CN109030821A - A kind of antibody, kit and its application method of lactobacillus plantarum bacteriocin - Google Patents
A kind of antibody, kit and its application method of lactobacillus plantarum bacteriocin Download PDFInfo
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- CN109030821A CN109030821A CN201810795938.5A CN201810795938A CN109030821A CN 109030821 A CN109030821 A CN 109030821A CN 201810795938 A CN201810795938 A CN 201810795938A CN 109030821 A CN109030821 A CN 109030821A
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention relates to antibody, kit and its application methods of a kind of lactobacillus plantarum bacteriocin.The antibody of lactobacillus plantarum bacteriocin of the present invention obtains through amino acid sequence protein immunization as shown in SEQ ID NO:1, can accurately and efficiently detect lactobacillus plantarum bacteriocin, has the advantages that specific good, high sensitivity.
Description
Technical field
The present invention relates to field of biological detection, in particular to antibody, the reagent of a kind of lactobacillus plantarum bacteriocin
Box and its application method.
Background technique
Bacteriocin is as a kind of safe and efficient natural biological bacteriostatic agent, in recent years in food preservation and antibacterial class drug
The application of the industries such as exploitation has been a great concern.Bacteriocin (bacteriocins) is certain bacteriogenic with suppression
Polypeptide, protein or the protein complex of bacterium bioactivity.Bacteriocin is initially from gramnegative bacterium Escherichia coli
It was found that.Produced bacteriocin is metabolized by lactic acid bacteria and is referred to as lactein, is a kind of safe protein matter, it can be by egg
White enzymic digestion, on eukaryocyte without influence.It can inhibit the growth of certain pathogens and spoilage organisms simultaneously, such as: inhibiting single in food
The pathogenic bacteria such as nucleus monocytogenes, staphylococcus aureus, helicobacter pylori and salmonella.
The development object that lactein can become natural sandy gravel is based on following characteristics: 1. its producing strains lactic acid
Bacterium growth and breeding is fast, with short production cycle, has industrial production prospect；2. having human protease's biological degradability, Bu Hui
Accumulation, will not have an impact the normal flora of human body intestinal canal in human body；3. being different from antibiotic meeting drug resistance, will not lure
Pathogenic microorganisms is sent out to the resistance of antibiotic；4. bacteriocin gene is modified and is cloned convenient for modern genetic engineering technology.
The separation source majority of lactic acid bacteria is therefore traditional fermented food and human body intestinal canal with probiotic properties have antibacterial activity
Lactic acid bacteria and its metabolite are considered as basic security.
Lactococcus lactis rhzomorph Nisin and pediocin Pediocin is the lactic acid of current large-scale commercial application
Bacterium bacteriocin is allowed to be mainly used in processed cheese, milk product and tank as food preservative by more than 80 a countries
In head product.
Applicant screens from natural fermented meat products and obtains a lactobacillus plantarum.It is demonstrate,proved by antibacterial experiment in vitro
Real, lactobacillus plantarum, which can be metabolized, generates a kind of bacterium fibroin, is named as lactobacillus plantarum bacteriocin, the bacterium fibroin
To Listeria monocytogenes (Listeria monocytogenes), enterococcus faecium (Enterococcus
Facealis), Escherichia coli (Escherichia coli), salmonella (Salmonella enteritidis), shiga
Bacterium (Shigella dysenteriae) etc. all has certain fungistatic effect.Since lactobacillus plantarum bacteriocin is to large intestine bar
The Gram-negative bacterias such as bacterium, salmonella, Shigella all have certain fungistatic effect, relative to Nisin in food antiseptic
Preservation field has more extensive application prospect.
And limiting lactobacillus plantarum bacteriocin greatest problem present in food fresh keeping application at present is that there are no build
The quantitative detecting method of lactobacillus plantarum bacteriocin in vertical food.Since lactobacillus plantarum bacteriocin is a kind of protein,
The immunology quantitative detecting method established for lactobacillus plantarum bacteriocin is particularly important.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of antibody of lactobacillus plantarum bacteriocin, the antibody can it is accurate,
Efficiently detect the lactobacillus plantarum bacteriocin.
The second object of the present invention is to provide a kind of kit, and the kit includes afore mentioned antibodies, can it is accurate,
Lactobacillus plantarum is efficiently detected, further, the kit is using antibody M301 as coated antibody, antibody M123 conduct
Antibody is detected, the two successful matching has the advantages that accuracy is good, high sensitivity when detecting lactobacillus plantarum.
The third object of the present invention is to provide a kind of detection method of lactobacillus plantarum bacteriocin, the method use
Afore mentioned antibodies or kit.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of antibody of lactobacillus plantarum bacteriocin, the antibody is through amino acid sequence albumen as shown in SEQ ID NO:1
It is immune to obtain.
In some specific embodiments, the antibody is M301 or M123, wherein the antibody M301 is by preservation
The hybridoma that number is CGMCC No.15194 generates, and the antibody M123 is the miscellaneous of CGMCC No.15193 by deposit number
Tumor is handed over to generate.
The invention further relates to a kind of kit, the kit includes afore mentioned antibodies.
In some specific embodiments, the kit includes antibody M301 and antibody M123, wherein described anti-
Body M301 is as coated antibody, and the antibody M123 is as detection antibody.
In some specific embodiments, the antibody M123 and horseradish peroxidase, it is preferable that pass through
The coupling of periodates oxidizing process.
In some specific embodiments, the kit at least further includes one or more of: ELISA Plate is washed
Wash liquid, confining liquid, developing solution and terminate liquid.
In some specific embodiments, the cleaning solution be include 0.03%v/v~0.07%v/v (preferably,
0.05%v/v) the PBS buffer solution of Tween20.
In some specific embodiments, the confining liquid be include 0.7%w/v~1.2%w/v (preferably, 1%w/
V) PBS buffer solution of bovine serum albumin(BSA), 0.07%v/v~0.12%v/v (preferably, 0.1%v/v) Tween20.
In some specific embodiments, the developing solution includes substrate colour developing A liquid and substrate colour developing B liquid, wherein
Every 500mL substrate colour developing A liquid includes 12~15g of sodium acetate (preferably 13.6g), 1~2g of citric acid (preferably 1.6g), often
500mL substrate colour developing B liquid includes: 0.15~0.25g of disodium ethylene diamine tetraacetate (preferably 0.2g), 0.8~1.0g of citric acid
(preferably 0.95g), 45~55mL of glycerine (preferably 50mL), 0.1~0.2g of TMB2HCl (preferably 0.15g).
In some specific embodiments, the terminate liquid is 1.5M~2.5M (preferably, 2M) sulfuric acid solution.
The invention further relates to a kind of method for detecting lactobacillus plantarum bacteriocin, the method uses afore mentioned antibodies or preceding
Kit is stated to detect lactobacillus plantarum albumen.
In some specific embodiments, which comprises
(1) antibody M301 is coated with to ELISA Plate；
(2) sample to be tested, negative control and positive control are separately added into ELISA Plate, constant-temperature incubation for a period of time after,
It is washed with cleaning solution；
(3) be added enzyme labelled antibody M123, constant-temperature incubation for a period of time after, washed with cleaning solution；
(4) substrate colour developing A liquid is mixed in equal volume with substrate colour developing B liquid, is added in ELISA Plate, room temperature is protected from light incubation one
Terminate liquid is added after the section time and terminates reaction, and measures its OD450Value.
In some specific embodiments, the method is specifically included:
(1) with the amount in 75~125 holes μ L/ (preferably 100 holes μ L/) by concentration be 0.1 μ of μ g/mL~30 g/mL (preferably
It is coated with for 5 μ g/mL) antibody M301 to ELISA Plate；
(2) sample to be tested, negative control and positive control are separately added into ELISA Plate with 75~125 holes μ L/, 35
DEG C~40 DEG C (preferably 37 DEG C) under, 25~35min of constant-temperature incubation (preferably 30min) is washed 2~4 times with cleaning solution later
(preferably 3 times)；
(3) by enzyme labelled antibody 123 by (1:5000)~(1:20000) dilution after (preferably 1:10000), 35 DEG C~
Under 40 DEG C (preferably 37 DEG C), 25~35min of constant-temperature incubation (preferably 30min) is washed with cleaning solution later；
(4) substrate colour developing A liquid is mixed in equal volume with substrate colour developing B liquid, with 75~125 hole μ L/ (preferably 100 μ L/
Hole) amount by mixed liquor be added ELISA Plate in, room temperature is protected from light 10~20min of incubation (preferably 15min) and terminate liquid 40 is added afterwards
The hole μ L/~60 holes μ L/ (preferably 50 holes μ L/) terminates reaction, and measures its OD450。
Detailed description of the invention
It, below will be to tool in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Body embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing be some embodiments of the present invention, for those of ordinary skill in the art, what is do not made the creative labor
Under the premise of, it is also possible to obtain other drawings based on these drawings.
Fig. 1 is the immunizing potency of 4 mouse described in embodiment 2；
Fig. 2 is the standard curve of dual anti-sandwich ELISA detection method described in embodiment 5.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
It will be appreciated that the following example is merely to illustrate the present invention, and it is not construed as limiting the scope of the invention.It is not specified in embodiment
Actual conditions person carries out according to conventional conditions or manufacturer's recommended conditions.Production firm is not specified in agents useful for same or instrument
Person is that can buy the conventional products obtained by city.
Following embodiments are specifically described the preparation method and double crush syndrome inspection of lactobacillus plantarum monoclonal antibody
The foundation of survey system.
The synthesis and coupling of 1 antigen of embodiment
Albumen shown in following sequence is synthesized according to lactobacillus plantarum bacterium avidin sequence, and is coupled KLH albumen respectively
(pKYCS-KLH) and BSA albumen (pKYCS-BSA):
KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATGGHQGNHKC (SEQ ID NO:1).
2 animal immune of embodiment
It is mixed for the first time with equivalent Freund's complete adjuvant, subcutaneously with " pKYCS-KLH " by 60 μ g albumen/mouse amount
The Balb/c female mice that 4 SPF grades of initial immunity.It every two weeks, is mixed with equivalent freund 's incomplete adjuvant, reinforces one
It is secondary immune, reinforce altogether 4 times, carries out eye socket later and take blood, survey its serum titer.
Bioactivity: with the pKYCS-BSA of 1 μ g/ml, 4 DEG C of 96 orifice plates of coating are stayed overnight；2% milk, 37 DEG C of closing 1h；Blood
2 times of gradient dilutions clearly since 200 times, blank control (blank) are PBS, and negative control (negative) is negative serum
200 times of dilutions.The result shows that lactobacillus plantarum bacteriocin potency highest (Fig. 1) in #4 mice serum, therefore select #4 as thin
Born of the same parents' fusion experiment material.
The experiment of 3 cell fusion of embodiment
It chooses impact #4 mouse and does cell fusion experiment, with " pKYCS-KLH " 50 μ g, #4 mouse is impacted in abdominal cavity, and step is such as
Shown in lower:
1) it is blown and beaten from culture bottle wall by sp2/0 cell in good condition is soft, is drawn into 50ml centrifuge tube
In.
2) mouse plucks eyeball and takes blood, then neck is drawn to put to death, is put into 75% alcohol and impregnates 5min.
3) DMEM that a small amount of serum-free is poured into plate, cell sieve and plunger are put into plate.Use scissors
The spleen that mouse is removed with tweezers, is put into cell sieve.Lightly spleen is sufficiently pulverized with the inner core of syringe, by what is ground
Cell is drawn into the centrifuge tube of dress sp2/0, and 1500r/min is centrifuged 5min.
4) thymus gland that mouse is removed with scissors and tweezers, pulverizes.By the thymocyte ground into 15ml centrifuge tube, then
The HAT of 1ml is added, is placed on spare in incubator.
5) cell that will be centrifuged outwells supernatant, and cell is carefully gently blown to even, centrifugation with the DMEM of serum-free
(1500r/min, 5min).
6) cell conditioned medium being centrifuged is outwelled as far as possible.The abundant suspension cell in centrifuge tube bottom is patted, centrifuge tube is put into 37
In DEG C warm water, it is slowly added to the PEG of 1ml in 1 minute, after adding, 1min is stood in warm water.Then slowly add in 2min
Enter the DMEM of the serum-free of 2ml, the DMEM of 8ml serum-free is then slowly added in 2min, 1000r/min is centrifuged 5min.
7) supernatant is outwelled, the serum of 10ml is added, careful blow cell is even, pours into the ready thymocyte in front.
The sterilized semisolid culturemedium of 25ml is added, is mixed well.Then it uniformly pours into 30 Tissue Culture Dish.By cell
Culture dish is put into wet box, is then placed in incubator and is cultivated.
Embodiment 4, which is chosen, clones and is screened
Choose 10 plate × 93 cell monoclonals, be incubated at 96 porocyte culture plates (in advance with thymocyte bed board,
The hole 100ul/).Experimental procedure is as follows:
1) " pKYCS-BSA " is diluted with coating buffer, final concentration of 2 μ g/ml, 100 holes μ L/, 4 DEG C, overnight；After use washing lotion
Washing 3 times.2) 2% milk confining liquid is closed, 200 holes μ l/, 37 DEG C of incubators, 2h；It is washed 3 times with washing lotion afterwards.3) primary antibody is added
(cells and supernatant), negative control (SP2/0 culture supernatant), blank control (PBS), positive control (positive serum PBS
1000 times of dilutions), it is 100 holes μ L/, 37 DEG C of incubators, 1h；It is washed 3 times with washing lotion afterwards.4) PBS is added and dilutes 20000 times
Secondary antibody, 100 holes μ L/, 37 DEG C of incubators, 1h；It is washed 3 times after taking-up with washing lotion.5) it develops the color, 100 hole μ L/ of developing solution, developing time
For 5min or so.6) every hole is added 50 μ L terminate liquids and terminates.7) dual wavelength (450,630) surveys light absorption value, and record saves data.
7 plants of positive cell strains are filtered out, it is final to determine that two plants of cells generate antibody M301 and M123 and match detection
Lactobacillus plantarum bacteriocin, and preservation is carried out to the hybridoma cell strain F301 and F123 that produce aforementioned two kinds of antibody and is specifically protected
It is as shown in table 1 to hide information:
1 hybridoma preservation information of table
The foundation of 5 double crush syndrome detection architecture of embodiment
1, the preparation of related solution
Cleaning solution (PBS containing 0.05%Tween 20): 500 μ L of Tween-20 is added in 1L PBS.Confining liquid (contains
1% bovine serum albumin(BSA), the PBS of 0.1%Tween 20): 100mgBSA, 10 μ LTween-20 are added in 10mLPBS.
Detection antibody: by horseradish peroxidase and and the monoclonal antibody for M123 is numbered using periodates oxidation
Method is coupled.
Developing solution: substrate colour developing A liquid and B liquid mixed in equal amounts are kept in dark place, matching while using.Substrate colour developing A liquid: sodium acetate
13.6g, citric acid 1.6g, 30% hydrogen peroxide 0.3mL, distilled water add to 500mL.Substrate colour developing B liquid: ethylenediamine tetra-acetic acid two
Sodium 0.2g, citric acid 0.95g, glycerine 50mL, TMB2HCl 0.15g, distilled water add to 500mL.
Terminate liquid: 2mol/L H2SO4。
2, the foundation of ELISA system
With Checkerboard titration method determine M301 as coated antibody and detect antibody M123 best operating condition, from
As can be seen that be coated with 5 μ g/mL as M301 in table 2, and enzyme labelled antibody using concentration for 1:10000 when, OD value (1.085)
Close to 1.0, and negative control is lower, and P/N value is bigger, so conditions above is ELISA system best effort amount.
2 Checkerboard titration experimental result of table
3, ELISA system detection limit measurement
(1) it is coated on plate in advance with 100 hole μ L/ 5 μ g/mL M301.
(2) add sample to be tested: 100 hole μ L/ of sample to be tested is added on coated plate.Positive control is the plant of purifying
Object lactobacillus bacteriocin, negative control are solvent.37 DEG C of insulating boxs incubate 30min.
(3) it is washed 3 times, is dried with cleaning solution.
(4) enzyme mark antiantibody: 1:10000 dilutes enzyme labelled antibody M123, and 100 holes μ L/ cover 37 DEG C of insulating boxs and incubate
30min。
(5) it is washed 5 times with cleaning solution.
(6) it develops the color: adding 100 hole μ L/ of developing solution of Fresh, room temperature dark place is placed 15min, is displayed in blue.
(7) reaction, colorimetric are terminated: adding 50 hole μ L/ terminate liquids.Colour changed into yellow.
(8) with the light absorption value, that is, OD450nm in each hole at microplate reader measurement 450nm.
Optimized and detection, the bacteriocin that the ELISA detection kit as shown in Figure 2 can at least detect are 2ppb.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；
Although present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that:
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of technologies
Feature is equivalently replaced；And these are modified or replaceed, the present invention that it does not separate the essence of the corresponding technical solution is each to be implemented
The range of example technical solution.
SEQUENCE LISTING
<110>Beijing Agricultural College
<120>a kind of antibody, kit and its application method of lactobacillus plantarum bacteriocin
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 44
<212> PRT
<213> Lactobacillus plantarum
<400> 1
Lys Tyr Tyr Gly Asn Gly Val Thr Cys Gly Lys His Ser Cys Ser Val
1 5 10 15
Asp Trp Gly Lys Ala Thr Thr Cys Ile Ile Asn Asn Gly Ala Met Ala
20 25 30
Trp Ala Thr Gly Gly His Gln Gly Asn His Lys Cys
35 40
Claims (10)
1. a kind of antibody of lactobacillus plantarum bacteriocin, which is characterized in that the antibody is through amino acid sequence such as SEQ ID NO:1
Shown protein immunization obtains.
2. antibody according to claim 1, which is characterized in that the antibody is M301 or M123, wherein the antibody
M301 is generated by the hybridoma that deposit number is CGMCC No.15194, and the antibody M123 is CGMCC by deposit number
The hybridoma of No.15193 generates.
3. a kind of kit, which is characterized in that the kit includes antibody as claimed in claim 1 or 2.
4. kit according to claim 3, which is characterized in that the kit includes antibody M301 and antibody M123,
Wherein, the antibody M301 is as coated antibody, and the antibody M123 is as detection antibody.
5. kit according to claim 4, which is characterized in that the antibody M123 and horseradish peroxidase,
Preferably, it is coupled by periodates oxidizing process.
6. kit according to claim 4, which is characterized in that the kit at least further includes following a kind of or more
Kind: ELISA Plate, cleaning solution, confining liquid, developing solution and terminate liquid.
7. kit according to claim 6, which is characterized in that the cleaning solution be include 0.03%v/v~0.07%
The PBS buffer solution of v/v (preferably, 0.05%v/v) Tween20；
The confining liquid be include 0.7%w/v~1.2%w/v (preferably, 1%w/v) bovine serum albumin(BSA), 0.07%v/v~
The PBS buffer solution of 0.12%v/v (preferably, 0.1%v/v) Tween20；
The developing solution includes substrate colour developing A liquid and substrate colour developing B liquid, wherein every 500mL substrate colour developing A liquid includes sodium acetate
12~15g (preferably 13.6g), 1~2g of citric acid (preferably 1.6g), every 500mL substrate colour developing B liquid includes: ethylenediamine tetraacetic
0.15~0.25g of acetic acid disodium (preferably 0.2g), 0.8~1.0g of citric acid (preferably 0.95g), 45~55mL of glycerine
(preferably 50mL), TMB2HCl0.1~0.2g (preferably 0.15g)；
The terminate liquid is 1.5M~2.5M (preferably, 2M) sulfuric acid solution.
8. a kind of method for detecting lactobacillus plantarum bacteriocin, which is characterized in that the method uses described in as claimed in claim 1 or 22
Any one of antibody or claim 1~7 kit detect lactobacillus plantarum albumen.
9. according to the method described in claim 8, it is characterized in that, which comprises
(1) antibody M301 is coated with to ELISA Plate；
(2) sample to be tested, negative control and positive control are separately added into ELISA Plate, constant-temperature incubation for a period of time after, with washing
Wash liquid washing；
(3) be added enzyme labelled antibody M123, constant-temperature incubation for a period of time after, washed with cleaning solution；
(4) substrate colour developing A liquid is mixed in equal volume with substrate colour developing B liquid, is added in ELISA Plate, room temperature is protected from light incubation a period of time
Terminate liquid is added afterwards and terminates reaction, and measures its OD450Value.
10. according to the method described in claim 9, it is characterized in that, the method specifically includes:
(1) with the amount in 75~125 holes μ L/ (preferably 100 holes μ L/) by concentration be 0.1 μ of μ g/mL~30 g/mL (preferably 5 μ g/
ML) antibody M301 is coated with to ELISA Plate；
(2) sample to be tested, negative control and positive control are separately added into ELISA Plate with 75~125 holes μ L/, 35 DEG C~40
Under DEG C (preferably 37 DEG C), 25~35min of constant-temperature incubation (preferably 30min) washs 2~4 times (preferably with cleaning solution later
3 times)；
(3) by enzyme labelled antibody M123 by (preferably 1:10000) after (1:5000)~(1:20000) dilution, at 35 DEG C~40 DEG C
Under (preferably 37 DEG C), 25~35min of constant-temperature incubation (preferably 30min) is washed with cleaning solution later；
(4) substrate colour developing A liquid is mixed in equal volume with substrate colour developing B liquid, with 75~125 holes μ L/ (preferably 100 holes μ L/)
Mixed liquor is added in ELISA Plate amount, and room temperature is protected from light 10~20min of incubation (preferably 15min) and 40 hole μ L/ of terminate liquid is added afterwards
~60 holes μ L/ (preferably 50 holes μ L/) terminate reaction, and measure its OD450。
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