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BIV indication cell line using enhanced green fluorescence protein as reporting gene

A technology of green fluorescent protein and indicator cell line, which is applied in the field of detection of cell lines infected by bovine immunodeficiency virus, can solve problems such as high subjectivity, and achieve the effects of simple and easy operation, reducing safety risks and easy operation.

Inactive Publication Date: 2011-01-19
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus titer determined by the cytopathic method depends on the naked eye observation of the experimenter, which is highly subjective, and different experimenters may get different results

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Establishment of BIV indicator cell line with EGFP as reporter gene

[0019] 1. Construction of indicator plasmids

[0020] Using the original BIV clone plasmid BIV127 as a template, the U3+TAR segment in the BIV LTR was amplified by PCR reaction, without the negative regulatory region U5, which could better respond to Tat activation. Connect it to the eukaryotic expression vector pEGFP-N1, which removes the CMV promoter, and construct a BIV indicator plasmid. The pEGFP-N1 vector contains the neo gene, which can make the cells transfected with the plasmid resistant to G418. In this plasmid, the U3+TAR region is upstream of the reporter gene EGFP and directly regulates the expression of the reporter gene. The BIV transactivator Tat can bind to TAR to initiate the transcription of the downstream reporter gene, and the level of BIV infection can be judged by observing the green fluorescence of the EGFP protein. The BIV Tat and indicator plasmids were transiently transfe...

Embodiment 2

[0024] Application of BIV indicator cell line to indicate the infection of BIV virus

[0025] Co-cultivate BIV R29-infected FBL and BIV indicator cell line at a ratio of 1:1, observe the green fluorescence with a fluorescence microscope, weak fluorescence can be seen 12 hours after infection, the intensity of the fluorescence increases with time, and the cells that emit green fluorescence The syncytia also increased gradually, and the green fluorescent cells and syncytia could be clearly seen after 24 hours. The indicator function of the BIV indicator cell line is effective for a long time. After long-term culture of the indicator cell line infected with BIV, green fluorescence can still be seen two weeks after infection.

Embodiment 3

[0027] Activation of transactivators indicated using BIV indicator cell lines

[0028] 1. Indicating the activation of BIV Tat

[0029] to 10 5 Inoculum amount Spread the BIV indicator cell line into two wells of a 12-well plate, transfect with PEI as the transfection reagent 24 hours later, and use 500ng of BIV Tat eukaryotic expression plasmid and pCDNA3.1 as the negative control, transfect after 12 hours Green fluorescence was observed in the wells transfected with BIV Tat, and the green fluorescence was clearly visible after 24 hours, while the negative control wells transfected with empty vector had no green fluorescence.

[0030] 2. Indicate the activation of JDV Tat

[0031] The JDV transactivator JDV Tat can also activate BIV LTR, and the activation effect of JDV Tat on BIV LTR is stronger.

[0032] In the same way as above, the eukaryotic plasmid of JDV Tat was transfected, pCDNA3.1 was used as negative control, and BIV Tat was used as positive control. The green f...

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Abstract

A Bovine Immunodeficiency Virus BIV infected cell system and use are disclosed. The process is carried out by expressing reinforced EGFP under BIV infectious specific condition, constructing respondent Bovine Immunodeficiency Virus BIV infected reporting plasmid with nucleotide acid sequence in sequence table, transfecting it to BHK-21 cell, screening expressed cell system, mono-cloning and selecting high-expression EGFP negative cloning. It can be used to indicate BIV infection and activate BIV trans factor.

Description

【Technical field】 [0001] The invention relates to a cell line for detecting bovine immunodeficiency virus infection, a method for establishing the cell line and its specific application. 【Background technique】 [0002] Bovine immunodeficiency virus (BIV) and human immunodeficiency virus (HIV) belong to the genus Lentivirus of the Retroviridae family, and are highly homologous to the HIV genome. The relationship, pathogenic mechanism, etc. are very similar to HIV. It is used as an AIDS research model to explore the regulation of lentiviral gene expression, the interaction between lentivirus and other viruses after superinfection, and between viruses and host cells. It has high security. The establishment of BIV indicator cell lines is a powerful tool for studying the molecular biology of BIV. [0003] The cell line that can be stably transfected by the virus with the indicator plasmid is called the indicator cell line. The genome of this cell line contains a reporter gene ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/65
Inventor 乔文涛姚雪陈启民耿运琪
Owner NANKAI UNIV
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