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Novel method for in vitro culturing and staining Neospora caninum tachyzoites

A technology for Neospora caninum and Neospora, which is applied in the field of life science, can solve the problems of difficulty in culturing Neospora tachyzoites, abortion of pregnant female animals, and no tachyzoite staining method, and achieves a simple, practical and fast cultivation method. The effect of cultivation

Inactive Publication Date: 2012-07-04
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It can cause miscarriage and stillbirth in pregnant dams, make newborns suffer from serious mental diseases, cause huge economic losses to animal husbandry, and cause serious harm, but so far there is no ideal control method. The reason is Neospora Tachyzoites are difficult to cultivate in vitro, and because the tachyzoites are small in size, they are not easy to be observed or misdiagnosed if they do not use staining methods when growing in cells, and there is no simple and practical Neospora intracellular tachyzoites at present. The staining method of the proteus, which is not conducive to the rapid detection of the disease, but also limits the research of the disease vaccine

Method used

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  • Novel method for in vitro culturing and staining Neospora caninum tachyzoites
  • Novel method for in vitro culturing and staining Neospora caninum tachyzoites
  • Novel method for in vitro culturing and staining Neospora caninum tachyzoites

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Experimental program
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Effect test

Embodiment 1

[0017] 1. Culture method of Neospora caninum (N.C-1)

[0018] 1.1 Recovery of MCF-7 cells, using RPMI-1640 (10% fetal bovine serum, 1% double antibody, 4mM glutamine, 2.30mg / ml NaHCO 3 , 2.38 mg / ml HEPES (pH 7.2), 50 U / ml penicillin, 50 mg / ml streptomycin), in an incubator at 37°C, 5% CO 2 continue to grow in the environment.

[0019] 1.2 Inoculate Neospora tachyzoites in the cultured MCF-7 cells, using medium RPMI-1640 (2% fetal bovine serum, 1% double antibody, 4mM glutamine, 2.30mg / ml NaHCO 3 , 2.38 mg / ml HEPES (pH 7.2), 50 U / ml penicillin, 50 mg / ml streptomycin), in an incubator at 37°C, 5% CO 2 Continue to culture in the environment, replace the fresh medium every day, count the tachyzoites in each field of view through an inverted phase contrast microscope (40×10), and draw the growth curve of the tachyzoites in different cells (such as figure 1 Shown), it can be seen that the number of tachyzoites can reach the maximum in about four days.

Embodiment 2

[0021] 2. Observation of intracellular tachyzoites by acridine orange staining

[0022] Neospora tachyzoites were fixed with 16% paraformaldehyde (12ml paraformaldehyde, 88ml distilled water) for 20 minutes, PBS (Na 2 HPO 4 12H 2 O 0.89g, NaCl 2g, KCl 0.06g, KH 2 PO 4 0.05g, 250ml distilled water), washed twice, stained with 10% acridine orange (10mg acridine orange, 100ml PBS) for 2 minutes, 1% triton (triton 1ml, 99ml distilled water) for 3~5 minutes, mounted Afterwards, photographs were taken by ordinary fluorescent microscope. Experimental results such as image 3 shown.

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Abstract

The invention relates to a novel method for in vitro culturing and staining Neospora caninum tachyzoites, which is characterized in that the concrete culturing steps are as follows: culturing MCF-7 breast cancer cells using a conventional method, inoculating in 1ml Neospora caninum at an inoculum size of 10<4> / ml after the cells grow to form a monolayer of cells, culturing with RPIM-1640 medium containing 2% fetal bovine serum, and observing the number of extracellular Neospora caninum tachyzoites with an inverted microscope everyday; and the concrete staining steps are as follows: fixing Neospora caninum tachyzoites with paraformaldehyde, staining with acridine orange, and observing and taking photos with a common fluorescence microscopy. The culturing method of the invention is simple and practical, and Neospora caninum tachyzoites can be rapidly cultured. In addition, after acridine orange staining, intracellular parasites can be rapidly, visually and clearly observed with the common fluorescence microscopy.

Description

Technical field [0001] The present invention involves a new method of in vitro culture and dyeing of new canine new spores, which belongs to the field of life science. Background technique [0002] The new spores of the dog are an intracellular parasitic, which is similar to the Gong Di Archworm. Several different forms can appear in all their life history, namely ascendant colonials, pockets, and oval sacs.It can cause abortion and death of pregnancy female animals, cause newborn to suffer from severe psychiatric diseases, and cause huge economic losses to animal husbandry and serious harm.Speed colony is more difficult to cultivate in vitro, and because the volume of speed colonials is small, it is not easy to be observed or misdiagnosed if it is unfavorable when growing in the cell, and there is no simple and practical new spores.The dyeing method of colonials is not conducive to the rapid detection of the disease, and it also limits the research of the disease vaccine. Inven...

Claims

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Application Information

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IPC IPC(8): C12N1/10C12Q1/02
Inventor 李建华吕强张西臣宫鹏涛张楠杨举李赫张国才
Owner JILIN UNIV
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