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Hepatitis B virus gene eukaryotic expression vector encoding tc chimeric core protein and its construction method

A technology of eukaryotic expression vector and hepatitis B virus, which is applied in the field of genetic engineering and can solve the problems of inability to dynamically observe the entry and intracellular transport of hepatitis B virus.

Inactive Publication Date: 2016-11-23
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

[0008] The purpose of the present invention is to fill the blank of hepatitis B virus labeling technology, provide the eukaryotic expression vector of hepatitis B virus gene encoding TC chimeric core protein and its construction method, and solve the problem of not being able to dynamically observe the entry and intracellular transport of hepatitis B virus

Method used

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  • Hepatitis B virus gene eukaryotic expression vector encoding tc chimeric core protein and its construction method
  • Hepatitis B virus gene eukaryotic expression vector encoding tc chimeric core protein and its construction method
  • Hepatitis B virus gene eukaryotic expression vector encoding tc chimeric core protein and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Obtain 1.3 times the hepatitis B virus gene

[0034] 1) Design primers as follows:

[0035] Primers are as follows:

[0036]

[0037] 2) Reaction system

[0038]

[0039] Mix well by flicking, and centrifuge briefly to collect the liquid droplets on the tube wall to the bottom of the tube.

[0040] 3) PCR amplification

[0041] Pre-denaturation 95℃, 5min

[0042] Cycle (30 times) 30s→95℃, 30s→58℃, 4min30s→72℃,

[0043] Final extension 72°C, 5min

[0044] 4) Electrophoresis recovery and purification

[0045] (1) Add 500 μL of equilibrium solution to the CA2 adsorption column, centrifuge at 12,000 g for 30 s, and discard the supernatant.

[0046] (2) Cut off the target band and put it into a clean centrifuge tube.

[0047] (3) Add 3 times the volume of sol solution PN, and bathe in 50°C water until the glue block dissolves.

[0048] (4) Transfer the solution into a CA2 adsorption column, centrifuge at 12000g for 30s, and discard the supernatant.

[0049...

Embodiment 2

[0160] The steps of this embodiment and embodiment 2 are basically the same, the difference is that the TC tag is inserted into the 49th position of the hepatitis B virus core protein, and the steps are as follows:

[0161] 1) Plasmid extraction

[0162] Extract pcHBV1.3 plasmid, the method is the same as embodiment 1.

[0163] 2), SOE-PCR amplified SOE fragment 1-2

[0164] (1) Design primers

[0165]

[0166] (2) Reaction system

[0167] SOE Fragment 1-2: Take a 0.2ml PCR tube, prepare the following reaction system, and prepare 4 tubes:

[0168]

[0169]

[0170] Flick to mix and centrifuge briefly;

[0171] (3) PCR amplification

[0172] Step is with embodiment 1

[0173] (4) Electrophoresis recovery and purification

[0174] Step is with embodiment 1

[0175] 3) SOE-PCR amplification of SOE fragment 2-2

[0176] (1) Design primers

[0177]

[0178] (2) Reaction system

[0179] SOE Fragment 2-2: Take a 0.2ml PCR tube, prepare the following reaction sy...

Embodiment 3

[0200] The steps of this embodiment and embodiment 2 are basically the same, the difference is that the TC tag is inserted into the 79th position of the hepatitis B virus core protein, and the steps are as follows:

[0201] 1) Plasmid extraction

[0202] Extract pcHBV1.3 plasmid, the method is the same as embodiment 1.

[0203] 2), SOE-PCR amplification of SOE fragments 1-3

[0204] (1) Design primers

[0205]

[0206] (2) Reaction system

[0207] SOE Fragment 1-3: Take a 0.2mL PCR tube, prepare the following reaction system, and prepare 4 tubes:

[0208]

[0209]

[0210] Flick to mix and centrifuge briefly;

[0211] (3) PCR amplification

[0212] Step is with embodiment 1

[0213] (4) Electrophoresis recovery and purification

[0214] Step is with embodiment 1

[0215] 3) SOE-PCR amplification of SOE fragments 2-3

[0216] (1) Design primers

[0217]

[0218] (2) Reaction system

[0219] SOE Fragment 1: Take a 0.2ml PCR tube, prepare the following reac...

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Abstract

The invention belongs to the field of genetic engineering and in particular relates to a hepatitis B virus gene eukaryotic expression vector for coding TC-tagged core proteins and a construction method of the vector. According to the hepatitis B virus gene eukaryotic expression vector for coding the TC-tagged core proteins, the eukaryotic expression vector is named pcHBV1.3-TC and takes pcDNA3.1+ as a basic skeleton, the hepatitis B virus gene eukaryotic expression vector contains 1.3-factor hepatitis B virus genes, and TC tags are contained in the 1.3-factor hepatitis B virus genes. The pcHBV1.3-TC can be stably combined to a host cell genome to form stable expression cells, and the whole life cycle process of the hepatitis B virus is conveniently researched.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the construction of eukaryotic expression vector of hepatitis B virus gene encoding TC chimeric core protein. Background technique [0002] Hepatitis B virus (HBV) is a small enveloped virus containing double-stranded DNA, which can infect liver cells, cause acute and chronic hepatitis, and eventually lead to liver cirrhosis and hepatocellular carcinoma. HBV virions are double shell-like particles with a diameter of 40nm to 42nm. The outer lipid protein envelope of the virion contains three related envelope glycoproteins. In the outer membrane of the virion, there is a regular 20-hedron-shaped core particle composed of core protein (21kD), with a diameter of 34nm. The core particle contains viral DNA polymerase and viral DNA genome. In addition to virion particles, cells infected with HBV also secrete two different subviral particles: 17-25nm spherical and cast H...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/79C12N15/66
Inventor 林菊生孙淑珍廖家智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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