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Chilo suppressalis larva midgut cell line with high yield of baculovirus

A technology of baculovirus and diploid borer, applied in the direction of animal cells, viruses/phages, microorganism-based methods, etc., to achieve the effect of strong repeatability and good culture effect

Inactive Publication Date: 2015-12-23
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, there are few related reports on the in vitro culture technology of C.

Method used

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  • Chilo suppressalis larva midgut cell line with high yield of baculovirus
  • Chilo suppressalis larva midgut cell line with high yield of baculovirus
  • Chilo suppressalis larva midgut cell line with high yield of baculovirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Establishment of cell lines in the midgut of Chilo suppressalis larvae

[0038] (1) Prepare cell primary culture medium and cell subculture medium;

[0039] (2) Under sterile conditions, immerse the larvae of C. borer in 75% ethanol solution, disinfect the surface for 10 minutes, wash the larvae twice with sterile distilled water, dry the larvae with sterile filter paper or use Dry the worms with a hair dryer;

[0040] (3) Place the worm body in a dissecting wax dish sterilized with 75% ethanol, fix the head and tail with a dissecting needle, and take out the midgut of the larvae;

[0041] (4) Wash the tissue with normal saline (3) for 3 times, then wash the tissue twice with HBSS cleaning solution, and finally wash the midgut once with cell culture medium TNM-FH, the HBSS cleaning solution contains 200U / ml penicillin , 0.2 μg / ml streptomycin, 0.5 μg / ml amphotericin B;

[0042] (5) Cut the washed midgut tissue into small pieces and place it in a T-25 cm me...

Embodiment 2

[0046] The biological characteristic observation and determination of embodiment 2 cell lines

[0047] (1) Morphological characteristics: Observed under a microscope, the cell line grows adherently, and there are three main cell shapes: most are round, a few are spindle-shaped, and less macrophage-like ( figure 1 ). Round cells account for 90%, with an average diameter of 12.49±1.15 μm; spindle cells are about 21.0-40 μm long, and the average width is about 11 μm; macrophages are between 20-22 μm in diameter;

[0048] (2) Cell growth: the growth curve is a typical "S", and the population doubling time is 78 hours ( figure 2 );

[0049] (3) Karyotype analysis: the chromosome number n=29 of Chilo suppressalis, while the chromosome number of the cell line established by the present invention is 136-147, therefore, the newly established cell line is composed of pentaploid cells ( image 3 );

[0050] (4) DAF-PCR identification: use interleukin-1β to design primers, and use D...

Embodiment 3

[0051] The mensuration of embodiment 3 virus sensitivity and output

[0052] Determination of sensitivity of cell lines to Brassica cabbage nuclear polyhedrosis virus: Take cells in the logarithmic growth phase and prepare 1x10 6 / mL concentration, inoculated into culture flasks, 10mL per bottle. After culturing for 3 hours, the medium was discarded, leaving the adherent cells, and the prepared virus liquid was inoculated on the cells, 100uL per bottle. After cultivating for 7 days, harvest cells and virus polyhedrons, count the number of virus polyhedrons under a microscope with a hemocytometer, and as a result, the polyhedron concentration produced by the cell line of the present invention reaches 16.5×10 6 / PIB / mL. 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 5 ).

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Abstract

The invention discloses a cell line that is derived from an insect midgut tissue and is highly sensitive to baculovirus. The invention also discloses an establishment method of the cell line, and application of the cell line in baculovirus large-scale growth. The cell line can be used for replication of the virus, large-scale production of baculovirus insecticides, and construction of a baculovirus expression vector system to express protein with commercial or scientific value. Therefore, the chilo suppressalis larva midgut cell line has extremely high commercial and the scientific research value.

Description

technical field [0001] The invention relates to the technical field of insect cell culture, in particular to a midgut cell line of Chilo suppressalis larvae with high yield of baculovirus. Background technique [0002] With the rapid development of life science, cell engineering has been paid more and more attention in the field of bioengineering technology. Cultivating insect cells as research materials has always been an important aspect of scientific research in cell biology, molecular biology and biochemistry, and insect toxicology. [0003] Since Grace first established the celestial moth cell line in 1962, insect cell culture technology has developed rapidly, and more than 800 insect cell lines have been established at present, mainly focusing on Lepidoptera and Diptera (Pan Lizhen et al., 1980, Zeng Qingtao et al., 1998), Coleoptera (Lynnetal., 1995; Iwabuchi, 1999; Stiles, 1992), Orthoptera (Heinandez-Crespoetal., 2000), Hymenoptera, Homoptera and Hemiptera, etc., m...

Claims

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Application Information

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IPC IPC(8): C12N5/07C12N7/00C12R1/91
Inventor 刘光富许益鹏杨倩倩
Owner CHINA JILIANG UNIV
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