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Method for establishing in-vitro culture model of cryptosporidium baileyi

A technology of Cryptosporidium bainii and in vitro culture, applied in protozoa and other directions, can solve the problem of less Cryptosporidium in avian species

Inactive Publication Date: 2012-07-25
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] But so far, there are few studies on the in vitro culture of avian Cryptosporidium, only isolated from patients C. meleagridis Cultured study on bovine kidney cells (MDBK)

Method used

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  • Method for establishing in-vitro culture model of cryptosporidium baileyi

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Experimental program
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Effect test

Embodiment 1

[0015] After 24 hours of culture, tracheal rings with ciliary motor activity above 80% and no impurities inside the tracheal ring were inoculated with about 5×10 4 , 5×10 5 indivual C. bailey Mixture of oocysts and sporozoites (refers to the number of complete oocysts before decystification), 5×10 5 , 1×10 6 , 2×10 6 10 purified sporozoites were inoculated into 10 tracheal loops, and another 10 tracheal loops were inoculated with oocysts inactivated at 80 °C for 10 min 5×10 4 As a control of the cleaning process, 24 hours after the inoculation of the above-mentioned worms, the culture solution in each culture well was removed with a pipette, and then the tracheal ring was washed 3 times by sucking PBS and then transferred to another new 24-well culture plate ( Add 1 mL of DMEM maintenance solution containing 200 IU / mL double antibody to each well), then place at 37 ℃ 5% CO 2 Cultivate in an incubator, and observe the appearance of parasites (new oocysts, sporozoites, sc...

Embodiment 2

[0017] After 24 hours of culture, tracheal rings with ciliary motor activity above 80% and no impurities inside the tracheal ring were inoculated with about 5×10 4 , 5×10 5 indivual C. bailey Mixture of oocysts and sporozoites (refers to the number of complete oocysts before decystification), 5×10 5 , 1×10 6 , 2×10 6 10 purified sporozoites were inoculated into 10 tracheal loops, and another 10 tracheal loops were inoculated with oocysts inactivated at 80 °C for 10 min 5×10 4 As a control of the cleaning process, 24 hours after the inoculation of the above-mentioned worms, the culture solution in each culture well was removed with a pipette, and then the tracheal ring was washed 3 times by sucking PBS and then transferred to another new 24-well culture plate ( Add 1 mL of DMEM maintenance solution containing 200 IU / mL double antibody to each well), and then place in 38 ℃ 5% CO 2 Cultivate in an incubator, and observe the appearance of parasites (new oocysts, sporozoi...

Embodiment 3

[0019] After 24 hours of culture, tracheal rings with ciliary motor activity above 80% and no impurities inside the tracheal ring were inoculated with about 5×10 4 , 5×10 5 indivual C. bailey Mixture of oocysts and sporozoites (refers to the number of complete oocysts before decystification), 5×10 5 , 1×10 6 , 2×10 6 10 purified sporozoites were inoculated into 10 tracheal loops, and another 10 tracheal loops were inoculated with oocysts inactivated at 80 °C for 10 min 5×10 4 As a control of the cleaning process, 24 hours after the inoculation of the above-mentioned worms, the culture solution in each culture well was removed with a pipette, and then the tracheal ring was washed 3 times by sucking PBS and then transferred to another new 24-well culture plate ( Add 1 mL of DMEM maintenance solution containing 200 IU / mL double antibody to each well), then place at 39 ℃ 5% CO 2 Cultivate in an incubator, and observe the appearance of parasites (new oocysts, sporozoites, ...

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Abstract

The invention discloses a method for establishing an in-vitro culture model of cryptosporidium baileyi. The method comprises the following steps of: purifying cryptosporidium baileyi oocysts, and then carrying out excystation on the cryptosporidium baileyi oocysts at 37-40 DEG C; then innocualting mixture of 5*10<4>-5*10<5> C. baileyi oocysts and sporozoites or 5*10<5>-2*10<6> purified sporozoites into embryonated embryo trachea tissues, and culturing at 37-40 DEG C. According to the method provided by the invention, the growth of the chicken-original cryptosporidium baileyi is researched by using an embryonated embryo trachea tissue culture model; the history of the C. baileyi part is completed in the system; and a new in-vitro C. baileyi culture model is established. The model. can be used for carrying out polypide growth analysis, toxicity test, internal and external gene expression, drug screening and the like.

Description

technical field [0001] The invention relates to the establishment of a cryptosporidium trachea tissue culture model, and at the same time, it also relates to the preparation of cryptosporidium vaccine antibody, toxicity measurement, parasite metabolism detection, drug screening, endogenous and exogenous gene expression and the like by using the model. Background technique [0002] Cryptosporidium ( Cryptosporidium ) is an important pathogen of water-borne diseases, and can cause persistent fatal diarrhea in immunosuppressed patients and acute diarrhea in immune-normal populations. It can spread through water, food, air, etc., causing severe diarrhea in children and immunosuppressed patients, as well as digestive tract diseases in young animals and respiratory diseases in young birds, resulting in decreased production performance or death. 24 effective species of Cryptosporidium have been identified, among which Cryptosporidium beinii ( C.baileyi ) is the dominant insect s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/10
Inventor 张龙现张素梅黄磊菅复春赵金凤宁长申王荣军
Owner HENAN AGRICULTURAL UNIVERSITY
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