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Construction method for fat body cell line of chilo suppressalis larva

A construction method and a technology for Diploidus chinensis are applied in the field of construction of a larval fat body cell line of Diploidus chinensis, and achieve the effect of good culture effect and strong repeatability.

Inactive Publication Date: 2014-05-28
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports about the in vitro culture technology of C. borer cells and cell lines of C.

Method used

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  • Construction method for fat body cell line of chilo suppressalis larva
  • Construction method for fat body cell line of chilo suppressalis larva
  • Construction method for fat body cell line of chilo suppressalis larva

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Preparation of cell culture medium TNM-FH

[0034] Go through the following steps:

[0035] (1) Add 700mL deionized water to 47.5g Grace dry powder medium, stir with a magnetic stirrer to accelerate dissolution;

[0036] (2) Add 0.35g NaHCO in sequence 3 , 3.0g of hydrolyzed milk protein, 3.0g of yeast extract;

[0037] (3) Measure the pH value with a precision acidity meter, and adjust the pH value to 6.20-6.30 with 1N NaOH solution prepared with deionized water;

[0038] (4) Dilute to 1000mL with deionized water;

[0039] (5) 0.45μm membrane filtration;

[0040] (6) Use a 0.22μm filter membrane to filter the culture medium on the Super Workbench and distribute it;

[0041] (7) Store in a 28°C incubator for 1 week, and store at 4°C after checking for contamination.

Embodiment 2

[0043] Preparation of Primary Cell Culture Medium and Passage Cell Culture Medium

[0044] Primary cell culture medium (500mL prepared):

[0045] Ingredient Quantity

[0046] TNM-FH medium 400mL

[0047] Fetal bovine serum 50.0mL

[0048] Phenylthiourea saturated solution 50.0mL

[0049] Penicillin 100U

[0050] Streptomycin 0.1μg

[0051] Amphotericin B 0.25μg

[0052] In the aseptic operating table, mix the above ingredients and store at 4°C for later use.

[0053] Passage cell culture medium (prepare 500mL):

[0054] Ingredient Quantity

[0055] TNM-FH medium 400mL

[0056] Fetal bovine serum 100.0mL

[0057] In the aseptic operating table, mix the above ingredients and store at 4°C for later use.

Embodiment 3

[0058] Example 3 Primary culture of fat body cells of Chilo suppressalis

[0059] (1) Under sterile conditions, immerse the larvae of Chilo stem borer in 75% ethanol solution, and carry out surface disinfection for 10 minutes; wash the larvae of Chilo borer 3 times with sterile distilled water; blot the larvae with sterile filter paper;

[0060] (2) Place the worm body in a 75% sterilized dissection wax dish, fix the head and tail with a dissecting needle; dissect the Chilo borer, and absorb the fat body with sterilized elbow tweezers;

[0061] (3) Put the fat body into a petri dish filled with HBSS cleaning solution (containing 200U / ml penicillin, 0.2μg / ml streptomycin, 0.5μg / ml amphotericin B), and wash the fat body with HBSS 2 times, and then wash the fat body 1 time with cell culture medium TNM-FH;

[0062] (4) Place the washed fat body in a T-25cm container filled with 1ml of primary cell culture medium. 2 Cell culture flasks were placed in a cell culture incubator at ...

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Abstract

The invention discloses a construction method for a fat body cell line of a chilo suppressalis larva. The construction method for the fat body cell line of the chilo suppressalis larva includes the following steps: (1) a cell primary culture solution and a cell subculture solution are prepared; (2) a chilo suppressalis larva is soaked in an ethanol solution, surface sterilization is carried out, and the larva body is cleaned and dried through absorption; (3) the larva body is placed in a wax dissection tray disinfected with ethanol, and the chilo suppressalis larva is dissected; (4) a fat body tissue block is placed in a cell culture bottle filled with an HBSS cleaning solution, the cell culture bottle with the fat body tissue block stands still, and the process of pipetting the HBSS and adding fresh HBSS is repeated for three times after fat body cells become adherent; (5) the cell primary culture solution is added, the cell culture bottle is placed into a non-illuminated cell culture box for culture through the night, and then the cell subculture solution is added; (6) the cell subculture solution is pipetted and replaced every 7 days till the culture bottle is filled with expanded and reproduced cells. The construction method for the fat body cell line of the chilo suppressalis larva provided by the invention facilitates chilo suppressalis researches under an isolated culture condition, so as to provide help for the research and development of chilo suppressalis prevention and control methods.

Description

technical field [0001] The invention relates to the technical field of insect cell culture, in particular to a method for constructing a fat body cell line of Chilo suppressalis larvae. Background technique [0002] With the rapid development of life science, cell engineering has been paid more and more attention in the field of bioengineering technology. Cultivating insect cells as research materials has always been an important aspect of scientific research in cell biology, molecular biology and biochemistry, and insect toxicology. [0003] Since Grace first established the cell line of the celestial moth in 1962, insect cell culture technology has developed rapidly, and more than 800 insect cell lines have been established at present, mainly focusing on Lepidoptera and Diptera (Pan Lizhen et al., 1980,1989 ; Zeng Qingtao et al., 1998), Coleoptera (Lynn et al., 1995; Iwabuchi, 1999; Stiles, 1992), Orthoptera (Heinandez-Crespo et al., 2000), Hymenoptera, Homoptera and Hemi...

Claims

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Application Information

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IPC IPC(8): C12N5/07C12R1/91
Inventor 刘光富俞晓平
Owner CHINA JILIANG UNIV