditional identification method it is quick, accurate, direct, to the morphologic observation of bullfrog antibody and muscle rabphilin Rab, the protein-bonded quick screening of citrinin have important practical significance.
Description
Technical field
The present invention utilizes fluorescence microscope tangerine according to the absorption principle between fluorescent material and special rabphilin Rab matter
Bullfrog rabphilin Rab, belongs to food safety detection technology after mycin is immune.
Background technique
Citrinin (Citrinin) is a kind of mycotoxin.The molecular structure of citrinin is the planar structure of conjugation, is had
Natural fluorescent characteristic.Rabbit, rat, mouse are the animals of the common immune antiboidy for preparing citrinin.In order to identify antibody
Whether protein is in conjunction with citrinin, and someone is using sepectrophotofluorometer respectively to the citrinin solution of antibody and citrinin mark
Quasi- solution carries out fluorescent scanning, by comparing the variation of wavelength corresponding to fluorescence intensity maximum emission peak in fluorescence spectrum, with
This determine antibody whether with citrinin covalent bond.Its principle is the light of the substituent structure that connects on material molecule to compound
Large effect can be generated by learning performance, and wavelength corresponding to compound fluorescence spectrum maximum emission peak is made to change.This side
Method may determine that the two binding ability, but this method can not observe the form of citrinin rabphilin Rab in bullfrog.At present
Research for Amphibia animal immune globulin molecule is mainly based on the research to Africa xenopus.It has IgM, lgX,
Five class of IgY and IgD, IgF.IgM is earliest caused, most important immunoglobulin in the Xenopus laevis history of life.IgX in structure with
IgM is similar, exists in the form of polymeric, is functionally similar to the IgA of mammal, mainly expresses in enteron aisle, in blood
Content is very low in clear.IgY occurs later in the Xenopus laevis history of life.IgD and IgF is two kinds of immunoglobulins point of latest find
Son, IgD only have faint expression in spleen, and structure is identical as other animal IgD；IgF genome and other immunoglobulins point
Sub different, heavy chain gene only has there are two structure, and function does not have research also.By electrophoretic analysis, there are two for batrachia antibody
Class, respectively low molecular weight 7S and high molecular weight 18S, existing research prove that batrachia 18S antibody is similar to people IgM, and about 7S
There are no unified final conclusions for the function of antibody.The antibody of amphibian animal and the antibody of mammal have very big difference, antibody
So far it is not yet fully apparent from.Bullfrog antibody is not reported even more.In addition medically there is super quick phenomenon, it is considered that be because of certain
The haptens of kind small molecule forms comlete antigen with in-vivo tissue protein binding in vivo, thus the generation of induction of antibodies.But
It is the report of never which kind of specific histone, also lacks evidence of the histone in conjunction with haptens.This method provides
A kind of method for directly observing bullfrog antibody protein and histone.
Summary of the invention
The technical problem to be solved by the present invention is to overcome deficiency in the prior art, provide a kind of using fluorescence microscope
The method for observing the immune bullfrog rabphilin Rab of citrinin, for observing rabphilin Rab whether in conjunction with citrinin, combination degree,
And the form of rabphilin Rab.
In order to solve the technical problem, solution of the invention is:
There is provided a kind of method of rabphilin Rab being immunized using fluorescence microscope citrinin comprising following steps:
(1) each 1mL of protein extract from citrinin antibody mediated immunity group bullfrog and control group bullfrog is fetched, in extracting solution
Protein content is 2.4~3.6mg/mL；The citrinin standard items that 1mL concentration is 1.5 μ g/mL are respectively added into extracting solution, mix
3h is stood in 4 DEG C of refrigerators afterwards；Then it is respectively charged into bag filter, and the PBS that 0.01M, pH7.4 of 20mL is respectively added is used as dialysis
Liquid, in 4 DEG C of refrigerators dialysis 13h；
(2) the reservation liquid in 10 μ L bag filters is respectively taken, drop is placed in light microscope in glass slide center and covered
In；Filter plate 490-520nm is excited using blue green light, microscope magnification is 40 × 10 times, and scale bar is 100 μm；It uses
The size of 6.0 software analysis of fluorescence spot of Image-Pro Plus；
As the fluorescence spot area of immune group haemocyanin extracting solution is greater than 77 times of citrinin titer fluorescence spot area
More than, show that the antibody protein of immune group bullfrog can be in conjunction with citrinin；If fluorescence spot area ratio is not met
Condition is stated, shows that the antibody protein of immune group bullfrog cannot be in conjunction with citrinin；Such as immune group muscle rabphilin Rab extracting solution
Fluorescence spot area be greater than 6 times of citrinin titer fluorescence spot area or more, show the affine egg of the muscle of immune group bullfrog
White matter can be in conjunction with citrinin；If fluorescence spot area ratio does not meet above-mentioned condition, show the muscle of immune group bullfrog
Rabphilin Rab matter cannot be in conjunction with citrinin.
In the present invention, the protein extract is haemocyanin extracting solution or muscle protein extracting solution.
Invention realization principle:
Fluorescence microscopy can carry out highly sensitive detection to cell component by fluorescent marker, in life science
It is widely used.Material molecule during generating fluorescence, wherein excitation photon and transmitting photon between energy difference
(i.e. Stokes shift) is the key that fluorescent microscopic imaging.Pass through color filter array (i.e. excitation filter plate and the pressure of fluorescence microscope
Filter plate processed) exciting light is filtered off completely and does not block the fluorescence launched, it can observe the substance for only generating fluorescence.Use fluorescence
Microscope pair is observed with the citrinin after protein combination, analyzes its imaging contexts, can observe rabphilin Rab
Form and judge whether citrinin can be in conjunction with antibody protein.
Compared with prior art, the beneficial effects of the present invention are:
The present invention in the form of the bullfrog haemocyanin and muscle protein after fluorescence microscopy observation citrinin is immune,
And citrinin binding protein is identified by measurement fluorescence spot area, belong to pionerring research.Relative to traditional identification side
Method, this method have the characteristics that quick, accurate, direct.Morphologic observation of the present invention to bullfrog antibody and muscle rabphilin Rab, tangerine
The protein-bonded quick screening of mycin has important practical significance.
Detailed description of the invention
The case where Fig. 1 is fluorescence microscope citrinin standard items.
The case where Fig. 2 is 33% supernatant of fluorescence microscope (bullfrog) protein combination citrinin.
Fig. 3 is the case where fluorescence microscope 33% precipitates (bullfrog) protein combination citrinin.
Fig. 4 is the case where fluorescence microscope 33% precipitates (bullfrog) protein combination citrinin.
The case where Fig. 5 is fluorescence microscope bullfrog muscle protein combination citrinin.
Illustrate: green fluorescence (light speckle regions in figure) are presented in citrinin, and standard items light color spot is small (Fig. 1).
It is main in the supernatant of 33% ammonium sulfate precipitation in mammalian serum according to the principle of ammonium sulfate precipitation protein purification
It is IgM, the beta Globulins such as IgA.Observe that a kind of huge energy combines tangerine in the supernatant of 33% ammonium sulfate precipitation of bullfrog serum
The spherical antibody (Fig. 2) of mycin, which is IgM.It is mainly the γ such as IgY in 33% ammonium sulfate precipitation in mammalian serum
Globulin.There are two types of antibody in 33% ammonium sulfate precipitation of bullfrog serum, and one is the lesser spherical antibody of diameter, remaining tangerine is mould
Plain binding ability is weaker (Fig. 3).There is the antibody combined strongly with citrinin there are also a kind of Y type, which is IgY (Fig. 4).
Also there is a kind of little albumen that can combine citrinin in bullfrog muscle, light spot is smaller (Fig. 5).
Specific embodiment
With reference to the accompanying drawing, realization process of the invention is described in detail.
(1) prepared by red yeast rice culture solution:
Using isolated monascus anka mould from Hongqu powder (red colouring agent), (Hongqu powder (red colouring agent) has from Fujian Province's Gutian County brightness honor industry and trade
Limit company), it is inoculated into MSG liquid culture medium and cultivates.Every bottle takes 50mL MSG culture medium, is inoculated with 20 μ L thallus Monascus ankas
Mould, the isolation of bottleneck gauze ball cultivates 5d at 30 ± 1 DEG C.The content of citrinin reaches 40.4 μ g/mL.
(2) bullfrog is immune:
Commercially available bullfrog is supported in bucket, every barrel of 3.8-4.2kg bullfrog, and water and bullfrog weight ratio are 1:1, and addition 1mL tangerine is mould
Cellulose content is the red yeast rice culture solution of 40.4 μ g/mL, room temperature cultivation.It can natural death successively cultivating later period bullfrog.After immune
8-24d takes bullfrog of being at death's door, and focuses on one barrel, and every 10min observation takes just dead bullfrog, adopts in 15min after death
Bullfrog ventrimeson blood vessel is selected in blood, blood sampling position.And after blood sampling in 1h with the speed centrifugal treating 15ml of 3000r/min, collect
Supernatant.
(3) extraction purification of serum proteins
Immunoglobulin is extracted using saturated ammonium sulfate substep salting out method.The preparation of saturated ammonium sulfate solution: sulfuric acid is weighed
Ammonium 450g is dissolved in 500mL pure water, until being heated to most solute dissolutions, is filtered while hot, is set ambient temperature overnight, then use
28% ammonium hydroxide tune pH to 7.0.The preparation of 0.01M PBS (pH7.4): 2.9g Na is weighed2HPO4·12H2O, 0.27g K2HPO4,
0.2g KCl, 8.0g NaCl, ultrapure water dissolution, is settled to 1000mL, and heating is boiled, cooling stand-by.
To adopt blood sample is placed in 10mL centrifuge tube, be stored at room temperature 1h, take supernatant after 3000r/min centrifugation 15min, this
For serum to be checked.Purified using ammonium sulfate precipitation method to serum to be checked: 20mL PBS (0.01M is added in serum 20mL to be checked
PH7.4), it is gently mixed mixing with glass bar on ice, saturated ammonium sulfate solution 40mL is added dropwise while stirring, keeps ammonium sulfate dense eventually
Degree is 50%, and 4 DEG C of refrigerators stand 3h or more, 8000r/min, 4 DEG C, are centrifuged 30min, abandons supernatant；Precipitating 20mL PBS
(0.01M pH7.4) dissolution, saturated ammonium sulfate solution 13.33mL is added dropwise while stirring, makes ammonium sulfate final concentration of 40%, and 4 DEG C
Refrigerator stands 1h or more, 8000r/min, 4 DEG C, is centrifuged 30min, abandons supernatant；Precipitating is molten with 20mL PBS (0.01M pH7.4)
Solution, saturated ammonium sulfate solution 9.85mL is added dropwise while stirring makes ammonium sulfate final concentration of 33%, and 4 DEG C of refrigerators stand 1h or more,
8000r/min 4 DEG C, is centrifuged 30min, retains supernatant and precipitating.It is dissolved and is precipitated with 15mL PBS (0.01M pH7.4), precipitating
Bag filter is filled respectively with supernatant, sets 4 DEG C of refrigerator dialysis desalting 3h.Take bag filter internal protein.
(4) muscle tissue proteins extract
Method before reference takes the bullfrog of just natural death during immune cultivation 8-24d, after death in 15min
Leg muscle tissue 10g is taken, shreds, is put into triangular flask, 100mL PBS (0.01M pH7.4,1mMEDTA, 1mM coke phosphorus is added
Sour sodium, 1%Tween-20), ultrasonication 30min takes supernatant, as musculature is total after 10000r/min is centrifuged 20min
Protein extract, 4 DEG C of refrigerators are sealed spare.
(5) fluorescence microscope of citrinin conjugated protein
The supernatant protein of 33% ammonium sulfate precipitation of control bullfrog and citrinin immune group bullfrog, 33% sulphur are taken respectively
The muscle tissue proteins 1mL that sour ammonia-sinking is formed sediment is put in 10mL glass tube that (protein content in extracting solution is 2.4~3.6mg/
ML), the citrinin standard items that 1mL concentration is 1.5 μ g/mL are added thereto respectively and are put in 4 DEG C of ice after vortex oscillator mixes
Case acts on 3h, and mixed liquor is packed into bag filter, is put in 50mL centrifuge tube, then 20mL 0.01M PBS is added to centrifuge tube
(pH7.4) it is used as dialyzate, is put in 4 DEG C of refrigerator dialysis, dialysis time 13h.Clean glass slide and coverslip are taken, pipettor is used
After drawing dialysis, 10 μ L of liquid is retained in bag, glass slide center is placed in, is put in fluorescence microscope.100 μm of use ratio ruler.
Observation excites filter plate 490-520nm using blue green light, and microscope magnification is 40 × 10 times.
Size through Image-Pro Plus6.0 software analysis of fluorescence spot, the maximum of citrinin titer fluorescence spot
Diameter is 17 μm, and globular protein matter IgM combination citrinin fluorescence spot diameter is 277 μ in the supernatant of 33% ammonium sulfate precipitation
M, the fluorescence spot diameter of globular protein matter combination citrinin is 168 μm in 33% ammonium sulfate precipitation, Y shape protein I gY combination tangerine
The fluorescence spot of mycin is long and width is 265 μm and 66 μm.The fluorescence spot diameter of muscle protein combination citrinin is 43 μm.This
The combination of a little protein and citrinin, shows the stronger fluorescence signal more concentrated.And it compares with bovine serum albumin(BSA) to tangerine
Mycin does not generate suction-operated, will not generate the green fluorescence of blotch.Antibody diameter is generally 3-15nm, nothing under microscope
Method observation.But antibody or rabphilin Rab absorption have the citrinin of fluorescence, generate huge light amplification, it can be glimmering
Under light microscope, observing protein form and bonding state.The Supernatant protein combination citrinin fluorescence of 33% ammonium sulfate precipitation
Speck area is 265 times of citrinin titer speck area, protein binding citrinin fluorescence spot face in 33% ammonium sulfate precipitation
Product is 97 times and 77 times of citrinin titer speck area.Muscle protein combination citrinin fluorescence spot area is citrinin standard
6.4 times of liquid speck area.
Therefore, the method for the invention may finally be judged according to following principles:
As the fluorescence spot area of immune group haemocyanin extracting solution is greater than 77 times of citrinin titer fluorescence spot area
More than, show that the antibody protein of immune group bullfrog can be in conjunction with citrinin；If fluorescence spot area ratio is not met
Condition is stated, shows that the antibody protein of immune group bullfrog cannot be in conjunction with citrinin.Such as immune group muscle rabphilin Rab extracting solution
Fluorescence spot area be greater than 6 times of citrinin titer fluorescence spot area or more, show the affine egg of the muscle of immune group bullfrog
White matter can be in conjunction with citrinin；If fluorescence spot area ratio does not meet above-mentioned condition, show the muscle of immune group bullfrog
Rabphilin Rab matter cannot be in conjunction with citrinin.
Claims (2)
1. utilizing the immune rear bullfrog antibody of fluorescence microscope citrinin and affine method, which is characterized in that including following
Step:
(1) each 1 mL of protein extract from citrinin antibody mediated immunity group bullfrog and control group bullfrog, the egg in extracting solution are fetched
Bai Hanliang is 2.4 ~ 3.6 mg/mL；The citrinin standard items that 1 mL concentration is 1.5 μ g/mL are respectively added into extracting solution, mix
3 h are stood in 4 DEG C of refrigerators after even；Then it is respectively charged into bag filter, and the PBS of 0.01 M, pH7.4 of 20 mL is respectively added
As dialyzate, in 4 DEG C of 13 h of refrigerators dialysis；
(2) the reservation liquid in 10 μ L bag filters is respectively taken, drop is placed in fluorescence microscope in glass slide center and covered；
Filter plate 490-520 nm is excited using blue green light, microscope magnification is 40 × 10 times, and shooting picture scale bar is 100 μm；
Use the size of Image-Pro Plus6.0 software analysis of fluorescence spot；
If the fluorescence spot area of immune group haemocyanin extracting solution is 77 times of area of citrinin titer fluorescence spot or more, table
The antibody protein of bright immune group bullfrog can be in conjunction with citrinin；If fluorescence spot area ratio does not meet above-mentioned condition,
Show that the antibody protein of immune group bullfrog cannot be in conjunction with citrinin；Such as the fluorescent spot of immune group muscle rabphilin Rab extracting solution
Selecting area is 6 times of area of citrinin titer fluorescence spot or more, shows that the muscle rabphilin Rab matter of immune group bullfrog can be with
Citrinin combines；If fluorescence spot area ratio does not meet above-mentioned condition, show the muscle rabphilin Rab matter of immune group bullfrog
It cannot be in conjunction with citrinin.
2. the method according to claim 1, wherein the protein extract is haemocyanin extracting solution or muscle
Protein extract.
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| WO2000045165A1 (en) * | 1999-02-01 | 2000-08-03 | Cytovia, Inc. | Methods of identifying therapeutically effective antineoplastic agents with cultured cells having intact cell membranes and corresponding products |
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| CN105738629A (en) * | 2015-12-28 | 2016-07-06 | 中华人民共和国上海出入境检验检疫局 | Indirect immuno-fluorescence detection method of yellow fever virus IgG antibody |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130045185A1 (en) * | 2011-08-18 | 2013-02-21 | Dupont Nutrition Biosciences Aps | Strains and methods useful for mycotoxins |
| SG11201500370SA (en) * | 2012-07-20 | 2015-02-27 | Agency Science Tech & Res | <i>IN VITRO</i> ASSAY FOR PREDICTING RENAL PROXIMAL TUBULAR CELL TOXICITY |
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2017
- 2017-04-20 CN CN201710259433.2A patent/CN107219201B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000045165A1 (en) * | 1999-02-01 | 2000-08-03 | Cytovia, Inc. | Methods of identifying therapeutically effective antineoplastic agents with cultured cells having intact cell membranes and corresponding products |
| CN1715924A (en) * | 2005-07-25 | 2006-01-04 | 福建农林大学 | Method for detecting tobacco mosaic virus using phycoerythrin fluorescent probe |
| CN105738629A (en) * | 2015-12-28 | 2016-07-06 | 中华人民共和国上海出入境检验检疫局 | Indirect immuno-fluorescence detection method of yellow fever virus IgG antibody |
Non-Patent Citations (3)
| Title |
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| Fluorescence spectroscopic investigation of the interaction of citrinin with native and chemically modified cyclodextrins;Miklós Poór,et al;《Journal ofLuminescence》;20151130;第172卷;第23-28段 * |
| 免疫荧光球;程晓光 等;《湖南医学院学报》;19800630;第5卷(第2期);第181页 * |
| 大鼠储精囊分泌蛋白Ⅱ在精子表面的荧光定位研;张胜 等;《生殖与避孕》;19901231;第10卷(第2期);第200页 * |
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