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Transgenic fat body cell line of high-yield baculovirus and preparation method as well as application thereof

A baculovirus, fat body technology, applied in viruses/phages, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as immortalized cell lines of unseen insects

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

AI Technical Summary

Problems solved by technology

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 gene to obtain immortalized insect cell lines

Method used

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  • Transgenic fat body cell line of high-yield baculovirus and preparation method as well as application thereof
  • Transgenic fat body cell line of high-yield baculovirus and preparation method as well as application thereof
  • Transgenic fat body cell line of high-yield baculovirus and preparation method as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Establishment of Transgenic Beet Spodoptera Larvae Fat Body Cell Line

[0057] Get the last instar larvae of beet armyworm and immerse them in 3% hypochlorous acid solution for 5 minutes and in 75% ethanol solution for 10-20 minutes to carry out surface disinfection. Dissect the insect to remove the fat body tissue, keeping it as intact as possible during the manipulation. 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 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 solution I, and culture under the same conditions. Note that the key to the successful establishment of cell lines by this method is to make the tissue block stick to the bottom of the cell culture flask and not suspen...

Embodiment 2

[0058] Embodiment 2. human telomerase reverse transcriptase (hTERT) gene recombinant plasmid pIZT-hTERT and Construction of pIB-hTERT

[0059] 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, hTERT gene (its sequence is shown in SEQ ID NO 1) (from plasmid pBABE-puro-hTERT) is subjected to SalI restriction enzyme digestion, dNTP After fill-in and EcoRI digestion, clone into vector plasmid to obtain hTERT recombinant plasmid pIZT-hTERT.

[0060] 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-Blasticidin gen...

Embodiment 3

[0061] Example 3. Observation and determination of biological characteristics of IOZCAS-SpexX

[0062] (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.

[0063] (2) Cell growth: at 27°C, the 12th generation 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 56.98h. Such as Figure 5 As shown, the highest cell density can reach about 1.3×106 cells / mL.

[0064] (3) Karyotype analysis: such as Image 6 As shown, the 22nd generation cells of IOZCAS-Spex X are tetraploid cells, and the number of chromosomes ranges from 116 to 131 (2n=62).

[0065] (4) DAF-PCR iden...

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Abstract

The invention provides a transgenic fat body cell line of high-yield baculovirus and a preparation method as well as application thereof. The cell line which is named as IOZCAS-Spex X has a preservation number of CGMCC No. 4505; and the preparation method of the transgenic fat body cell line comprises following steps of: culturing asparagus caterpillar fat body cells; transforming genes containing human telomerase reverse transeriptase into a known expression vector to obtain a recombinant expression vector; leading the recombinant expression vector into the asparagus caterpillar fat body cells; and enabling the asparagus caterpillar fat body cells to express human telomerase reverse transeriptase so as to obtain the transgenic fat body cell line. In addition, the invention discloses application of the transgenic fat body cell line to production of baculovirus.

Description

technical field [0001] The invention relates to a transgenic insect cell line, in particular to a high-yield baculovirus transgenic fat body cell line and its preparation method and application, belonging to the technical field of transgenic animals. Background technique [0002] Insects are the most diverse biological group in the world with a rich cytogenetic system. 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. According to reports, since the first insect cell line was successfully established in 1965, mo...

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 张寰李瑄秦启联王雁玲王红托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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