Chimeric virus of complete structural protein of hepatitis C virus and GB virus B

A hepatitis C virus, structural protein technology, applied in the direction of virus/bacteriophage, biochemical equipment and methods, applications, etc. Status, inability to indicate HCV-host interaction, etc.

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A hepatitis C virus, structural protein technology, applied in the direction of virus/bacteriophage, biochemical equipment and methods, applications, etc. Status, inability to indicate HCV-host interaction, etc.

CN102154227AActive Publication Date: 2011-08-17SOUTHERN MEDICAL UNIVERSITY

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Embodiment 1

[0033] Embodiment 1, prepare the chimeric virus of hepatitis C virus complete structural protein and GB virus B

[0034] (1) Preparation of the complete structural protein gene of hepatitis C virus

[0035] (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.

[0036] (b) Design and synthesize the upstream outer primers, downstream outer primers, upstream inner primers and downstream inner primers for amplifying the complete structural protein gene of hepatitis C virus type 1b; reverse the extracted hepatitis C virus RNA by RT-PCR It was recorded as cDNA; the complete structural protein gene fragment of hepatitis C virus was amplified in two rounds by nested PCR. The amplified complete structural protein gene of the hepatitis C virus is connected with the pMD-20T vector, transformed with TOP10 competent cells, and...

Embodiment 2

[0075] Example 2. Infectivity evaluation of chimeric virus in marmoset

[0076] 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.

[0077] The method of infecting marmosets can be intrahepatic injection of chimeric virus and intravenous injection of primary marmoset serum containing chimeric virus to infect marmosets. The specific infection steps and evaluation are as follows:

[0078] 1. Intrahepatic injection of chimeric viral RNA and determination of viral load in marmoset serum

[0079] (1) Intrahepatic injection of chimeric virus RNA

[0080]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 v...

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Abstract

The invention relates to a virus, a composition and the field of preparation or purification thereof, in particular to a chimeric virus of the complete structural protein of a hepatitis C virus and a GB virus B. The chimeric virus is formed by connecting the sequence of a non-coding region on the 5' terminal of the GB virus B, the gene sequence of the complete structural protein of the hepatitis C virus, a nonstructural protein of the GB virus B and the sequence of a non-coding region on a 3' terminal in turn, wherein the gene sequence of the complete structural protein of the hepatitis C virus is the gene sequence of the complete structural protein of 1b genotype hepatitis C virus. The chimeric virus can be used to infect marmoset effectively through the intrahepatic or intravenous injection of the chimeric virus-containing serum of a primary marmoset. The chimeric virus simulates the infection and immune state of the hepatitis C virus in bodies of primates, a platform for testing and evaluating the immunity to the complete structural protein of the hepatitis C virus is provided, and the scientific problems such as limitation on the research on immunity, prevention and control ofhepatitis C virus and vaccine evaluation due to lack of small infected models of primates are solved.

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] At present, there are about 170 million people infected with hepatitis C virus (Hepatitis C Virus, HCV) worldwide. Hepatitis C virus is one of the main pathogenic factors of acute / chronic hepatitis transmitted by blood. The prevalence of HCV infection varies greatly around the world, especially in developing countries. 50-85% of hepatitis C virus infected patients develop into chronic hepatitis, 10-20% of them further develop into complex chronic liver diseases such as liver cirrhosis, and 1-5% develop into liver cancer after 20 or 30 years. However, there is still a lack of very effective therapeutic drugs for hepatitis C virus infection at present, and what is more serious is that there is no hepatitis C preventive vaccine in the world so...

Claims

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Application Information

Patent Timeline

17 Aug 2011

Publication

CN102154227A

IPC: C12N7/01; C12N15/51; C12N15/40; C12N15/63

Inventors: 黎诚耀; 李婷婷