Orient armyworm pupal ovary cell line with high production rate of baculovirus and its preparation method and application

A baculovirus, ovarian cell technology, applied in the biological field, can solve the problem of cell lines without oriental armyworm ovary and so on

Active Publication Date: 2022-05-17
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Although there are relevant reports on oriental armyworm cell lines in the aforementioned literature, so far, there is no cell line derived from the ovary of oriental armyworm

Method used

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  • Orient armyworm pupal ovary cell line with high production rate of baculovirus and its preparation method and application
  • Orient armyworm pupal ovary cell line with high production rate of baculovirus and its preparation method and application
  • Orient armyworm pupal ovary cell line with high production rate of baculovirus and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The establishment of embodiment 1 oriental armyworm pupal ovary cell line

[0051] The pupae of Oriental Armyworm at the final stage (raised in the Key Laboratory of Comprehensive Management of Agricultural Pests and Rodents, Institute of Zoology, Chinese Academy of Sciences) was immersed in an ethanol solution with a volume fraction of 75% for 10 minutes to carry out surface disinfection. The insect was dissected to remove the ovarian tissue, which was kept as intact as possible during the manipulation. Wash the tissue 2-3 times with physiological saline, then use cell culture medium (Insect-XPRESS, containing 100U / mL penicillin, 100U / mL streptomycin and 10% (v / v) fetal bovine 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 ...

Embodiment 2I

[0052] Example 2. Observation and determination of biological characteristics of IOZCAS-Myse-1

[0053] (1) Morphological characteristics: Observed under a microscope, the cell line is usually suspended in the culture medium, and there are two types of cell shapes: round and spindle ( figure 2 ). In IOZCAS-Myse-1, round cells accounted for 77.09%, with an average diameter of 10.23±0.296 (mean±SD) μm; spindle cells accounted for 22.91%, with a length of 16.77±0.604 μm and a width of 9.33±0.226 μm.

[0054] (2) Cell growth: at 27°C, the 10th generation of the cell line was in TNM-FH medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100U / mL streptomycin, and the cell population doubling time was 67.95 hours ( image 3 ). The highest cell density can reach about 3.61×10 6 cells / mL.

[0055] (3) DAF-PCR identification: the cell line IOZCAS-Myse-1 is indeed derived from oriental armyworm, not the pollution of other cell lines ( Figure 4 ). The DNA extracte...

Embodiment 3I

[0057] Example 3. Determination of IOZCAS-Myse-1 Sensitivity to Baculovirus

[0058] IOZCAS-Myse-1 against oriental armyworm nuclear polyhedrosis virus (MyseNPV), oriental armyworm granulovirus (MyseGV), Autographa californica nuclear polyhedrosis virus (AcMNPV), cabbage armyworm nuclear polyhedrosis virus (MabrNPV), Spodoptera celery nucleopolyhedrosis virus (AnfaNPV) sensitive: inoculate IOZCAS-Myse-1 with AcMNPV, MabrNPV, and AnfaNPV budding virion BV at a concentration of 0.001 larvae equivalent / ml, and after culturing for 10 days, the cells can be harvested and viral polyhedrons, under an inverted phase-contrast microscope, typical cytopathological features can be observed, that is, the nucleus is enlarged and contains a large number of polyhedron particles ( Figure 5-Figure 9 ). Each cell can produce 5-20 polyhedrons, comparable to commercial Sf21.

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Abstract

The present invention provides a high-yielding baculovirus pupae ovary cell line of Oriental Armyworm. The name of the cell line is IOZCAS-Myse-1, and its preservation number is CGMCC No.17282. The present invention provides a method for constructing the O. orientalis pupal ovary cell line of the high-yielding baculovirus. The method comprises the following steps: step 1) obtaining the ovary tissue of the oriental armyworm; step 2) cultivating the ovary tissue of the oriental armyworm, until The proliferated cells are filled with the culture flask, wherein the tissue is closely attached to the bottom of the cell culture flask; step 3) take the newly proliferated single cell and continue to culture until the cell is passed down; the whole process is carried out under sterile conditions . The oriental armyworm pupal ovary cell line with high yield of baculovirus provided by the present invention can be used to replicate baculovirus for large-scale production of baculovirus or baculovirus insecticide; the cell line can also be applied to construct Baculovirus expression vector system for expressing proteins of commercial or scientific value.

Description

technical field [0001] The invention belongs to the field of biotechnology. In particular, the present invention relates to an insect cell line. More specifically, the present invention relates to an ovarian cell line of an oriental armyworm pupae. The cell lines provided by the present invention are highly sensitive to baculovirus. The present invention also relates to the establishment method of the cell line and the application of the cell line in the large-scale growth of baculovirus. Background technique [0002] According to reports, since the first insect cell line was successfully established in 1965, more than 700 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07C12N7/00C12R1/91C12R1/93
CPCC12N5/0601C12N7/00C12N2710/14151
Inventor 李瑄秦启联张寰佟岩张继红孟茜舒锐豪于旭鹏
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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