CN104846051B - A kind of method of use Vero cell detections toxigenic pasteurella multocida toxin formalin-inactivated effect - Google Patents
A kind of method of use Vero cell detections toxigenic pasteurella multocida toxin formalin-inactivated effect Download PDFInfo
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Abstract
The invention belongs to biological technical field, more particularly to a kind of method of use Vero cell detections toxigenic pasteurella multocida toxin formalin-inactivated effect, including：Step 1：It is prepared by toxigenic Pasteurella multocida bacterium solution；Step 2：Inactivated bacterial liquid is standby；Step 3：It is prepared by toxigenic pasteurella multocida toxin liquid；Step 4：After the digestion of cultured Vero cell monolayer is counted, cell suspension is diluted with containing 2% hyclone MEM culture mediums, adds 100 μ l MEM culture mediums per hole into porous experiment orifice plate；By verticillium toxin liquid, the method diluted according to equimultiple is added in hole；100 μ l Vero cell suspensions are added per hole, rearmounted 36 ~ 38 DEG C, CO are mixed2Incubator culture observes result after seven days；Cell wire drawing lesion is judged to the positive, and bacterium agglomerate is judged to suspicious, and no lesion is judged to feminine gender.It can more accurately detect whether toxigenic pasteurella multocida toxin complete inactivation or inactivates to greatest extent using this method, so as to ensure that the safety of related vaccines well.
Description
Technical field
It is more particularly to a kind of to use Vero cell detection toxigenic Pasteurella multocidas the invention belongs to biological technical field
The method of toxin formalin-inactivated effect.
Background technology
Toxigenic Pasteurella multocida (Toxigenic pasteurellamultocida, T+Pm) it is pig transmissible atrophy
The main pathogenic fungi of property rhinitis (Atrophicrhinitis, AR).Atrophic rhinitis can cause pig concha osteanabrosis, destruction
Nasal mucosal immune barrier, and then secondary other breathing problems, cause the heavy economic losses of pig industry.Research shows production poison
Pasteurella multocida can cause progressive atrophoderma rhinitis, wherein being caused a disease by main of toxigenic pasteurella multocida toxin
The factor.Pasteurella multocida is divided into 6 serotypes such as A, B, D, E, F by pod membrane, has been reported that and shows, T+Pm using D types produce poison as
It is main, also there is the production of A types malicious, and T+Pm is mainly located away from the serious pigs of AR.
Atrophic rhinitis is prevented and treated at present mainly by vaccine immunity, in atrophic rhinitis inactivated vaccine production process
In, toxigenic Pasteurella multocida is mainly using formalin inactivation, and the quality of bacterial strain inactivating efficacy directly influences vaccine
Security and immune effect.At present, toxigenic Pasteurella multocida bacterium solution inactivating efficacy is detected, bacterial strain is predominantly detected and whether can
Inactivation, and ignore whether toxigenic pasteurella multocida toxin inactivates.Toxigenic pasteurella multocida toxin is examined according to the literature
Testing is detected by guinea pig skin necrosis experiment, so someone tests to determine toxigenic Pasteurella multocida using cutaneous necrosis
Whether toxin inactivates.Because the strain culturing time is different, cavy individual difference and human users' error can cause false negative, simultaneously
Guinea pig skin necrosis experiment is that, with the bacterium solution after inactivation, the ratio that thalline is accounted for is smaller, it is impossible to which it is many that reasonable reaction goes out production poison in thalline
Whether killing property Pasteurella toxin inactivates.At present, it can be obtained by way of ultrasonic degradation and slightly carry toxigenic Pasteurella multocida
Toxin, a small amount of thick toxigenic pasteurella multocida toxin that carries can produce typical lesion to Vero cell monolayers.Production poison is more
The virulence of killing property Pasteurella toxin is strong, to the LD of mouse50Only 0.2ug.
The content of the invention
It is an object of the invention to：Using Vero cells (African green monkey kidney cell) detection toxigenic Pasteurella multocida poison
Plain formalin-inactivated effect, this method more traditional can pass through guinea pig skin necrosis detection toxigenic pasteurella multocida toxin formaldehyde
Inactivating efficacy method is sensitiveer, the inactivating efficacy of toxin can be directly reflected, so that the toxigenic Pasteurella multocida system of reduction
Standby vaccine side reaction, improves the security of vaccine.
Technical scheme：One kind Vero cell detection toxigenic pasteurella multocida toxin formalin-inactivated effects
Method, including：Step 1：Toxigenic Pasteurella multocida is inoculated in brain-heart infusion medium, 36~38 DEG C of constant temperature is placed in and shakes
Incubator is swung, is cultivated 12~18 hours with 200r/min, bacterium solution is harvested；
Step 2：Formalin is added in the bacterium solution prepared in step 1 according to 0.3% final concentration (v/v), 36 are put
~38 DEG C of constant-temperature shaking incubators, after being acted on 48 hours with 200r/min, inactivated bacterial liquid is standby；
Step 3：The inactivated bacterial liquid prepared in step 2 is taken under the conditions of 4 DEG C, is centrifuged 30 minutes with 10000r/min, is gathered
Collect thalline, after being rinsed 4 times with the sterile PBS of inactivated bacterial liquid same volume, suspended with the sterile PBS of the volume of inactivated bacterial liquid 1/5, will
Inactivation bacteria suspension is transferred to centrifuge tube, centrifuge tube is put ultrasonic instrument interior room is put into mixture of ice and water, 40% power output,
Often work 4 seconds, be spaced 15 seconds, ultrasonication 30 minutes；Under the conditions of 4 DEG C, centrifuged 30 minutes with 10000r/min and collect supernatant
Verticillium toxin liquid, supernatant verticillium toxin liquid is through 0.22 membrane filtration, after steriling test is qualified, standby；
Step 4：After the digestion of cultured Vero cell monolayer is counted, with containing 2% hyclone MEM culture mediums by cell
Suspension is diluted to 2 × 105Individual/ml, adds 100 μ l MEM culture mediums into porous experiment orifice plate per hole；By step 3 prepare it is upper
Clear verticillium toxin liquid, the method diluted according to equimultiple is sequentially added in the hole of experiment orifice plate；Then, 100 μ l dilutions are added per hole
Good Vero cell suspensions, mix rearmounted 36~38 DEG C, CO2Incubator culture observes result after seven days；Cell wire drawing lesion is sentenced
For the positive, bacterium agglomerate is judged to suspicious, and no lesion is judged to feminine gender.Toxigenic Pasteurella multocida includes A types production poison killing property bars more
Family name bacillus and D type toxigenic Pasteurella multocidas.
Beneficial effects of the present invention：It can more accurately detect whether toxigenic pasteurella multocida toxin is complete using this method
Full inactivation is inactivated to greatest extent, so as to ensure that the safety of related vaccines well.
Brief description of the drawings
The present invention is further illustrated below in conjunction with the accompanying drawings.
Accompanying drawing 1 causes Vero cytopathic effect inhibition assays for the thick toxigenic pasteurella multocida toxin liquid that carries；
Accompanying drawing 2 is different concentration of formaldehyde, and inactivation different time inactivated bacterial liquid prepares vaccine immunity test pig turbinate pathology
Change.
Embodiment
The preparation of bacterium solution：Toxigenic Pasteurella multocida is inoculated in brain-heart infusion medium, 36~38 DEG C of constant temperature are placed in
Shaken cultivation case, is cultivated 12~18 hours with 200r/min, harvests bacterium solution.
The inactivation of bacterium solution：Formaldehyde is separately added into production poison according to 0.2%, 0.25% and 0.3% final concentration (v/v) to kill more
Property Pasteurella bacterium solution, puts 36~38 DEG C of constant-temperature shaking incubators, acts on 36 respectively with 200r/min, 48 and 60 hours.Each
Time point takes 10ml inactivated bacterial liquids standby.
The each concentration of guinea pig skin necrosis detection, each period take 0.1ml bacterium solutions, back intracutaneous injection cavy, injection
Observe 72 hours afterwards, and measure the size in injection point cutaneous necrosis area.Necrotic zone diameter is not less than 0.5cm and is judged to the positive, small
Be judged in 0.5cm it is suspicious, it is reactionless to be judged to feminine gender.
Vero cytopathies are detected
The preparation of verticillium toxin liquid：Each concentration, each period take 5ml inactivated bacterial liquids under the conditions of 4 DEG C, with 10000r/
Min centrifuges 30 minutes aggregation thalline, after being rinsed 4 times with the sterile PBS of inactivated bacterial liquid same volume, with the nothing of the volume of bacterium solution 1/5
Bacterium PBS is suspended, and bacteria suspension is transferred into 50ml centrifuge tubes, centrifuge tube is put ultrasonic instrument interior room is put into mixture of ice and water,
40% power output, often works 4 seconds, is spaced 15 seconds, ultrasonication 30 minutes.Under the conditions of 4 DEG C, 30 are centrifuged with 10000r/min
Minute collects supernatant verticillium toxin liquid, and supernatant verticillium toxin liquid is through membrane filtration, after steriling test is qualified, standby.
Verticillium toxin liquid causes the experiment of Vero cytopathies
The preparation of Vero cell suspensions：After the digestion of cultured Vero cell monolayer is counted, with containing 2% hyclone
Cell suspension is diluted to 2 × 10 by MEM culture mediums5Individual/ml；
Verticillium toxin liquid causes Vero cytopathies to determine into 96 orifice plates per hole plus 100 μ l MEM culture mediums.H behaviors are negative
Control row, A~G behavior sample rows.A~G sample rows, first row adds verticillium toxin liquid sample prepared by 100 μ l, according to equimultiple
The method of dilution is diluted to the 12nd row from first row.Each sample, does two repetitions.Add the cell that 100 μ l have diluted per hole
Suspension, mixes rearmounted 36~38 DEG C, the culture of CO2 incubators observes result after seven days.Cell wire drawing lesion is judged to the positive, and bacterium gathers
Group is judged to suspicious, and no lesion is judged to feminine gender.
Each concentration, each period are obtained bacterium solution and verticillium toxin liquid, non-inactivated bacterial liquid and brain by small white mouse lethal test
Heart infusion medium takes 0.2ml that small white mouse is injected intraperitoneally respectively, observes seven, records dead mouse situation.
48 hours bacterium solutions of 0.2% formalin-inactivated, 60 hours bacterium solutions and 0.3% formalin-inactivated are selected in vaccine safety detection experiment
Univalent vaccine is prepared by mixing into proportion with aluminium glue adjuvant respectively.With the susceptible pig 9 of 3~4 week old health, neck flesh after 3 ears
Meat injects the formalin-inactivated of neck intramuscular injection 0.3% after univalent vaccine prepared by 0.2% formalin-inactivated, 60 hours bacterium solutions, 3 ears
Univalent vaccine prepared by 48 hours bacterium solutions, another 3 are not injected as control, under equal conditions isolated rearing, with phase after 14 days
Two are carried out with approach and same dose to exempt from.
The observation vaccine one of clinical symptoms is exempted from, two exempt from latter continuous 14 days fixed point determination test temperature of pig body, observes injection site
It is whether red and swollen and whether there is other adverse reactions
Test pig turbinate pathological change two is exempted from 14 days afterwards, and by all test pig cut open inspections, observation turbinate whether there is atrophy, nose
In be separated with no deformation.
As a result：Guinea pig skin necrosis experiment and Vero cytopathy result of the tests are shown in Table 1 and Fig. 1.
The different formalin-inactivated concentration of table 1, different inactivation time guinea pig skin necrosis experiments and Vero cytopathies experiment knot
Really
Note：A represents guinea pig skin necrosis experiment.B represents the experiment of Vero cytopathies.+ represent the result of the test positive, ± generation
Watch test result is suspicious ,-represent result of the test feminine gender.
It can be seen from table 1, toxigenic pasteurella multocida toxin inactivation is judged by guinea pig skin necrosis experiment most
Good condition is that formalin-inactivated concentration 0.2% is inactivated 48~60 hours；And judge that production poison is more by the experiment of Vero cytopathies
The optimum condition of killing property Pasteurella toxin inactivation is that formalin-inactivated concentration 0.3% is inactivated 36~48 hours.
Small white mouse lethal test the results are shown in Table 2.
The different formalin-inactivated concentration of table 2, different inactivation time mouse lethal result of the tests
It can be seen from table 2, when concentration of formaldehyde is 0.3%, inactivate 48 hours, bacterium solution and verticillium toxin liquid are to the lethal of mouse
It is 0%.
Vaccine safety detection experiment
After the observation vaccine immunity piglet of clinical symptoms 0.3% formalin-inactivated, 48 hours bacterium solution univalent vaccine immune groups and
Control group experiment temperature of pig body is normal, and the state of mind is good, has no adverse reaction, injection site have no red and swollen, itch and it is local by
The symptoms such as tenderness.The 0.2% bacterium solution univalent vaccine immune group of formalin-inactivated 60 hours, body temperature is normal after being immunized, and injection site has no
Red and swollen, itch and local by symptoms such as tenderness, one exempt from after test pig on the 20th clinical symptoms such as occur sneezing.
0.3% formalin-inactivated, 48 hours bacterium solution univalent vaccines are exempted from after test pig turbinate pathological change vaccine immunity piglet
Epidemic disease group and control group test pig turbinate are normal, and the 0.2% bacterium solution univalent vaccine immune group turbinate of formalin-inactivated 60 hours goes out
Existing atrophy, is shown in Fig. 2.
Conclusion：1st, when guinea pig skin necrosis experiment result be negative, the experiment of Vero cytopathies is the positive when, can still cause
60%~80% dead mouse.When guinea pig skin necrosis experiment and the experiment of Vero cytopathies are feminine gender, mouse is good for
It is living.The result illustrates that, when guinea pig skin necrosis experiment result is feminine gender, toxigenic pasteurella multocida toxin does not go out completely yet
It is living, can cause dead mouse, but the experiment of Vero cytopathies for it is negative when, it was demonstrated that Pasteurella toxin complete inactivation.
2nd, when guinea pig skin necrosis experiment result be negative, the experiment of Vero cytopathies is the positive when, the vaccine of preparation is exempted from
Epidemic disease test pig, can cause test pig the clinical symptoms such as sneeze and concha osteanabrosis occur.As guinea pig skin necrosis experiment and Vero
When cytopathy experiment is feminine gender, the vaccine immunity test pig of preparation, test pig is without clinical symptoms, and turbinate is normal.
Therefore, toxigenic pasteurella multocida toxin inactivating efficacy is detected using the experiment of Vero cytopathies, more accurately.
Claims (2)
1. a kind of method of use Vero cell detections toxigenic pasteurella multocida toxin formalin-inactivated effect, it is characterised in that：
Including：
Step 1:Toxigenic Pasteurella multocida is inoculated in brain-heart infusion medium, 36 ~ 38 DEG C of constant-temperature shaking incubators are placed in,
Cultivated 12 ~ 18 hours with 200r/min, harvest bacterium solution；
Step 2:By formalin according to 0.3% final concentration（V/v) add in the bacterium solution prepared in step 1, put 36 ~ 38 DEG C
Constant-temperature shaking incubator, after being acted on 48 hours with 200r/min, inactivated bacterial liquid is standby；
Step 3:The inactivated bacterial liquid prepared in step 2 is taken under the conditions of 4 DEG C, is centrifuged 30 minutes with l0000r/min, assembles bacterium
Body, after being rinsed 4 times with the sterile PBS of inactivated bacterial liquid same volume, is suspended with the sterile PBS of the volume of inactivated bacterial liquid 1/5, will inactivated
Bacteria suspension is transferred to centrifuge tube, centrifuge tube is put ultrasonic instrument interior room is put into mixture of ice and water, 40% power output, per work
Make 4 seconds, be spaced 15 seconds, ultrasonication 30 minutes；Under the conditions of 4 DEG C, centrifuged 30 minutes with l0000r/min and collect supernatant bacterium
Plain liquid, supernatant verticillium toxin liquid is through 0.22 membrane filtration, after steriling test is qualified, standby；
Step 4:After the digestion of cultured Vero cell monolayer is counted, with containing 2% hyclone MEM culture mediums by cell suspension
It is diluted to 2 × 105Individual/ml, adds 100 μ l MEM culture mediums into porous experiment orifice plate per hole；The supernatant bacterium that step 3 is prepared
Toxin solution, the method diluted according to equimultiple is sequentially added in the hole of experiment orifice plate；Then, add what 100 μ l had diluted per hole
Vero cell suspensions, mix rearmounted 36 ~ 38 DEG C, CO2Incubator culture observes result after seven days；Cell wire drawing lesion is judged to sun
Property, bacterium agglomerate is judged to suspicious, and no lesion is judged to feminine gender.
2. according to the method described in claim 1, it is characterised in that：Toxigenic Pasteurella multocida includes A types production poison killing property more
Pasteurella and D type toxigenic Pasteurella multocidas.
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Inventor after: Hao Chengwu Inventor after: Han Rui Inventor after: He Hai Inventor after: He Sun Inventor after: Li Yantao Inventor after: He Chuanyu Inventor after: He Yuxian Inventor after: Zhang Shuqin Inventor before: Hao Chengwu Inventor before: Han Duan Inventor before: He Hai Inventor before: He Sun Inventor before: Li Yantao Inventor before: He Chuanyu Inventor before: He Yuxian Inventor before: Zhang Shuqin |
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Address after: 830000 No. 528 South Changchun Road, Urumqi hi tech Industrial Development Zone, Xinjiang Applicant after: Tiankang biological Limited by Share Ltd Address before: 830032, 221, Lantian Road, North Zone, Urumqi high tech Zone, the Xinjiang Uygur Autonomous Region Applicant before: Xinjiang Tecon Animal Husbandry Biological Technology Co., Ltd. |
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Effective date of registration: 20210420 Address after: Room 201, building 1, No.46, Weixing Road, phase II, Xinjiang Urumqi Economic Development Zone (Toutunhe District), Urumqi City, Xinjiang Uygur Autonomous Region 830000 Patentee after: Tiankang biopharmaceutical Co.,Ltd. Address before: 830000 No. 528 South Changchun Road, Urumqi hi tech Industrial Development Zone, Xinjiang Patentee before: TECON BIOLOGICAL Co.,Ltd. |