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Human liver cancer cell line for observing life period of hepatitis B virus in cells and applications thereof

A liver cancer cell line, hepatitis B virus technology, applied in the direction of microorganism-based methods, cells modified by introducing foreign genetic material, microorganism measurement/testing, etc., can solve the problems that cannot be truly confirmed, lack sensitive cell lines and virus visualization Methods and other issues

Inactive Publication Date: 2014-03-26
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The outer membrane of the hepatitis B particles is in contact with the host cells, and the S protein used to attach and invade the cells, but how the virus particles attach and invade the cells is still poorly understood. There are many reports so far, but they cannot be truly confirmed
Studying the mechanism of HBV infecting the human body is the key to solving the problem, but due to the lack of sensitive cell lines and virus visualization methods, the research on how HBV enters cells, how it transports, replicates, expresses and exits cells is still unclear. confirmed by the method

Method used

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  • Human liver cancer cell line for observing life period of hepatitis B virus in cells and applications thereof
  • Human liver cancer cell line for observing life period of hepatitis B virus in cells and applications thereof
  • Human liver cancer cell line for observing life period of hepatitis B virus in cells and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Construction of eukaryotic expression vector pcHBV1.3-TC-1

[0050] 1. Obtain 1.3 times the hepatitis B virus gene

[0051] 1) Design primers as follows:

[0052] Primers are as follows:

[0053]

[0054] 2) Reaction system

[0055]

[0056] Mix well by flicking, and centrifuge briefly to collect the liquid droplets on the tube wall to the bottom of the tube.

[0057] 3) PCR amplification

[0058] Pre-denaturation at 95°C, 5min

[0059] Cycle (30 times) 30s→95℃, 30s→58℃, 4min30s→72℃,

[0060] End extension 72°C, 5min

[0061] 4) Electrophoresis recovery and purification

[0062] (1) Add 500 μL of equilibrium solution to the CA2 adsorption column, centrifuge at 12,000 g for 30 s, and discard the supernatant.

[0063] (2) Cut off the target band and put it into a clean centrifuge tube.

[0064] (3) Add 3 times the volume of sol solution PN, and bathe in 50°C water until the glue block dissolves.

[0065] (4) Transfer the solution into a CA2 adsorption column...

Embodiment 2

[0301] Taking the eukaryotic expression vector pcHBV1.3-TC-1 as an example, it was transfected into human liver cancer cells

[0302] 1. Cell culture: Take out the frozen TC2 cells from the liquid nitrogen tank (there is no transfection here, how is it TC-2), shake it in a water bath at 37°C for 1 minute to dissolve it, add about 5mL of DMEM medium and centrifuge it in 10mL centrifuge at 1000rpm / min for 5min, remove the supernatant, add to a 75mL culture bottle containing 10mL medium, and place at 37°C, 5% CO 2 in the incubator.

[0303] After 24 hours, the medium was changed once. After the cells grew to about 90% confluent, they were passaged according to the ratio of 1:2. Wait until there are enough cells for subsequent experiments.

[0304] Observing the cells under a microscope (*100), it was found that the cells could adhere to the wall 8 hours after thawing, and the cells were observed after 24 hours, and the cells were densely grown, growing in clusters, with abundan...

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Abstract

The invention belongs to the field of animal cell line, and specifically relates to a human liver cancer cell line for observing life period of hepatitis B virus in cells and applications thereof. The human liver cancer cell line is characterized in that the human liver cancer cell line can secrete hepatitis B virus particles, and different loci of core antigens of hepatitis B virus particles all carry a TC label. The human liver cancer cell line can directly combine with bi-arsenic dye, so the processes that hepatitis B virus enters a cell, is transported, duplicated and expressed in the cell, and finally is transported out of the cell can be continuously and stably observed, and thus the characteristics of hepatitis B virus can be more visually observed.

Description

technical field [0001] The invention belongs to the field of animal cell lines, in particular to a human liver cancer cell line for observing the intracellular life cycle of hepatitis B virus and its application. technical background [0002] There are people infected with hepatitis B virus in various regions of the world. In Asia, the South Pacific region, Africa, New Zealand and other regions, hepatitis B virus HBV is one of the main pathogens that endanger human health. China is a high-incidence area of ​​hepatitis B, and the prevention and treatment of hepatitis B is an arduous and important task. [0003] Hepatitis B virus belongs to the Hepadnaviridae family and has only a narrow host range. For example, human hepatitis B virus can only infect humans or primate orangutans and baboons, and duck hepatitis B virus cannot naturally infect all species of domestic ducks. . The replication cycle of hepatitis B virus has been studied in vivo and in vitro in animals, especial...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/70C12R1/91
Inventor 林菊生廖家智孙淑珍闫静君何星星常莹田德安赵秋但自立
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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