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High-yield baculovirus cell line induced by carcinogen, preparation method and application

A technology of baculovirus and carcinogen, applied in the field of insect cell lines and its preparation, can solve the problem of induced transformation of Lepidoptera insect cells that have not been seen with methylnitrosonitrosoguanidine

Inactive Publication Date: 2014-05-21
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] There is no report on the use of methylnitrosonitrosoguanidine (MNNG) for the induction and transformation of lepidopteran insect cells

Method used

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  • High-yield baculovirus cell line induced by carcinogen, preparation method and application
  • High-yield baculovirus cell line induced by carcinogen, preparation method and application
  • High-yield baculovirus cell line induced by carcinogen, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Establishment of MNNG Induced Pupa Ovary Cell Line of Beet Spodoptera

[0058] Get the 5-day-old female pupae of the last instar beet armyworm (from the State Key Laboratory of Comprehensive Management of Agricultural Pests and Rodents, Institute of Zoology, Chinese Academy of Sciences, the beet armyworm larvae use artificial diet at 26 ± 1.5% ℃, relative humidity 65% ​​- Raise under the condition of 75%.) Immerse in 3% hypochlorous acid solution for 5 minutes, and in 75% ethanol solution for 10-20 minutes to carry out surface disinfection. The pupae were dissected to remove the ovarian tissue, keeping it as intact as possible during the operation. Wash the tissue 2-3 times with normal saline, and then use cell culture medium I (based on TNM-FH, containing 100 U / mL penicillin, 100 U / mL streptomycin and 10% (v / v) fetal calf Serum, pH=6.2) was washed 2-3 times, and placed in a 25cm 2Cell culture flasks were placed in a dark cell culture incubator at 27°C for...

Embodiment 2

[0059] Example 2 Observation and determination of biological characteristics of IOZCAS-Spex12

[0060] (1) Morphological characteristics: such as figure 1 As shown, by microscopic observation, the cells of this cell line are easy to form cell clusters and can tolerate a high-density growth environment, breaking through contact inhibition. There are 3 types of cell shape: round, fusiform and oval. Most cells adhere to the wall.

[0061] (2) Cell growth: at 27°C, the 11th generation of the cell line was treated with 10% fetal bovine serum, 100 U / mL penicillin, 100 U / mL streptomycin, 3 μg / mL methylnitrosonitrosoguanidine ( In the TNM-FH medium of MNNG), the population doubling time was 71.06h. Such as Figure 4 As shown, the highest cell density can reach about 1.6×10 6 cells / mL.

[0062] (3) Karyotype analysis: the 12th generation cells of IOZCAS-Spex12 are tetraploid cells, the number of chromosomes ranges from 116-131 (2n=62), see Figure 5 .

[0063] (4) DAF-PCR ide...

Embodiment 3

[0065] Example 3. Virus susceptibility

[0066] IOZCAS-Spex12 is sensitive to a variety of nuclear polyhedrosis viruses: SeNPV, AcMNPV and SpltNPV budding virion BV (both from the State Key Laboratory of Integrated Management of Agricultural Pests and Rodents, Institute of Zoology, Chinese Academy of Sciences) were treated with 0.001 larval equivalent / After inoculating IOZCAS-Spex12 at a concentration of 1 ml, after 7 days of culture, typical cytopathological features can be observed through an inverted microscope, such as Figure 2-3 , that is, the nucleus is enlarged and contains a large number of polyhedron particles. And it can be passed down continuously for at least 3 generations. The viral infection rates were 91.4%, 81.4% and 31.9%, respectively.

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Abstract

The invention provides a high-yield baculovirus ovary cells line induced by a carcinogen, a preparation method and an application, a name of the cell line is IOZCAS-Spex12, and a preservation number is CGMCC No. 4804, the preparation method comprises the following steps: culturing beet armyworm ovary cells; and using the carcinogen to induce and converse the beet armyworm ovary cells obtained in the step 1 to obtain the high-yield baculovirus ovary cells line induced and conversed by the carcinogen; and the invention also relates to the application of high-yield baculovirus ovary cells line in baculovirus production.

Description

technical field [0001] The present invention relates to an insect cell line and its preparation method and application, in particular to a cell line derived from insect pupal ovary tissue, which is induced and transformed by the carcinogen methylnitrosonitrosoguanidine (MNNG), and obtained by artificial in vitro The method for inducing transformation to obtain an immortalized cell line belongs to the technical field of transgenic animals. Background technique [0002] Insects are the most diverse group of organisms in the world, with a rich cytogenetic system. Insect cell lines are increasingly valued in both scientific research and practical applications. Insect cell line has always been an important tool for scientific research in physiology, developmental biology, cell biology, molecular biology and biochemistry; it is the main component of the baculovirus expression vector system, one of the four major expression systems, and can express Exogenous proteins of great econ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07C12N7/00C12R1/91
Inventor 李瑄张寰王红托秦启联张继红苗麟张宁孟茜朱未周桂灵杨青
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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