Chimeric virus of complete envelope protein of HCV (hepatitis C virus) and GB virus B

A technology of hepatitis C virus and envelope protein, which is applied in the field of viruses modified by introducing foreign genetic materials, composition and its preparation or purification, and can solve the problem of incomplete imitation of the immune response of hepatitis C virus and the inability to express Interaction between hepatitis C virus and host, inability to fully simulate hepatitis C virus infection and immune status

Inactive Publication Date: 2011-08-17
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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  • Chimeric virus of complete envelope protein of HCV (hepatitis C virus) and GB virus B
  • Chimeric virus of complete envelope protein of HCV (hepatitis C virus) and GB virus B
  • Chimeric virus of complete envelope protein of HCV (hepatitis C virus) and GB virus B

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1. Preparation of chimeric virus of hepatitis C virus complete envelope protein and GB virus B

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

[0035] (a) Using the Roche nucleic acid extraction kit to extract viral RNA from 200 μl of serum from HCV type 1b serum samples according to the instructions, dissolve in 50 μl of Elution buffer.

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

Embodiment 2

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

[0077] Chimeric virus infectivity evaluation criteria: ① Infectious: the virus replicates, and the viral load is detected in the serum of the marmoset and verified correctly; ② Non-infectious: the virus does not replicate, and the viral load cannot be detected in the serum of the marmoset.

[0078] The methods of infecting marmosets can be by 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:

[0079] 1. Intrahepatic injection of chimeric viral RNA and determination of viral load in marmoset serum

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

[0081]Select healthy adult experimental animal marmosets (with normal liver enzyme activities such as ALT and AST, and negative for GB virus B) (about 300-400 g per animal), and divide them into three gr...

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Abstract

The invention relates to the field of a virus, a compound and preparation or purification of the virus, in particular to a chimeric virus of a complete envelope protein of an HCV (hepatitis C virus) and a GB virus B. The chimeric virus is formed by sequentially connecting a 5'-end non-coding region and core protein gene sequence of the GB virus B, a complete envelope protein gene sequence of the HCV and a nonstructural protein and 3'-end non-coding region sequence of the GB virus B, wherein the complete envelope protein gene sequence of the HCV is a complete envelope protein gene sequence of a 1b-genotype HCV. A marmoset monkey can be successfully infected by the virus by carrying out intrahepatic injection and intravenous injection of a primary marmoset monkey serum containing the chimeric virus. The chimeric virus simulates infection and immune states of the HCV in a primate body. A platform for carrying out immunoassay and evaluation on the complete envelope protein of the HCV is provided. The scientific problems of limitation on HCV immunity prevention and treatment research, vaccine evaluation and the like due to shortage of a small primate infection model are solved.

Description

technical field [0001] The present invention relates to the field of viruses, compositions and their preparation or purification, in particular to the field of viruses modified by introducing foreign genetic materials. Background technique [0002] Hepatitis C Virus (HCV) is one of the main pathogenic factors that cause acute / chronic hepatitis through blood transmission. The prevalence of HCV infection varies greatly around the world, and the most serious in developing countries. Worldwide, approximately 170 million people are currently infected with the hepatitis C virus. 50-85% of HCV-infected patients develop chronic hepatitis, 10-20% of them further develop complex chronic liver diseases such as cirrhosis, and 1-5% develop liver cancer after 20 to 30 years. However, there is still a lack of very effective therapeutic drugs for hepatitis C virus infection. What is more serious is that there is no hepatitis C preventive vaccine in the world so far. Until today, the resea...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/40C12N15/51C12N7/01
Inventor 黎诚耀李婷婷王文敬张玲
Owner SOUTHERN MEDICAL UNIVERSITY
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