Young beet armyworms fat body cell system for producing baculiform virus with high yields

A technology of baculovirus and beet armyworm, applied in the field of insect cell lines, can solve the problem of no cell lines

Inactive Publication Date: 2008-07-23
INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Although the aforementioned literature has related reports on cell lines of beet armyworm, so far, there is no cell line derived from the fat body of beet armyworm

Method used

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  • Young beet armyworms fat body cell system for producing baculiform virus with high yields
  • Young beet armyworms fat body cell system for producing baculiform virus with high yields
  • Young beet armyworms fat body cell system for producing baculiform virus with high yields

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Establishment of fat body cell lines of beet armyworm larvae

[0038] Take the last instar beet armyworm larvae and immerse them in 70% ethanol solution for 10 minutes to carry out surface disinfection. Dissect the insect to remove the fat body tissue, keeping it as intact as possible during the manipulation. Wash the tissue 2-3 times with normal saline, and then use cell culture medium (based on TNM-FH, containing 100U / mL penicillin, 100U / mL streptomycin and 10% (v / v) fetal calf serum , pH=6.2) wash 1-2 times, put into a 25cm 2 Cell culture flasks were placed in a dark cell culture incubator at 27°C for 24 hours. Then add 3mL of the above-mentioned cell culture medium, and culture under the same conditions. Note that the key to the successful establishment of cell lines by this method is to make the tissue block stick to the bottom of the cell culture flask and not suspend the tissue block in the cell culture medium. After that, suck out half the amount ...

Embodiment 2

[0039] The biological characteristic observation and determination of embodiment 2.Spex II

[0040] (1) Morphological characteristics: Observed under a microscope, the cell line is usually suspended in the culture medium, and there are three types of cell shapes: round, spindle and macrophage-like ( figure 1 ). In Spex II, round cells accounted for 85.7%, with an average diameter of 14.7 μm; spindle cells accounted for 12.7%, with a length of about 16.0-41.9 μm (average 25.3 μm), a width of about 8.4-16.0 μm (average 11.2 μm); about 1.6% of The cells were macrophage-like and ranged in diameter from 18.2-31.4 μm.

[0041] (2) Cell growth: at 27°C, the population doubling time of the 10th generation of the two cell lines in TNM-FH medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin 108.8hr and 156.8hr respectively. The highest cell density can reach about 1.3×10 6 cells / mL.

[0042] (3) Karyotype analysis: the 10th generation cells of Sp...

Embodiment 3

[0045] Example 3. Determination of virus sensitivity and yield

[0046] Spex II is sensitive to beet armyworm nuclear polyhedrosis virus: inoculate Spex II with SeNPV budding virion BV at a concentration of 0.001 larvae equivalent / ml, and after culturing for 10 days, harvest cells and virus polyhedrons, and use a hemocytometer under a microscope The number of viral polyhedrons was counted. As a result, Spex II amplified SeNPV could produce 1.8×10 8 Production of PIBs. At the same time, typical cytopathological features can be observed, that is, the nucleus is enlarged and contains a large number of polyhedron particles ( Figure 4 ). And it can be passed down continuously for at least 3 generations.

[0047] Bioassays were performed on late 2nd instar larvae of Spodoptera exigua using viral polyhedrons produced by the cell line as described above. Its method is as follows: after Spex II inoculates SeNPV virus 10 days, collect cell and culture fluid thereof, add the 10% sod...

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Abstract

A high-yield rod-shaped virus beet noctuids larval fatty somatic system, its construction and use are disclosed. It can be used to copy this kind of virus, construct rod-shaped virus expression carrier system and for scaled production.

Description

technical field [0001] The present invention relates to an insect cell line, especially a cell line derived from insect fat body tissue and highly sensitive to baculovirus. Use in large-scale growth. technical background [0002] According to reports, 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. They come from more than 170 kinds of insects including Lepidoptera, Diptera, Homoptera, Hymenoptera, Orthoptera and Coleoptera, most of which come from Lepidoptera and Diptera. 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 in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N7/00C12N5/07
Inventor 张永安张寰秦启联王玉珠曲良建
Owner INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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