Mythimna separata pupa ovary cell line capable of highly yielding baculovirus, and preparation method and applications thereof

A technology of baculovirus and ovarian cells, applied in the biological field, can solve the problem of cell lines without O. orientalis ovaries

Active Publication Date: 2021-05-07
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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  • Mythimna separata pupa ovary cell line capable of highly yielding baculovirus, and preparation method and applications thereof
  • Mythimna separata pupa ovary cell line capable of highly yielding baculovirus, and preparation method and applications thereof
  • Mythimna separata pupa ovary cell line capable of highly yielding baculovirus, and preparation method and applications thereof

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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 granulosa virus (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, 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 invention provides a mythimna separata pupa ovary cell line capable of highly yielding baculovirus. The cell line is named as IOZCAS-Myse-1 and assigned the accession number CGMCC No.17282. A construction method of the cell line is provided. The method includes the following steps: step 1) obtaining mythimna separata ovary tissue; step 2) culturing the mythimna separata ovary tissue until a culture flask is filled with proliferative cells, wherein the tissue is closely attached to the bottom of the cell culture flask; and step 3) taking newly proliferative single cells, performing continuous culture until cell passage so as to obtain the cell line. The whole process is conducted under an aseptic condition. The provided cell line can be used for copying baculovirus, so that the baculovirus or baculovirus insecticide can be produced in large scale; and the cell line can also be applied to express the protein having commercial or scientific values by using a constructed baculovirus expression vector system.

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 Applications(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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