Culture method and application of plutella xylostella embryonic cell line

A technology of embryonic cells and culture methods, applied in cell dissociation methods, animal cells, tissue culture, etc., can solve the problems of too large deviation and unsuitable drug screening, etc., and achieve easy-to-use, stable and reliable results, and simplified operation steps Effect

Inactive Publication Date: 2016-12-14
ZHEJIANG UNIV
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Problems solved by technology

[0006] At present, the existing cell line BCIRL-PX2-HNU3 of Plutella xylostella is a cell line developed from the pupa of Plutella xylostella. The cells and tissues in the pupal stage of insects will transform into organs unique to the adult stage. The cell composition is similar to that in the larval stage. The deviation of an important hazard period is too large, so it is not suitable for drug screening

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  • Culture method and application of plutella xylostella embryonic cell line
  • Culture method and application of plutella xylostella embryonic cell line
  • Culture method and application of plutella xylostella embryonic cell line

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Embodiment 1

[0035] The preparation of embodiment 1 diamondback moth embryonic cell line

[0036] Plutella xylostella embryonic cell line culture method, concrete steps are as follows:

[0037] 1. Take 200-300 healthy diamondback moth eggs that are in the embryonic stage within 15 to 24 hours after laying eggs;

[0038] 2. Soak eggs with 75% alcohol for 8-10 minutes to disinfect the surface; then wash twice with sterile water, and then wash twice with serum-free medium;

[0039] 3. In the TNM-FH medium containing 20% ​​fetal bovine serum FBS (Fetal Bovine Serum) (Gibco product number 10099-141), the eggs of diamondback moth were broken one by one, and passed through a 100-mesh cell sieve to obtain diamondback moth embryonic cells;

[0040] 4. Transfer the filtered diamondback moth embryo cells to a cell culture dish (Φ3.5 cm) containing 1 ml of culture medium (20% FBS TNM-FH), and cultivate them at 27°C;

[0041] 5. Then add 250 μl of fresh medium containing 1% antibiotic (Gibco model 1...

Embodiment 2

[0047] 1, utilize the diamondback moth embryonic cell line prepared in embodiment 1 to carry out drug screening, the method steps are as follows:

[0048] A. Resuscitate the frozen cell line in Example 1 by conventional methods; collect the cells in the logarithmic growth phase, adjust the concentration of the cell suspension and place it in a 96-well cell culture plate, and adjust the cell concentration to 50 cells / μl, per well Add 100 μl; culture at 27°C, and observe the growth of the cells under an inverted microscope every 12 hours.

[0049] B. Dissolve the original drug with methanol, and use the culture medium to prepare the mother solution of the drug to be tested with a concentration of 1000mg / L, and dilute the drug twice in a two-fold gradient, and set up 8 gradients in total.

[0050] After the cells adhered to the wall (generally cultured for about 24 hours), the medium was aspirated, and diluted drugs of different concentrations were added, and three biological rep...

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Abstract

The invention discloses a culture method and application of a plutella xylostella embryonic cell line. The culture method comprises the following steps that moth eggs in the embryonic period of plutella xylostella are taken and subjected to disinfection treatment, crushing and screening, and embryonic cells of the plutella xylostella are obtained; the embryonic cells are put into a cell culture medium for primary culture and subculture, and the plutella xylostella embryonic cell line is obtained. According to the culture method and application of the plutella xylostella embryonic cell line, the moth eggs in the embryonic period of the plutella xylostella are adopted for primary culture and subculture, the obtained plutella xylostella embryonic cell line can be used for judging the level of toxicity of different insecticides to the plutella xylostella, screening of the insecticides is achieved, the screening steps of existing insecticides are simplified, and the false positive rate of the insecticides is decreased.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing embryonic cell lines of diamondback moth and its application. Background technique [0002] Diamondback moth (Plutella xylostella) belongs to Lepidoptera and Plutellaidae. It is one of the main pests of cruciferous vegetables worldwide. It has been widely distributed in Asia, Europe, America, Africa and Oceania. The economic losses caused by it are due to The expansion of the scope of occurrence and the increase of the frequency of occurrence are increasing day by day. Diamondback moth has become a major obstacle to cruciferous vegetable production worldwide (Bai, S.F., Chen, X.X., Cheng, J.A., Fu, W.J., He, J.H., 2002.Characterization of Cotesia plutellae polydna virus and its physiological effects on the diamondback moth, Plutella xylostella larvae. Acta Entomologica Sinica 46, 401-408. Bai, S.F., Chen, X.X., Cheng, J.A., Fu, W.J., He, J.H., 2005. P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C12Q1/04
CPCC12N5/0601C12N2509/00G01N33/5014G01N2333/43552
Inventor 陈学新王泽华时敏邹佳妮
Owner ZHEJIANG UNIV
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