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Transgenic insect cell line for high-yield baculovirus, and preparation method and application thereof

A technology of ovarian cells and baculoviruses, applied in microorganism-based methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problems of obtaining immortalized insect cell lines, etc.

Inactive Publication Date: 2012-12-05
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although there are many reports on the application of telomerase gene to mammalian cell immortalization, there are no reports on the use of transfection of human telomerase reverse transcriptase gene (hTERT) to obtain immortalized insect cell lines

Method used

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  • Transgenic insect cell line for high-yield baculovirus, and preparation method and application thereof
  • Transgenic insect cell line for high-yield baculovirus, and preparation method and application thereof
  • Transgenic insect cell line for high-yield baculovirus, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Establishment of transgenic beet armyworm pupal ovary cell line

[0061] The 5-day-old female pupae of the last instar beet armyworm were immersed 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 1-2 times, and placed in a 25cm 2Cell culture flasks were placed in a dark cell culture incubator at 27°C for 24 hours. Then add 3mL of cell culture solution I, and culture under the same conditions. Note that the key to the successful establishment of cell lines in this method is to make the tissue stick to the bottom of the cell culture flask and not suspend the tissue in t...

Embodiment 2

[0062] Embodiment 2. Construction of human telomerase reverse transcriptase (hTERT) gene recombinant plasmid pIZT-hTERT and pIB-hTERT

[0063] The vector plasmid pIZT-V5-His was used to construct the recombinant plasmid pIZT-hTERT. Such as Figure 8 As shown, the vector plasmid pIZT-V5-His has a multiple cloning site, a baculovirus early promoter, a V5 epitope and an anti-Zeocin gene. The vector plasmid pIZT-V5-His is subjected to EcoR I and EcoR V restriction enzyme digestion, and the hTERT gene (its sequence is shown in SEQ ID NO 1) (from plasmid pBABE-puro-hTERT) is subjected to Sal I restriction enzyme digestion, After dNTP filling and EcoR I digestion, clone into vector plasmid to obtain hTERT recombinant plasmid pIZT-hTERT.

[0064] The recombinant plasmid pIB-hTERT was constructed using the vector plasmid pIB-V5-His. Such as Figure 9 As shown, the vector plasmid pIB-V5-His has a multiple cloning site, a baculovirus early promoter, a V5 epitope and an anti-Blastic...

Embodiment 3I

[0065] The biological characteristic observation and determination of embodiment 3.IOZCAS-Spex IX

[0066] (1) Morphological characteristics: Observed under a microscope, the cells of this cell line are easy to form cell clusters and can tolerate high-density growth environments, breaking through contact inhibition. Such as Figure 1-2 As shown, there are 3 types of cell shapes: round, fusiform, and elliptical. Most cells adhere to the wall.

[0067] (2) Cell growth: at 27°C, the 10th passage of the cell line was cultured in TNM-FH containing 10% fetal bovine serum, 100 U / mL penicillin, 100 U / mL streptomycin, and 50 μg / mL bleomycin solution, the population doubling time was 70.09h. Such as Figure 5 As shown, the highest cell density can reach about 2.7×10 6 cells / mL.

[0068] (3) Karyotype analysis: such as Image 6 As shown, the 12th generation cells of IOZCAS-Spex IX are tetraploid cells, and the number of chromosomes ranges from 116 to 131 (2n=62).

[0069] (4) D...

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Abstract

The invention provides a transgenic insect cell line for high-yield baculovirus, and a preparation method and the application thereof. The name of the transgenic insect cell line is IOZCAS-Spex IX and the preservation number of the cell line is CGMCC No.4506. The preparation method of the transgenic insect cell line comprises the steps as follows: spodoptera exigua ovary cells are cultured; humantelomerase reverse transcriptase genes are transformed into known expression vectors to obtain recombinant vectors; the recombinant vectors are introduced into the spodoptera exigua ovary cells; and the spodoptera exigua ovary cells are enabled to express human telomerase reverse transcriptase, so as to obtain the transgenic insect cell line. The transgenic insect cell line is applied to the production of the baculovirus.

Description

technical field [0001] The invention relates to a transgenic insect cell line and its preparation method and application, in particular to a high-yield baculovirus-producing transgenic ovarian cell line and its preparation method and application, belonging to the technical field of transgenic animals. Background technique [0002] The main uses of insect cell lines are as follows: As a research material, it has always been an important tool for scientific research in physiology, developmental biology, cell biology, molecular biology and biochemistry; as an important part of the baculovirus expression vector system, It can express foreign proteins with great economic value and scientific significance; as a bioreactor, it can amplify insect baculoviruses, especially recombinant baculoviruses containing foreign genes, and produce biopesticides. Insect cell lines have important economic and application values, but their establishment often requires a lot of time and effort. Acc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N7/00C12R1/91C12R1/93
Inventor 李瑄张寰秦启联王雁玲王红托张继红苗麟孟茜张宁朱未周桂灵杨青
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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