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A chimeric virus of nonstructural proteins of hepatitis C virus and gb virus b

A technology of hepatitis C virus and non-structural protein, applied in the field of virus, composition and its preparation or purification, to introduce foreign genetic material modified virus, it can solve the problem of inability to show that hepatitis C virus interacts with the host, cannot completely Simulate the immune response of hepatitis C virus, cannot fully simulate the infection and immune status of hepatitis C virus, etc.

Active Publication Date: 2011-12-07
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, GB virus B is still different from hepatitis C virus, and cannot completely simulate the infection and immune status of hepatitis C virus in primates. Therefore, if the GB virus B genome is used as the backbone, the function of hepatitis C virus The gene replaces the corresponding functional region of GB virus B to construct the HCV / GBV-B chimeric virus, which is an ideal solution to study the immunity of hepatitis C virus by using the infection model
However, there are certain difficulties in replacing the complete functional protein gene of hepatitis C virus into the GB virus B genome, and there are uncertainties and risks in whether the constructed chimeric virus with complete functional protein can infect small primates Therefore, the current research using HCV / GBV-B chimeric virus has only been found in the replacement of the 5'-NCR, p7 or HVR1 of the hepatitis C virus with smaller gene segments into the GB virus B genome, which only contains C Chimeric viruses with very short gene segments of hepatitis virus can only be used to study the function of these specific gene segments, and cannot show the interaction between hepatitis C virus and the host, and cannot completely simulate the interaction of hepatitis C virus in primates immune response

Method used

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  • A chimeric virus of nonstructural proteins of hepatitis C virus and gb virus b
  • A chimeric virus of nonstructural proteins of hepatitis C virus and gb virus b
  • A chimeric virus of nonstructural proteins of hepatitis C virus and gb virus b

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, prepare the nonstructural protein of hepatitis C virus and the chimeric virus of GB virus B

[0033] (1) Preparation of nonstructural protein 2 to 4A genes of hepatitis C virus

[0034] (a) The viral RNA in the serum sample of hepatitis C virus type 1b was extracted from 200 μl of serum with the Roche nucleic acid extraction kit according to the instructions, and dissolved in 50 μl of Elution buffer.

[0035] (b) Design and synthesize the upstream outer primers, downstream outer primers, upstream inner primers and downstream inner primers for amplifying hepatitis C virus type 1b nonstructural protein 2 to 4A genes; Viral RNA is reverse-transcribed into cDNA; then the non-structural protein 2 to 4A gene fragments of hepatitis C virus are amplified in two rounds by nested PCR. The non-structural protein 2 to 4A gene of the amplified hepatitis C virus is connected with the pMD-20T carrier, and TOP10 competent cells are used for transformation to obtain the p...

Embodiment 2

[0076] Example 2. Infectivity evaluation of chimeric virus in marmoset

[0077] Evaluation criteria for chimeric virus infectivity: ① Infectious: virus replicates, and the viral load is detected in marmoset serum and verified to be correct; ② Non-infectious: virus does not replicate, and viral load cannot be detected in marmoset serum.

[0078] The method of infecting marmosets is to infect marmosets by intrahepatic injection of chimeric virus. The specific infection steps and evaluation are as follows:

[0079] 1. Determination of virus load in marmosets infected with chimeric virus and marmoset serum

[0080] (1) Intrahepatic injection of chimeric virus RNA

[0081]Choose healthy adult marmosets (with normal liver enzyme activity such as ALT, AST, and GB virus B negative) (about 300-400g / monkey), divide them into three groups, expose the liver by surgery, and inject the prepared chimeric virus RNA into the liver (dissolved in phosphate buffer before injection), GB virus B...

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Abstract

The invention relates to the fields of viruses, compositions and preparation or purification thereof, particularly a hepatitis C virus (HCV) nonstructural protein / GB virus B (GBV-B) hybrid virus. The hybrid virus is formed by sequentially connecting 5' terminal noncoding area and structural protein gene sequences of GBV-B, nonstructural proteins 2 to 4a gene sequences of HCV, nonstructural proteins 4B to 5B and 3' terminal noncoding area sequences of GBV-B, wherein the nonstructural proteins 2 to 4a gene sequences of HCV are nonstructural proteins 2 to 4a gene sequences of 1b genotype HCV. The virus provided by the invention can successfully infect a marmoset by intrahepatic injection. After the hybrid virus infects the marmoset, the active state of HCV protease in a primate body is simulated, thereby providing technical support for screening, inspection and evaluation of HCV antiviral agents.

Description

technical field [0001] The present invention relates to the field of virus, composition and its preparation or purification, in particular to the field of virus modified by introducing foreign gene material. Background technique [0002] According to a survey, 170 million people in the world are infected with hepatitis C virus (HCV), and there are at least 3-4 million new infections every year. The HCV infection rate of the natural population in my country is about 3%, which is a high prevalence country. Studies have confirmed that more than 70% of acute HCV infection is transformed into persistent infection, 10%-20% of which further develop into complex chronic liver diseases such as liver cirrhosis, and 1-5% of patients develop liver cancer after 20 to 30 years. Currently, there is no vaccine available for the prevention of hepatitis C, and the effect of antiviral therapy is not good. Since HCV can only infect humans and chimpanzees, there is no appropriate small animal ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/51C12N15/40C12N15/63
Inventor 黎诚耀李婷婷陈姿喧张玲
Owner SOUTHERN MEDICAL UNIVERSITY
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