Cell strain with sterol carrier protein (SCP) stable transformation of prodenia litura and preparation method and application of cell strain

A sterol and transporter technology of Spodoptera litura, which is applied to biochemical equipment and methods, genetically modified cells, and cells modified by introducing foreign genetic material, etc. Cells, the effect of blocking the growth of insects

Inactive Publication Date: 2017-03-08
SOUTH CHINA NORMAL UNIVERSITY
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Problems solved by technology

Although the mechanism of cholesterol transport in vitro cannot explain the transport in cells and tissues, it can at least explain several problems: (1) The transport of cholesterol is very fast, and the transport involved by the SCP-2 protein is necessary
(4) There may be multiple competing pat

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Embodiment

[0058] Before setting forth the method of the present invention, the following abbreviations are explained and described:

[0059] Ham's F-12 medium:

[0060] NCBI: National Center for Biotechnology Information

[0061] SlSCPx: Spodoptera litura SCPx

[0062] pro-SlSCPx-2: Spodoptera litura SCPx-2 precursor

[0063] ORF: open reading frame

[0064] DH5α: bacterial strain name (Escherichia coli competent cells)

[0065] Opti-MEM: Medium

[0066] Hygromycin B: hygromycin

[0067] trypsin-EDTA: trypsin

[0068] DMSO: Dimethylsulfoxide

[0069] SCP-2 Gene Number: FJ986464

[0070] Literature source:

[0071] Guo, X.R., Zheng, S.C., Liu, L. and Feng, Q.L. The sterol carrier protein 2 / 3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgutof Spodoptera litura: gene cloning, expression, localization and functional analyzes .BMC Mol.Biol.10,102-120(2009)

[0072] Guo Xingrong, Zheng Sichun, Liu Lin, Feng Qili Sterol transporter 2 / 3 acyl-CoA thiolase (S...

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Abstract

The invention discloses a preparation method of a cell strain with sterol carrier protein (SCP) stable transformation of prodenia litura. The preparation method sequentially includes the steps of 1), cloning cDNA (complementary deoxyribonucleic acid) of SCP (single-cell protein)-2 gene from prodenia litura; 2), cloning the cDNA of the SCP-2 encoded protein into pcDNA5/FRT plasmid to form recombined pcDNA5/FRT-SCP2 plasmid; 3), applying the pcDNA5/FRT-SCP2 plasmid to transform colon bacillus and obtaining transformed colon bacillus via cultivation; 4), extracting recombined plasmid DNA from the transformed colon bacillus and performing purification; 5), transfecting Chinese hamster ovary cells with purified and recombined pcDNA5/FRT-SCP2 plasmid DNA; 6), selecting transfected CHO cells in a culture medium containing antibiotic hygromycinB and performing continuous subculture to obtain the cell strain. The invention aims to provide the cell strain with SCP stable transformation of the prodenia liture, the preparation method of the cell strain and an application of the cell strain. The cell strain has the advantages of high screening accuracy, simpleness in operation and accuracy in result.

Description

technical field [0001] The invention relates to the field of pesticide screening preparations, in particular to a stable transformed cell line of Spodoptera litura sterol transporter and its preparation method and application. Background technique [0002] At present, there are three main methods for the screening of chemical pesticides: 1) biological assay; 2) small molecule simulated docking analysis; 3) cell screening. The bioassay method is to directly place the pest larvae in the artificial culture medium containing the compound to be tested for screening. If the compound in the medium is toxic, the growth and development of the insect will be inhibited, and the compound may be a candidate drug for the insecticide . This method is currently the most effective screening method for most chemical pesticides. However, this method has high culture medium and labor costs, a long period, and takes up a lot of space, making it difficult to simultaneously screen thousands of c...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12Q1/02
CPCC12N15/85C07K14/43563C12N5/0682C12N2503/02C12N2510/00G01N33/5008G01N2500/10
Inventor 刘琳冯启理万星张丽丽郑思春梁丽宁
Owner SOUTH CHINA NORMAL UNIVERSITY
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