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
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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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