Method for constructing cell line by using insect egg

A technology of insect cells and cell lines, applied in the field of establishing cell lines with insect eggs

Inactive Publication Date: 2009-05-06
INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, whether the establishment of cell lines is successful or not is very random and has great uncertainty. In most cases, it ends

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the realization of the cell line of the moth moth

[0034] Put fertilized eggs of Clostera anachorta Fabricius that have been born for about four days in a 10ml test tube in a sterile operating table (the following operations are all under sterile conditions, and all instruments used must be sterilized or disinfected) , then immerse in 10% sodium hypochlorite solution for about 2-5 minutes and shake lightly, pour off the sodium hypochlorite solution and wash the eggs with sterile water, and then disinfect the eggs with 75% ethanol solution for 10-20 minutes after pouring off the sterile water. Constantly oscillate gently, pour off the ethanol and wash the eggs with Ringer's; pour or pipette the eggs into 1-2ml of insect cell culture medium (mainly TNM-FH, containing 100U / mL Penicillin, 100 U / mL streptomycin and 10% (v / v) fetal bovine serum, pH=6.3), gently squeeze the eggs one by one with elbow ophthalmic forceps under a dissecting microscope to release th...

Embodiment 2

[0036] Embodiment 2: Realization of American white moth cell line

[0037] Put fertilized eggs of Hyphantria cunea Drury (Hyphantria cunea Drury) that have been born for about four days into a 10ml test tube in a sterile operating table (the following operations are under sterile conditions, and all instruments used must be sterilized or disinfected) , then immerse in 10% sodium hypochlorite solution for about 2-5 minutes and shake lightly, pour off the sodium hypochlorite solution and wash the eggs with sterile water, and then disinfect the eggs with 75% ethanol solution for 10-20 minutes after pouring off the sterile water. Constantly oscillate gently, pour off the ethanol and wash the eggs with Ringer's; pour or pipette the eggs into 1-2ml of insect cell culture medium (mainly TNM-FH, containing 100U / mL Penicillin, 100 U / mL streptomycin and 10% (v / v) fetal bovine serum, pH=6.5), gently squeeze the eggs one by one with elbow ophthalmic tweezers under a dissecting microscope ...

Embodiment 3

[0038] Embodiment 3: the realization of spring looper cell line

[0039] Put fertilized eggs of spring inchworm (Apocheima cinerarius Erschoff) that have been born for about four days in a 10ml test tube in a sterile operating table (the following operations are under sterile conditions, and all instruments used must be sterilized or disinfected) , then immerse in 10% sodium hypochlorite solution for about 2-5 minutes and shake lightly, pour off the sodium hypochlorite solution and wash the eggs with sterile water, after pouring off the sterile water, disinfect the eggs with 75% ethanol solution for 10-20 minutes. Gently shake, pour off the ethanol and wash the eggs with Ringer's; pour the eggs or use a pipette into the insect cell culture medium containing 1-2ml (mainly TNM-FH, containing 100U / mL penicillin , 100U / mL streptomycin and 10% (v / v) fetal bovine serum, pH=6.5), under a dissecting microscope, gently squeeze the eggs one by one with elbow ophthalmic tweezers to relea...

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PUM

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Abstract

The invention relates to a method for establishing a cell line of insect eggs, which comprises the following steps: 1) dipping an insect oosperm sheet which has been laid for 90 to 100 hours in a sodium hypochlorite solution or a formaldehyde solution, and then disinfecting and washing the oosperm sheet; 2) pouring the treated eggs into an insect cell culture solution, and extruding the eggs one by one to release embryos; 3) cutting the embryos into tissue blocks to be cultured in an incubator; 4) adding the cell culture solution into the incubator to culture the tissue blocks continuously; 5) replacing the culture solution periodically until the proliferating cells are full of the whole culture bottle; 6) sucking out half of the cell culture solution containing newly proliferated single cells, putting the cell culture solution into a new culture bottle which is added with the same amount of a new cell culture solution, and putting the new culture bottle back to the incubator to culture the cells continuously; and 7) repeating the step 5) and the step 6) periodically to cause the cells to begin to passage so as to establish the cell line successfully, wherein the whole process is performed under a sterile condition. The insect eggs can be lepidoptera insect eggs. The method shortens the time from the prior 1.5 to 2 years to 1 to 3 months, thereby having quite obvious superiority and strong repeatability.

Description

technical field [0001] The invention relates to a biotechnology, in particular to a method for establishing cell lines with insect eggs. Background technique [0002] Since the first insect cell line was successfully established in 1965, more than 500 insect cell lines have been established in the past 40 years. As a research material, insect cell lines have always been an important tool for scientific research in physiology, developmental biology, cell biology, molecular biology and biochemistry, and insect cells, as an important part of the baculovirus expression vector system, express a large number of exogenous proteins of great economic or scientific significance; at the same time as a bioreactor to amplify insect baculoviruses for use as biopesticides, especially to amplify recombinant baculovirus insecticides containing exogenous genes, insects Cells also play an important role. [0003] A large number of commercially available cell lines from Lepidoptera insects ha...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N5/07
Inventor 张永安温发园王玉珠曲良建王文欢
Owner INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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