CN104312922A - Separation method of neospora caninum and in-vitro culture method of neospora caninum - Google Patents
Separation method of neospora caninum and in-vitro culture method of neospora caninum Download PDFInfo
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Abstract
The invention relates to a separation method of neospora caninum and an in-vitro culture method of the neospora caninum. In order to reach the aim, the separation method of the neospora caninum is completed according to the technical scheme as follows: a double-antibody which has a certain volume concentration and contains 10kU/mL penicillin and 10mg/ml streptomycin is used; animal brain tissues infected with neospora caninum are sterilely collected by adopting a disposable syringe; the volume of the animal brain tissues infected with neospora caninum is twice as large as the volume of the double-antibody; the double-antibody is added into the collected animal brain tissues infected with neospora caninum; and disposable syringes with different specifications are used for repeatedly sucking and blowing the collected animal brain tissues infected with neospora caninum according to the diameter reduction sequence of syringe needles. The separation method of the neospora caninum has the advantages of simplicity and convenience, rapidness, time saving, short multiplication time, strong practicability and the like.
Description
Technical field
The present invention relates to a kind of separation method and extracorporeal culturing method of Demodiosis canis.
Background technology
Demodiosis canis (Neospora caninum) is special sexual cell endoparasitism protozoon, is under the jurisdiction of Protozoa (Protozoa), Apicocomplexa (Apicomplexa), sporozoa (Sporozoa), Neosporidia (Neosporidia), neospora genus (Neospora).1988, the people such as Dubey were separated to this polypide first in the dog body of a lower extremity paralysis, and by its called after Demodiosis canis.The final host of Demodiosis canis is dog, and the multiple Mammalss such as ox, sheep, pig, horse, rabbit can as intermediate host, and its parasitic site is central nervous system, brain, liver, muscle and other viscera tissue.Neosporosis (Neosporiasis) parasitizes by Demodiosis canis a kind of protozoal disease caused in the multiple mammalian cell such as dog, ox, sheep, this disease mainly causes dam miscarriage, stillborn foetus or neonatal dyskinesia and nervous system disorders, brings huge financial loss to livestock industry.The domestic research to Demodiosis canis still belongs to the starting stage, still lacks the technological method such as the separation of Demodiosis canis body, in-vitro multiplication of independent intellectual property right.
The separation method of the Demodiosis canis of report in original method (Yamane et al.1997), realized by following steps: the human fetal brain of the fresh death of aseptic collection, weigh 10 grams and put into mortar, adding trypsinase makes final concentration be 0.5%, hatch 20min for 37 DEG C, add appropriate liquid nitrogen to grind rapidly, obtain homogenate centrifugal after cellular filter filters, and be suspended in and add in the dual anti-aseptic PBS such as microbiotic, finally the suspension containing polypide is inoculated on the Vero cell monolayer (African green monkey kidney cell line) containing 10% foetal calf serum substratum, every 24h changes nutrient solution, until observe Demodiosis canis tachyzoite.This separation method very complicated, very easily pollutes, and the liquid nitrogen that tissue abrasion splashes easily injures operator, and trypsinase also can affect growing of the polypide that is separated in tissue and culturing cell, and separation efficiency is lower, and mortality is high.
It is the passage cells such as Vero cell line that vitro culture Demodiosis canis commonly uses cell, the method of the vitro culture Demodiosis canis of report in original method (Davison et al.1999), Demodiosis canis tachyzoite is cultivated for Vero continuous cell line, the Vero cell of recovery is inoculated in the DMEM nutrient solution containing 8% calf serum, after Vero cell grows up to individual layer, inoculates 10
4individual left and right polypide, changed nutrient solution every 12 hours, and Demodiosis canis tachyzoite proliferative conditions observed by application inverted microscope, and after inoculation 4-5 day, polypide propagation peaks the phase.The Secondary Culture program of Vero cell is loaded down with trivial details, and Demodiosis canis in Vero cell line incubation growth and propagation slowly, especially frozen polypide recovery, its recovery of inoculation Vero cell and generation time are longer, strongly limit the progress being badly in need of polypide test.
Patent " a kind of vitro culture of new Demodiosis canis tachyzoite and dyeing process " adopts MCF-7 mammary gland cell to be host cell, RPIM-1640 substratum is adopted to cultivate, inverted microscope is adopted to observe the number of the outer neospora tachyzoite of born of the same parents, slowly increased from the 2nd day, 3rd day starts to increase rapidly, peaks at the 4th day.Still there is the long problem of generation time in the method.
At present, the report that primary human embryonic kidney cell-293 cell proliferation in vitro cultivates Demodiosis canis is not still applied.
Summary of the invention
The present invention makes in view of the above-mentioned problems in the prior art, the object of the present invention is to provide a kind of separation method of easy, quick, practical Demodiosis canis.
For achieving the above object, the separation method of Demodiosis canis of the present invention is realized by such technical scheme: getting certain volume concentration is the dual anti-of the penicillin of 10kU/ml and the Streptomycin sulphate of 10mg/ml, the animal brain that application disposable syringe aseptic collection Demodiosis canis infects, animal brain's volume that this Demodiosis canis infects is more than the twice of this dual anti-volume, by the animal brain that this dual anti-this Demodiosis canis joining collection infects;
Adopt the disposable syringe of different size to inhale according to the reiteration that needle diameter reduces to blow.
The separation method of Demodiosis canis of the present invention, is characterized in that, described suction is blown in step, repeatedly inhales and blows more than at least 10 times.
The separation method of Demodiosis canis of the present invention, is characterized in that, described disposable syringe, and the size of needle diameter is not less than 27G.
The separation method of Demodiosis canis of the present invention, is characterized in that, described disposable syringe, is selected from any one above syringe needle in 12G syringe needle, 20G syringe needle, 27G syringe needle.
The separation method of Demodiosis canis of the present invention, is characterized in that, also comprises: be inoculated in the Vero cell of logarithmic phase by treated described Demodiosis canis infection animal cerebral tissue and cultivate.
The separation method of Demodiosis canis of the present invention, is characterized in that, described cultivation adopts DMEM complete culture solution.
The separation method of Demodiosis canis of the present invention, is characterized in that, described nutrient solution contains 1% dual anti-and 8% calf serum.
The separation method of Demodiosis canis of the present invention, is characterized in that, described nutrient solution was changed once every 24 hours.
In order to realize another above-mentioned object, the extracorporeal culturing method of Demodiosis canis of the present invention is realized by such technical scheme: adopt the DMEM nutrient solution containing 8% calf serum to cultivate primary human embryonic kidney cell-293 cell; After primary human embryonic kidney cell-293 cell grows up to monolayer cell, in every bottle of primary human embryonic kidney cell-293 cell, inoculate 10
3individually be separated the Demodiosis canis tachyzoite (or other strain isolateds of Demodiosis canis) obtained by the present invention, change nutrient solution every 12 hours; And adopt inverted microscope to observe Demodiosis canis tachyzoite proliferative conditions, cultivate within 36 hours, namely can be observed polypide propagation peak the phase, cultivating more than 90% primary human embryonic kidney cell-293 cell after 48 hours to rise brokenly disintegration, is all almost Demodiosis canis tachyzoite in nutrient solution.
The technical scheme of the extracorporeal culturing method of Demodiosis canis of the present invention, the quantity being inoculated into polypide in primary human embryonic kidney cell-293 cell is only 1/10 of the quantity of the polypide be inoculated in Vero cell, cultivate 36 hours polypides can peak the phase, cultivate 48 hours polypides and can reach maximum, be only and adopt 1/2 of Vero cell generation time used.Vero cell is the clone that adherent property is strong in addition, and Secondary Culture needs the steps such as tryptic digestion, and complex operation is complicated; And primary human embryonic kidney cell-293 cell attachment is weak, pipettor gun head piping and druming is adopted to take off wall during Secondary Culture, do not need the complex steps such as tryptic digestion, therefore, adopt primary human embryonic kidney cell-293 to cultivate Demodiosis canis tachyzoite and there is the advantages such as time saving and energy saving, easy, quick, practical.
Beneficial effect:
The separation of Demodiosis canis of the present invention and extracorporeal culturing method have easy, quick, save time, generation time is short, the advantage such as practical.
Accompanying drawing explanation
For being illustrated more clearly in the specific embodiment of the present invention, the accompanying drawing used in the description below to embodiment part is briefly described.
Description of reference numerals is as follows:
Fig. 1 is the microgram that the first embodiment of the present invention is separated the Demodiosis canis obtained;
Fig. 2 is the microgram that the second embodiment of the present invention is separated the Demodiosis canis obtained;
Fig. 3 is the microgram of primary human embryonic kidney cell-293 cell adopted in the third embodiment of the present invention;
Fig. 4 is the microgram of Demodiosis canis tachyzoite when cultivating 36h in the third embodiment of the present invention;
Fig. 5 is the microgram of Demodiosis canis tachyzoite when cultivating 48h in the third embodiment of the present invention.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below, by reference to the accompanying drawings concrete embodiment of the present invention is described in detail.
First embodiment
Get 1.5mL centrifuge tube one, add the dual anti-0.5mL that volumetric concentration is 10kU/ml penicillin and 10mg/ml Streptomycin sulphate wherein.The agonal stage that the tool scissors such as scissors, tweezers, bone saw, scalpel of application sterilization infects except Demodiosis canis or death time are calf brain skin in 6 hours, skull is pried open under aseptic condition, adopt the disposable syringe aseptic collection cerebral tissue 1mL of 12G syringe needle, join this 1.5mL centrifuge tube, repeatedly inhale and blow about 10 times, then adopt the disposable syringe of the disposable syringe of 20G syringe needle and 27G syringe needle repeatedly to inhale successively respectively and blow each 10 times.The ox source cerebral tissue infected by treated Demodiosis canis is all inoculated in the Vero cell of logarithmic phase to be cultivated, nutrient solution is DMEM (Hyclone Products) that is dual anti-containing 1% and 8% calf serum, changes a nutrient solution every 24 hours.
Employing inverted microscope is observed, until observe Demodiosis canis.Collect the Vero cell containing Demodiosis canis strain isolated, centrifugal 5 minutes of 2000r/min, taking precipitate crosses 27G syringe needle, removes ox source cerebral tissue and Vero cell, filtered liquid centrifugal 5 minutes with 10000r/min, and precipitation is the Demodiosis canis of separation, as shown in Figure 1.
Second embodiment
Get 1.5mL centrifuge tube one, add the dual anti-0.5mL that volumetric concentration is 10kU/ml penicillin and 10mg/ml Streptomycin sulphate wherein.The agonal stage that the tool scissors such as scissors, tweezers, scalpel of application sterilization infects except Demodiosis canis or death time are mouse (gerbil jird, Balb/c mouse, small white mouse) brain skin in 6 hours, skull is pried open under aseptic condition, adopt the disposable syringe aseptic collection cerebral tissue 1mL of 12G syringe needle, join this 1.5mL centrifuge tube, repeatedly inhale and blow more than 10 times, then adopt the disposable syringe of the disposable syringe of 20G syringe needle and 27G syringe needle repeatedly to inhale successively respectively and blow each 10 times.The mouse source cerebral tissue infected by treated Demodiosis canis is all inoculated in the Vero cell of logarithmic phase to be cultivated, nutrient solution is DMEM (Hyclone Products) that is dual anti-containing 1% and 8% calf serum, changes a nutrient solution every 24 hours.
Employing inverted microscope is observed, until observe Demodiosis canis.Collect the Vero cell containing Demodiosis canis strain isolated, centrifugal 5 minutes of 2000r/min, taking precipitate crosses 27G syringe needle, removes ox source cerebral tissue and Vero cell, filtered liquid centrifugal 5 minutes with 10000r/min, and precipitation is the Demodiosis canis of separation, as shown in Figure 2.
3rd embodiment
Adopt DMEM nutrient solution (Hyclone Products, dual anti-and 8% calf serum containing 1%) in 37 DEG C of CO
2primary human embryonic kidney cell-293 cell is as shown in Figure 3 cultivated in incubator.After primary human embryonic kidney cell-293 cell grows up to monolayer cell, in every bottle of primary human embryonic kidney cell-293 cell, inoculate 10
3the Demodiosis canis tachyzoite that individual first embodiment obtains, changes a nutrient solution every 12h.Inverted microscope is adopted to observe Demodiosis canis tachyzoite proliferative conditions.As shown in Figure 4, cultivate within 36 hours, namely can be observed polypide propagation peak the phase.As shown in Figure 5, cultivating more than 90% primary human embryonic kidney cell-293 cell after 48h and to rise brokenly disintegration, is all almost Demodiosis canis tachyzoite in nutrient solution.
The separation of Demodiosis canis of the present invention and extracorporeal culturing method have easy, quick, save time, generation time is short, the advantage such as practical.
With embodiment as above only for illustrating spirit of the present invention; protection scope of the present invention is not limited thereto; for those skilled in the art; certainly can according to technology contents disclosed in this specification; by changing, displacement or the mode of modification make other embodiment easily, and these other embodiment all should be encompassed within protection scope of the present invention.
Claims (9)
1. a separation method for Demodiosis canis, is characterized in that, comprises the following steps:
Getting certain volume concentration is the dual anti-of the penicillin of 10kU/ml and the Streptomycin sulphate of 10mg/ml, the animal brain that application disposable syringe aseptic collection Demodiosis canis infects, animal brain's volume that this Demodiosis canis infects is more than the twice of this dual anti-volume, by the animal brain that this dual anti-this Demodiosis canis joining collection infects;
Adopt the disposable syringe of different size to inhale according to the reiteration that needle diameter reduces to blow.
2. the separation method of Demodiosis canis according to claim 1, is characterized in that, described suction is blown in step, repeatedly inhales and blows more than at least 10 times.
3. the separation method of Demodiosis canis according to claim 1, is characterized in that, described disposable syringe, and the size of needle diameter is not less than 27G.
4. the separation method of Demodiosis canis according to claim 1, is characterized in that, described disposable syringe, is selected from any one above syringe needle in 12G syringe needle, 20G syringe needle, 27G syringe needle.
5. the separation method of Demodiosis canis according to claim 1, is characterized in that, also comprises: be inoculated in the Vero cell of logarithmic phase by treated described Demodiosis canis infection animal cerebral tissue and cultivate.
6. the separation method of Demodiosis canis according to claim 5, is characterized in that, described cultivation adopts DMEM complete culture solution.
7. the separation method of Demodiosis canis according to claim 6, is characterized in that, described nutrient solution contains 1% dual anti-and 8% calf serum.
8. according to the separation method of the Demodiosis canis described in claim 6 or 7, it is characterized in that, described nutrient solution was changed once every 24 hours.
9. an extracorporeal culturing method for Demodiosis canis, is characterized in that, comprises the following steps:
The DMEM nutrient solution containing 8% calf serum is adopted to cultivate primary human embryonic kidney cell-293 cell;
After primary human embryonic kidney cell-293 cell grows up to monolayer cell, in every bottle of primary human embryonic kidney cell-293 cell, inoculate 10
3the individual Demodiosis canis tachyzoite obtained according to the separation method in claim 1-8 described in any one, changed nutrient solution every 12 hours; And
Inverted microscope is adopted to observe Demodiosis canis tachyzoite proliferative conditions.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009105355A2 (en) * | 2008-02-20 | 2009-08-27 | The United States Of America, As Represented By The Secretary Of Agriculture | A novel neospora caninum vaccine |
| US20100255577A1 (en) * | 2007-07-13 | 2010-10-07 | Universidad Complutense De Madrid | Use of a new isolate of neospora caninum for the development of diagnostic tests and for preparation of products for treatment and prevention of the infection caused by neospora |
| CN102533556A (en) * | 2010-12-17 | 2012-07-04 | 吉林大学 | Novel method for in vitro culturing and staining Neospora caninum tachyzoites |
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2014
- 2014-10-15 CN CN201410550101.6A patent/CN104312922A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100255577A1 (en) * | 2007-07-13 | 2010-10-07 | Universidad Complutense De Madrid | Use of a new isolate of neospora caninum for the development of diagnostic tests and for preparation of products for treatment and prevention of the infection caused by neospora |
| WO2009105355A2 (en) * | 2008-02-20 | 2009-08-27 | The United States Of America, As Represented By The Secretary Of Agriculture | A novel neospora caninum vaccine |
| CN102533556A (en) * | 2010-12-17 | 2012-07-04 | 吉林大学 | Novel method for in vitro culturing and staining Neospora caninum tachyzoites |
Non-Patent Citations (2)
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| 李琼章: "新孢子虫可在细胞培养基培养", 《畜牧兽医科技信息》 * |
| 赵占中: "新孢子虫NcSRS2 DNA疫苗的研究", 《中国优秀博硕士论文全文数据库.农业科技辑》 * |
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