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

A baculovirus and cell line technology, applied in the direction of virus/bacteriophage, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of immortalized cell lines of insects that have not been seen

Inactive Publication Date: 2014-04-30
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 armyworm larva fat body cell line

[0057] The last instar beet armyworm larvae were immersed in a 3% hypochlorous acid solution for 5 minutes and a 75% ethanol solution for 10-20 minutes to disinfect the surface. The insect was dissected to take out the fat body tissue, and the operation should try to keep it intact. Wash the tissue 2-3 times with saline, and then use cell culture medium I (mainly TNM-FH, containing 100U / mL penicillin, 100U / mL streptomycin and 10% (v / v) fetal cattle Serum, pH=6.2) Wash 1-2 times, put into 25cm rinsed with 1mL culture medium 2 Place the cell culture flask in the cell culture flask at 27°C without light for 24 hours. Then add 3 mL of the above-mentioned cell culture solution I and put it in the same condition for culture. Note that the key to the successful establishment of cell lines in this method is to make the tissue block close to the bottom of the cell culture flask, and do not suspend th...

Embodiment 2

[0058] Example 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 multiple cloning sites, baculovirus early promoter, V5 epitope and Zeocin resistance gene. The vector plasmid pIZT-V5-His was subjected to EcoR I and EcoR V restriction digestion, and the hTERT gene (the sequence shown in SEQ ID NO 1) (from the plasmid pBABE-puro-hTERT) was subjected to SalI restriction digestion, dNTP After filling and digesting with EcoRI, it was cloned into the vector plasmid to obtain the hTERT recombinant plasmid pIZT-hTERT.

[0060] The vector plasmid pIB-V5-His was used to construct the recombinant plasmid pIB-hTERT. Such as Picture 9 As shown, the vector plasmid pIB-V5-His has multiple cloning sites, baculovirus early promoter, V5 epitope and anti-Blasticidin gene. ...

Embodiment 3

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

[0062] (1) Morphological characteristics: According to microscope observation, the cells of this cell line are easy to form cell clusters and can tolerate high-density growth environment, breaking through contact inhibition. Such as Figure 1-2 As shown, there are 3 types of cell shapes: round, fusiform and oval. Most of the cells adhere to the wall.

[0063] (2) Cell growth: At 27°C, the 12th generation of the cell line was cultured in TNM-FH containing 10% fetal bovine serum, 1 OOU / mL penicillin, 100 U / mL streptomycin, and 50 μg / mL bleomycin In the solution, the population doubling time was 56.98h. Such as Figure 5 As shown, the highest cell density can reach approximately 1.3×106 cells / mL.

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

[0065] (4) DAF-...

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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 transgenic fat body cell line with high yield of baculovirus and a preparation method and application thereof, belonging to the technical field of transgenic animals. Background technique [0002] Insects are the most diverse group of organisms in the world and have a rich cytogenetic system. The main uses of insect cell lines are: 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 baculovirus, especially recombinant baculovirus containing foreign genes, and produce biological pesticides. According to reports, since the first insect cell line was successfully established in ...

Claims

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Application Information

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Patent Type & Authority Patents(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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