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American cotton bollworm yong-insect fatbody cell line of high yield stab-like virus, its constitution and use

A baculovirus, cotton bollworm technology, applied in the field of insect cell lines, can solve the problem of no cell line, etc., and achieve the effect of high commercial and scientific research value

Inactive Publication Date: 2007-11-14
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • American cotton bollworm yong-insect fatbody cell line of high yield stab-like virus, its constitution and use
  • American cotton bollworm yong-insect fatbody cell line of high yield stab-like virus, its constitution and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Establishment of fat body cell lines of cotton bollworm larvae

[0039] The last instar cotton bollworm larvae were immersed 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 of c...

Embodiment 2

[0040] The biological characteristic observation and determination of embodiment 2.IOZCAS-Ha-I

[0041] (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-shaped, and macrophage-like (Figure 1). In IOZCAS-Ha-I, round cells accounted for 85.6%, with an average diameter of 14.57 μm; spindle cells accounted for 9.5%, with a length of about 16.6-42.0 μm (average 23.95 μm), a width of about 8.0-15.2 μm (average 11.53 μm); 4.9% of the cells were macrophage-like, ranging in diameter from 21.7-28.2 μm.

[0042] (2) Cell growth: at 27°C, the 10th passage of the two cell lines was in TNM-FH culture medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100U / mL streptomycin, and the 9th passage The cell population doubling time was 65.2 hr. The highest cell density can reach about 8×10 5 cells / mL.

[0043] (3) Karyotype analysis: IOZCAS-Ha-I 7th g...

Embodiment 3

[0046] Example 3. Determination of virus sensitivity and yield

[0047] IOZCAS-Ha-I is sensitive to cotton bollworm nuclear polyhedrosis virus: inoculate IOZCAS-Ha-I with HaNPV budding virion BV at a concentration of 0.001 larvae equivalent / ml, cultivate for 10 days, harvest cells and virus polyhedrons, and use hemocytometer The counting board counts the number of viral polyhedrons under a microscope. As a result, IOZCAS-Ha-I can amplify HaNPV and produce 7.3×10 per milliliter of culture solution. 7 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.

[0048] Bioassays were performed on late 2nd instar larvae of H. armigera using viral polyhedrons produced by the cell line as described above. Its method is as follows: 10 days after IOZCAS-Ha-I was inoculated with HaNPV virus, ...

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Abstract

This invention publicized one kind to originate from the insect fat body organization, and to the rod-shaped viral high sensitive cell line, this invention has also publicized this cell line establishment method, as well as this cell line the use which grew into the rod-shaped viral scale metaplasia. This cell line may use for to duplicate this kind of virus, uses in the rod-shaped viral pesticide the formalization production, as well as the construction rod-shaped virus the expression vector system, the expression has commercial or the scientific value protein. Therefore it has the extremely high trade and the scientific research value.

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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IPC IPC(8): C12N5/06C12N15/866C12N5/07
Inventor 张寰秦启联李瑄苗麟殷珍仙张爱君丁翠
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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