CN105784984A - Preparation method and application of gram-positive bacterium ghost - Google Patents

Preparation method and application of gram-positive bacterium ghost Download PDF

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CN105784984A
CN105784984A CN201610116309.6A CN201610116309A CN105784984A CN 105784984 A CN105784984 A CN 105784984A CN 201610116309 A CN201610116309 A CN 201610116309A CN 105784984 A CN105784984 A CN 105784984A
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gram
positive bacteria
preparation
bacterium shadow
suspension
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CN105784984B (en
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鞠兴荣
都立辉
吴学友
袁建
何荣
陈正行
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Nanjing University of Finance and Economics
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Nanjing University of Finance and Economics
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    • 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
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria

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Abstract

The invention provides a preparation method and application of a gram-positive bacterium ghost, and belongs to the field of microbiology and immunology.The preparation method of the ghost includes the steps that NaOH, SDS and CaCO3 are added into gram-positive bacterium suspension, incubation is carried out at 30-40 DEG C, centrifugation is carried out, precipitate is collected, and after washing, resuspension A is prepared; H2O2 is added into the resuspension A, incubation is carried out at 30-40 DEG C, centrifugation is carried out, precipitate is collected, and after washing, resuspension B is prepared; the resuspension B is incubated at 20-30 DEG C, centrifugation is carried out, precipitate is collected, washing is carried out, and the gram-positive bacterium ghost is obtained.According to the preparation method, the gram-positive bacterium ghost can be rapidly and efficiently prepared with simple chemical reagents, and thus production cost is significantly reduced.As the integrity of a bacterium structure is retained in a Listeria monocytogenes ghost, antibodies for Listeria monocytogenes can be generated after immunization, and thus antibodies capable of efficiently and rapidly detecting Listeria monocytogenes can be obtained through screening.

Description

The preparation method and applications of gram-positive bacteria bacterium shadow
Technical field
The invention belongs to microbiology and field of immunology, be specifically related to the preparation side of gram-positive bacteria bacterium shadow Method.
Background technology
Bacteria simulacrum (Bacterial Ghost) is a kind of empty bacterial body not having cytoplasm and nucleic acid, and it remains The integrality of bacterial structure, remains former somatic surface antigen determinant, screens after therefore may be used for immune animal For the antibody of this bacterium, can efficiently, quickly detect the antibody of this bacterium with exploitation.At present, both at home and abroad In terms of the research of bacteria simulacrum, mostly use cracking system based on bacteriophage Φ X174 Lysis gene E Animal nutrition, and it is directed to gramnegative bacterium, such as: Escherichia coli, Salmonella typhimurium Bacterium, Bacterium enteritidis, haemophilus influenzae, Actinobacillus pleuropneumoniae, helicobacter pylori, pneumonia Klebsiella, haemolysis Pasteurella, pasteurella multocida, comma bacillus, Pseudomonas aeruginosa, stench vacation Monad etc..But, the method is for the preparation of gram-positive bacteria bacterium shadow inapplicable.
Summary of the invention
It is an object of the invention to provide one utilizes simple chemical reagent just can quickly, efficiently prepare leather orchid The method of family name's positive bacteria bacterium shadow.
The preparation method of gram-positive bacteria bacterium shadow, comprises the steps:
(1) in Gram-positive bacteria suspension, NaOH, SDS and CaCO are added3, under the conditions of 30-40 DEG C Hatch, centrifugal, collect precipitation, after washing, make re-suspension liquid A；
(2) in described re-suspension liquid A, H is added2O2, hatch under the conditions of 30-40 DEG C, centrifugal, collect precipitation, Re-suspension liquid B is made after washing；
(3) re-suspension liquid B is hatched under the conditions of 20-30 DEG C, centrifugal, collect precipitation, washing, obtain leather orchid Family name's positive bacteria bacterium shadow.
Preferably in technical scheme, the end of the NaOH added in Gram-positive bacteria suspension in step (1) is dense Degree is the final concentration of 0.1-0.3mmol/L of 20-40mmol/L, the SDS of addition, and every liter of gram-positive bacteria hangs CaCO in liquid3Addition be 9-11mmol；In step (1), in 80-120r/min condition when suspension is hatched Lower shaking, incubation time is 40-60min.
Preferably in technical scheme, the H added in re-suspension liquid A described in step (2)2O2Final mass dense Degree is 0.02-0.05%；In step (2), shake under the conditions of 80-120r/min when suspension is hatched, when hatching Between be 20-40min.
Preferably in technical scheme, in step (3), when described re-suspension liquid B is hatched under the conditions of 30-50r/min Shaking, incubation time is 20-40min.
Preferably in technical scheme, physiological saline is used to wash.
Preferably in technical scheme, in step (2), re-suspension liquid B is the second that described precipitation is resuspended in 55-65% Gained in alcohol solution.
Preferably in technical scheme, described re-suspension liquid A is that described precipitation is resuspended in gained after water.
Preferably in technical scheme, described gram-positive bacteria is Listeria monocytogenes.
Preferably in technical scheme, the OD of described Gram-positive bacteria suspension600It is 0.3～0.5.
The present invention also provides for bacterium shadow prepared by said method in preparation for detecting the anti-of described gram-positive bacteria Application in terms of body.
The present invention utilizes simple chemical reagent just can quickly, efficiently prepare gram-positive bacteria bacterium shadow, significantly Reduce production cost.Listeria monocytogenes (is abbreviated as Listeria monocytogenes, Listeria Monocytogenes) it is the pathogen that a kind of danger is serious, serious infectious diseases common to human beings and animals can be caused, Such as symptoms such as meningitis, septicemia, miscarriage and monocytosis, it the listeriosis caused is lethal Rate is more than 25%.Owing to Listeria monocytogenes bacterium shadow remains the integrality of bacterial structure, especially remain former bacterium Surface antigenic determinant, can produce the antibody for Listeria monocytogenes, thus screen energy after immunity Enough antibody efficient, quickly this bacterium of detection.
Accompanying drawing explanation
The SEM observed result of Fig. 1 Listeria monocytogenes bacterium shadow, in figure A, the object of observation is single increasing Liszt Bacterium living cells, in figure B, the object of observation is Listeria monocytogenes bacterium shadow.
The tem observation result of Fig. 2 Listeria monocytogenes bacterium shadow, in figure A, the object of observation is Listeria monocytogenes Living cells, in figure B, the object of observation is Listeria monocytogenes bacterium shadow.
The antibody titer that Fig. 3 Listeria monocytogenes bacterium shadow immune mouse produces.
Detailed description of the invention
Below in conjunction with embodiment, the present invention will be further described, it should be understood that these embodiments are only used In the purpose of illustration, it is in no way intended to limit protection scope of the present invention.Food-borne Gram-positive is chosen in this part Bacterium Listeria monocytogenes (Listeria monocytogenes) is specifically described as embodiment.
Embodiment 1 prepares Listeria monocytogenes bacterium shadow
1. thalline is cultivated and is collected
With 1% (v/v) inoculum concentration, Listeria monocytogenes (Listeria monocytogenes) CMCC54004 (is purchased From Chinese industrial Culture Collection) seed liquor access through 121 DEG C of high pressure steam sterilizations BHI train Support base (purchased from Beijing overpass technical concern Co., Ltd), in 37 DEG C, incubated overnight under the conditions of 150r/min. Bacterium solution after cultivating is centrifugal 20min, supernatant discarded under the conditions of 1500r/min, uses SPSS (0.9%NaCl, analyzes pure, and reagent is purchased from Nanjing Chemistry Reagent Co., Ltd.) washing bacterial sediment.Washing Concrete grammar: with the resuspended thalline of SPSS, under the conditions of 1500r/min, centrifugal 20min, abandons Remove supernatant.Washing step is repeated 3 times.
2. prepared by bacterium shadow cell
(1) take the thalline after washing in step 1, be prepared as OD with sterilized water600Be 0.3～0.4 initial Bacteria suspension, adds the NaOH of final concentration of 27.500mmol/L, the SDS of final concentration of 0.234mmol/L (lauryl sodium sulfate), adds powder CaCO of 10.490mmol in every liter of initial bacteria suspension3, at 37 DEG C In shaking table, hatching 50min under shaking state, shaking speed is 100r/min.Then in 1500r/min bar Under part, centrifugal 20min, supernatant discarded, collect precipitation, uses SPSS according to method in step 1 Wash 3 times, resuspended with sterilized water, obtain re-suspension liquid A.Re-suspension liquid A volume and initial bacteria suspension volume phase Deng.NaOH, SDS add the most in form of an aqueous solutions to initial bacteria suspension.The NaOH aqueous solution dense Degree is 1.170mmol/L for the concentration of 137.5mmol/L, the SDS aqueous solution.OD600Refer to that solution is at 600nm Light absorption value at wavelength.
(2) in re-suspension liquid A, add the H of final concentration of 0.035% (mass percentage concentration)2O2, in 37 In DEG C shaking table, hatching 30min under shaking state, shaking speed is 100r/min.Then in 1500r/min Under the conditions of centrifugal 20min, supernatant discarded, collect precipitation, use sterile physiological salt according to method in step 1 Water washing precipitation 3 times, is then resuspended in 60% (v/v) (concentration expressed in percentage by volume) ethanol water, obtains Re-suspension liquid B.H2O2Add in form of an aqueous solutions to re-suspension liquid A, H2O2The percent mass of the aqueous solution is dense Degree is 0.175%.
(3) by re-suspension liquid B in 25 DEG C of shaking tables, hatch 30min under shaking state, shaking speed is 40 r/min.Then centrifugal 20min, supernatant discarded under the conditions of 1500r/min, collect precipitation, according to step In 1, method uses SPSS to wash 3 times, i.e. obtains Listeria monocytogenes bacterium shadow.
The preparation effect of embodiment 2 Listeria monocytogenes bacterium shadow
In detection embodiment 1, the survival rate of Listeria monocytogenes bacterium shadow prepared by method, is scanned electricity simultaneously Mirror (Scanning Electron Microscope, SEM), transmission electron microscope (Transmission Electron Microscope, TEM) observe.
(1) survival rate
At MSC-AdvantageTMIn two stage biological safety cabinet (purchased from Thermo Scientific), by above-mentioned Preparation bacterium shadow cell sterilized water carry out gradient dilution, extension rate be respectively 100 times, 101Again with 102 Times, then coat (purchased from Beijing overpass technical concern Co., Ltd) on BHI solid medium, in 37 DEG C EYELA constant incubator (purchased from Shanghai lover Instrument Ltd.) in cultivate 5d, every day observes flat Colony growth situation on plate.In triplicate.The results are shown in Table 1, as can be seen from Table 1 prepared by embodiment 1 Listeria monocytogenes bacterium shadow survival rate after cultivating 5d is 0%, i.e. exists without viable bacteria.
Table 1 Listeria monocytogenes bacterium shadow survival rate
Note: in table, data are means standard deviation.
(2) SEM observes
Listeria monocytogenes bacterium shadow SEM (SEM) observed result such as Fig. 1.Can be seen that because of thin Endochylema and nucleic acid are discharged, and the most morphologically Listeria monocytogenes bacterium shadow cell with Listeria monocytogenes living cells is Different.
(3) tem observation
TEM (transmission electron microscope) observed result of Listeria monocytogenes bacterium shadow is as in figure 2 it is shown, can see Cytoplasm has not been had in Listeria monocytogenes bacterium shadow.
The above results illustrates, embodiment 1 is successfully prepared Listeria monocytogenes bacterium shadow.
The immune effect inspection of embodiment 3 Listeria monocytogenes bacterium shadow
Use the Listeria monocytogenes bacterium shadow immune mouse that prepared by method in embodiment 1, exempt from investigating this bacterium shadow Whether Listeria monocytogenes antibody can be produced after epidemic disease.
Physiological saline is used to adjust the cell concentration of Listeria monocytogenes bacterium shadow to 1 × 108Cfu/mL, obtains immunity Reagent.Take 10 6 week old Balb/c mouse, every subcutaneous multi-point injection 2mL immunoreagent.Immunity knot Shu Hou, measures immune mouse antibodies in blood titer.Testing result is fig. 3, it is shown that implement Antibody can be produced after the Listeria monocytogenes bacterium shadow immune mouse that in example 1 prepared by method, after immune 4 weeks, In mouse blood, antibody titer is up to 10000, respond well.

Claims (10)

1. the preparation method of gram-positive bacteria bacterium shadow, it is characterised in that comprise the steps:
(1) in Gram-positive bacteria suspension, NaOH, SDS and CaCO are added3, hatch under the conditions of 30-40 DEG C, centrifugal, collect precipitation, after washing, make re-suspension liquid A；
(2) in described re-suspension liquid A, H is added2O2, hatch under the conditions of 30-40 DEG C, centrifugal, collect precipitation, after washing, make re-suspension liquid B；
(3) re-suspension liquid B is hatched under the conditions of 20-30 DEG C, centrifugal, collect precipitation, washing, obtain gram-positive bacteria bacterium shadow.
The preparation method of gram-positive bacteria bacterium shadow the most according to claim 1, it is characterized in that the final concentration of 20-40mmol/L of the NaOH added in Gram-positive bacteria suspension in step (1), the final concentration of 0.1-0.3mmol/L, CaCO in every liter of Gram-positive bacteria suspension of the SDS added3Addition be 9-11mmol；In step (1), shaking when suspension is hatched under the conditions of 80-120r/min, incubation time is 40-60min.
The preparation method of gram-positive bacteria bacterium shadow the most according to claim 2, it is characterised in that the H added in re-suspension liquid A described in step (2)2O2Final mass percentage concentration be 0.02-0.05%；In step (2), shaking when suspension is hatched under the conditions of 80-120r/min, incubation time is 20-40min.
The most according to claim 3, the preparation method of gram-positive bacteria bacterium shadow, it is characterised in that in step (3), shake when described re-suspension liquid B is hatched under the conditions of 30-50r/min, and incubation time is 20-40min.
5. according to the preparation method of one of claim 1-4 described gram-positive bacteria bacterium shadow, it is characterised in that use physiological saline to wash.
The preparation method of gram-positive bacteria bacterium shadow the most according to claim 5, it is characterised in that in step (2), re-suspension liquid B is that described precipitation is resuspended in gained in the ethanol water of 55-65%.
The preparation method of gram-positive bacteria bacterium shadow the most according to claim 6, it is characterised in that described re-suspension liquid A is that described precipitation is resuspended in gained after water.
8. according to the preparation method of one of claim 1-7 described gram-positive bacteria bacterium shadow, it is characterised in that described gram-positive bacteria is Listeria monocytogenes.
The preparation method of gram-positive bacteria bacterium shadow the most according to claim 8, it is characterised in that the OD of described Gram-positive bacteria suspension600It is 0.3～0.5.
10. the bacterium shadow that prepared by one of claim 1-9 method is in preparation application in terms of detecting the antibody of described gram-positive bacteria.
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CN109852563A (en) * 2019-03-06 2019-06-07 杨凌职业技术学院 A kind of chemical preparation process of eggs crack detection bacterium shadow
CN114921373A (en) * 2022-05-25 2022-08-19 中国农业科学院农业资源与农业区划研究所 Application of rhizobium oryzae in improving salt tolerance of plants

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Publication number Priority date Publication date Assignee Title
CN109852563A (en) * 2019-03-06 2019-06-07 杨凌职业技术学院 A kind of chemical preparation process of eggs crack detection bacterium shadow
CN114921373A (en) * 2022-05-25 2022-08-19 中国农业科学院农业资源与农业区划研究所 Application of rhizobium oryzae in improving salt tolerance of plants

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