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Labeling and detecting method of 5-acetenyl-2'-deoxyuridine of genomic DNA of hepatitis B virus

A deoxyuridine and detection method technology, applied in the field of bioengineering, can solve the problems of infecting host cells, restricting the research and development of the mechanism, unclear, etc., and achieves the effects of simple operation, strong signal intensity and bright color.

Active Publication Date: 2017-07-07
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, many aspects such as the molecular mechanism of HBV infection and interaction with cells are still unclear, such as how HBV further infects host cells after binding to NTCP receptors, how cccDNA is formed, maintained, and transcriptional regulation, etc., severely limit HBV-related liver diseases Mechanism research and development of specific therapeutic targets for HBV
The establishment of HBV genomic DNA markers and detection methods is helpful for the study of molecular mechanisms of HBV infection and transcriptional regulation, and is of great significance for exploring potential targets for inhibiting and blocking HBV replication. However, so far there are few studies on HBV genomic DNA markers and reports on detection methods

Method used

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  • Labeling and detecting method of 5-acetenyl-2'-deoxyuridine of genomic DNA of hepatitis B virus
  • Labeling and detecting method of 5-acetenyl-2'-deoxyuridine of genomic DNA of hepatitis B virus
  • Labeling and detecting method of 5-acetenyl-2'-deoxyuridine of genomic DNA of hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Establishment of Huh7-NTCP cell line

[0061] 1. Amplify the NTCP gene, insert it into the vector pcDNA3.0, and construct the NTCP eukaryotic expression plasmid pcDNA-NTCP. Specifically, the amplified NTCP gene and the vector pcDNA3.0 are subjected to HindIII and XhoI double enzyme digestion reactions respectively, The product was detected by 1.5% agarose gel electrophoresis and the constructed NTCP-pcDNA plasmid was linearized using PvuI endonuclease to increase the efficiency of DNA integration and the proportion of positive cell clones; T4DNA ligase was used to connect the above-mentioned linear plasmid vector pcDNA3. 0 and NTCP genes. Primers for amplifying the NTCP gene are as follows:

[0062] pC3NTCP-F:cccAAGCTTgccaccatggaggcccacaacgcgtctg

[0063] pC3NTCP-R:ccgCTCGAGctaggctgtgcaaggggagcag

[0064] 2. Huh7 cells were transfected with pcDNA-NTCP and screened with 700ug / ml G418 for 4-6 weeks

[0065] 3. Pick G418 resistance-positive clones, detect NTC...

Embodiment 2

[0073] Example 2 Preparation of EdU-HBV

[0074] Material:

[0075] 5-ethynyl-2'-deoxyuridine(EdU)(Sigma)in DMSO

[0076] HepG2.2.15cell

[0077] HBV Genomic DNA Quantification Kit (QiaGen)

[0078] PEG8000 (Sigma)

[0079] step:

[0080] 1. 1x10 7 HepG2.2.15 cells were passaged in T75 culture flasks

[0081] 2. After culturing for 24 hours, replace the MEM containing 10 μM EdU and 2% FBS, collect the cell supernatant after 2 days, then add MEM containing 10 μM EdU and 2% FBS, and collect the cell supernatant again after 2 days

[0082] 3. Collect the cell supernatant for virus purification: first filter with a 0.45um filter membrane (Millipore) or centrifuge at 12000rpm for 2min to remove impurities such as cell debris

[0083] 4. Transfer the supernatant to another 50ml centrifuge tube, add PEG8000 at a final concentration of 5-10%, and mix slowly on ice for 4-6h

[0084] 5. Centrifuge at 3000-5000rpm 4°C for 20min

[0085] 6. Discard the supernatant, resuspend the ...

Embodiment 3

[0101] Example 3 virus infection

[0102] 1. Huh7-NTCP cells are passaged, and when the confluence is 50-80%, dilute EdU-HBV and HBV with DMEM containing 4% PEG8000 and 10% FBS and infect Huh7-NTCP cells (100-1000Geq / cell);

[0103] 2. After 2-12 hours of infection, discard the supernatant, wash with PBS 3 times, replace the 2% medium, detect HBsAg secretion and EdU-HBV fluorescence detection at the expected time point.

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Abstract

The invention discloses a labeling and detecting method of 5-acetenyl-2'-deoxyuridine of a genomic DNA of hepatitis B virus. The method specifically comprises the following steps: (1) amplifying an NTCP gene, constructing an NTCP eukaryotic expression plasmid pcDNA-NTCP, and shifting the pcDNA-NTCP to an Hhh7 hepatoma carcinoma cell to construct a Huh7-NTCP cell line with NTCP stably expressed; (2) treating a HepG2.2.15 cell by using EdU, obtaining a cell supernatant containing EdU-HBV, performing treatment with PEG8000, and quantifying the copy number of HBV genome after concentration; and (3) performing subculture on the HBV cells till the cell convergence degree is 50-80%, infecting the Huh7-NTCP cell with EdU-HBV, and then performing EdU-HBV fluorescence detection. Compared with the prior art, the method is simple to operate, the image result does not need to be processed in the later stage, and the dyeing results do not interfere with each other.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for labeling and detecting 5-ethynyl-2' deoxyuridine of hepatitis B virus genome DNA. Background technique [0002] Hepatitis B virus, referred to as Hepatitis B virus (HBV), is a DNA virus belonging to the hepadnaviridae family. HBV is only susceptible to humans and orangutans, and its chronic infection is closely related to liver cirrhosis and liver cancer, and is the most important inducing factor for hepatocellular carcinoma (HCC). Serological evidence has confirmed that 30% of the global population has ever been infected with HBV, of which about 250 million people are chronically infected with HBV, but the pathogenic mechanism of HBV has not yet been elucidated. HBV has strict species specificity, its natural hosts are limited to humans and orangutans, and although the HBV virus genome only contains 3200bp, it has an effective structure and a special replica...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12Q1/70G01N33/569C12R1/93
CPCC12N7/00C12N2730/10152C12Q1/02G01N33/56983G01N2333/02
Inventor 马春红武专昌梁晓红高立芬王泽华宋晓佳徐蕾琪王体潇宋洋杜宪红谭思雨
Owner SHANDONG UNIV
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