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Fast labeling and tracking method of recombinant human adenovirus type 5

An adenovirus and labeling technology, which is applied in the fields of virology and biological sciences, can solve the problems of unsatisfactory labeling effect of virus labeling materials, and achieve the effects of little influence on virus activity, simple labeling process, and overcoming fluorescence quenching

Pending Publication Date: 2019-08-02
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems existing in the prior art, the present invention provides a rapid labeling and tracing method of recombinant human adenovirus type 5, so as to solve the problem of unsatisfactory labeling effect of viral labeling materials in the prior art

Method used

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  • Fast labeling and tracking method of recombinant human adenovirus type 5
  • Fast labeling and tracking method of recombinant human adenovirus type 5
  • Fast labeling and tracking method of recombinant human adenovirus type 5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The amplification and purification steps of human adenovirus type 5 are as follows:

[0022] 1) Inoculate human kidney epithelial cells in good condition into a 75 cm2 cell culture flask until the cell density is 80-90%;

[0023] 2) Dilute the virus to control the multiplicity of infection within the range of 1 to 1000;

[0024] 3) Aspirate the diluted virus and mix it into the culture medium of the 75 square centimeter cell culture flask. After mixing, continue to culture for 4-8 hours and then replace with fresh complete medium;

[0025] 4) Continue to observe the lesion of the cells. When the lesion of the cells reaches 70-80%, scrape off the cells, collect them together with the supernatant into the virus collection tube, freeze and thaw three times at 37°C and -80°C, and centrifuge at 10,000 centrifugal force to remove the cells debris, transfer the supernatant to a new centrifuge tube;

[0026] 5) Filter out impurities with a 0.22 micron filter;

[0027] 6) Ta...

Embodiment 2

[0034] A rapid labeling and tracing method for human type 5 adenovirus based on aggregation-induced luminescent molecules, the specific steps are as follows:

[0035] 1) Inoculate HeLa cells in good condition into a 15mm glass-bottom culture dish until the cell density reaches 50%;

[0036] 2) Take the molecule n-tetraphenylethylene (TPE) with aggregation-induced emission (AIE) properties10 -6 Micro friction;

[0037] 3) Ultrasound with a power of 50 watts for 30 minutes;

[0038] 4) Centrifuge at 12,000 rpm for 10 minutes, and take 5 microliters of supernatant;

[0039] 5) Take a sterile 0.2 ml centrifuge tube, add 5 microliters of purified human adenovirus type 5 and 5 microliters of n-tetraphenylethylene supernatant and mix well, so that n-tetraphenylethylene is adsorbed on human type 5 Adenovirus surface.

[0040] 6) Take a sterile 1.5 ml centrifuge tube, add 10 microliters of human adenovirus type 5 labeled with aggregation-induced luminescent molecules and dilute to ...

Embodiment 3

[0044] A rapid labeling and tracing method for human type 5 adenovirus based on aggregation-induced luminescent molecules, the specific steps are as follows:

[0045] 1) Inoculate HeLa cells in good condition into a 15mm glass-bottom culture dish until the cell density reaches 50%;

[0046] 2) Take the molecule n-tetraphenylethylene (TPE) with aggregation-induced emission (AIE) properties10 -6 Micro friction;

[0047] 3) Ultrasound with a power of 50 watts for 30 minutes;

[0048] 4) Centrifuge at 12,000 rpm for 10 minutes, and take 5 microliters of supernatant;

[0049] 5) Take a sterile 0.2 ml centrifuge tube, add 5 microliters of purified human adenovirus type 5 and 5 microliters of n-tetraphenylethylene supernatant and mix well, so that n-tetraphenylethylene is adsorbed on human type 5 Adenovirus surface.

[0050] 6) Take a sterile 1.5 ml centrifuge tube, add 10 microliters of human adenovirus type 5 labeled with aggregation-induced luminescence molecule and dilute to ...

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Abstract

The invention discloses a fast labeling and tracking method of recombinant human adenovirus type 5. An aggregation-induced emission molecule is used as a fluorescence label, and through electrostaticinteraction, human adenovirus type 5 is labeled by the aggregation-induced emission molecule. Through a laser confocal imaging analysis system, a whole infection process of the human adenovirus type 5infecting a host cell can be tracked. The labeling method is simple and efficient, effects on adenovirus activity are small, and population dynamic infection behaviour of the adenovirus in the singlecell can be fully reflected. The multiobjective real-time long-time human adenovirus type 5 tracking method in combination with excellent optical property of the aggregation-induced emission moleculeis built, and a powerful tool is provided for globally and efficiently studying a human adenovirus type 5 infection mechanism.

Description

technical field [0001] The invention belongs to the fields of virology and biological sciences, and relates to a rapid labeling and tracing method of recombinant human type 5 adenovirus. Background technique [0002] In order to effectively prevent and control the spread of viral diseases, it is particularly important to have a deep understanding of the mechanism of virus infection host and other transmission processes. The interpretation of the dynamic process and mechanism of virus infecting host cells will lay an important theoretical foundation for understanding the pathogenic mechanism of viruses and preventing and treating viral diseases. The process of virus infecting host cells is very complex, often including many stages, multiple pathways and involving different subcellular structures. Single virus tracer technology mainly uses real-time dynamic imaging of fluorescence microscope to monitor the infection path and dynamic behavior of virus or the interaction betwee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N21/64C12R1/93
CPCC12Q1/02G01N21/6402
Inventor 王涛李显煌郑斌王志云
Owner TIANJIN UNIV
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