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Method for separating and purifying hepatitis B virus particles

A hepatitis B virus, separation and purification technology, applied in the direction of recovery/purification, can solve the problem of reducing the infection efficiency of tree shrew liver cells to hepatitis B virus, and achieve the effect of good hepatitis B virus characteristics and high yield

Active Publication Date: 2010-11-03
ARMY MEDICAL UNIV
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Problems solved by technology

Serum components reduce the infection efficiency of tree shrew hepatocytes to hepatitis B virus, a recent study revealed

Method used

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  • Method for separating and purifying hepatitis B virus particles
  • Method for separating and purifying hepatitis B virus particles
  • Method for separating and purifying hepatitis B virus particles

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Embodiment Construction

[0019] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0020] In a preferred embodiment, the experimental materials used are as follows: Hepatitis B virus DNA positive serum: Hepatitis B virus DNA positive patient blood (DNA copy number ≥ 10 7 , The Second Affiliated Hospital of Chongqing Medical University) was centrifuged at 1700g for 15 minutes, the serum was separated, and stored at -20°C; TNE buffer: Tris-HCl with a concentration of 20mM, with a concentration of 140mM sodium chloride and 1mM ethylenediaminetetraacetic acid (EDTA), pH 7.4; sucrose solution: using TNE buffer as solvent, the mass fractions were 60%, 45%, 35%, 25%, respectively. % and 15% solutions; adult male tree shrew weighing 135 g (Kunming Institute of Zoology, Chinese Academy of Sciences); tree shrew hepatocyte culture plate and medium Hepato-STIM Hepatocyte Defined Medium (BD Biosciences, USA); Hepatitis B virus DNA flu...

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Abstract

The invention discloses a method for separating and purifying hepatitis B virus particles, which comprises the following steps of: centrifuging 70,000 grams of hepatitis B virus DNA positive serum for 5 hours with discontinuous sucrose density gradients (60 percent, 45 percent, 35 percent, 25 percent and 15 percent), absorbing centrifugal liquid from the bottom of a centrifugal tube section by section, collecting the centrifugal liquid into different containers respectively, merging the colorless transparent collected liquid, and removing sucrose by ultra-filtration and centrifugation to obtain the hepatitis B virus particles. The method can efficiently separate and purify the hepatitis B virus particles in short time and has high virus particle yield; and the obtained virus particles have typical hepatitis B virus characteristics and good biological infection activity, and can effectively infect primary liver cells of tree shrews.

Description

technical field [0001] The invention relates to a method for separating and purifying virus particles, in particular to a method for separating and purifying hepatitis B virus (HBV) particles. Background technique [0002] Hepatitis B is a major global health problem. Studying the infection mechanism of hepatitis B virus is of great significance to the prevention and treatment of hepatitis B and the research and development of anti-hepatitis B drugs. The research on the mechanism of HBV infection requires a large amount of HBV particles. Although HBV particles can be extracted from the HepG.2.2.15 culture system, the virus titer is less than 10 5 copies / ml, far from meeting the research needs. At present, hepatitis B virus particles are mainly isolated and purified from the serum of hepatitis B virus DNA (HBV-DNA) positive patients by discontinuous sucrose density gradient ultracentrifugation. low cost and high cost. The tree shrew (Tupaia Belangeri), which is closely re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02
Inventor 曾兴光吴玉章刘宏利韩俊峰邹丽云谢谆怡
Owner ARMY MEDICAL UNIV
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