Hepatitis C virus and its external cell culture method

Abstract

An external culture system of hepatitis C totivirus for culturing complete multiplicative HCV virus by the molecular biology and gene recombination technique is disclosed. Its culture steps include amplifying the full-length genom containing 98 nucleotides after 3' PolyA in the serum of hepatitis C patient; artificial site-directed mutation to NS5A and NS5B in HCV genon; inserting the expression box of marker gene IRES-GFP at NS5B3' terminal in the mutated HCV genom; transfecting sensitive cell and culturing to obtain progeny HCV virus with infection activity.

Description

technical field [0001] The invention relates to the use of molecular biology and gene recombination technology to construct a complete and suitably mutated hepatitis C virus (HCV) genome, and to establish a whole hepatitis C virus in vitro culture system through transfection of sensitive cells. Background technique [0002] Human hepatitis C is an infectious disease caused by hepatitis C virus (hereinafter referred to as HCV). After HCV infects susceptible persons, it can not only cause acute infection, but also more likely to form chronic infection, leading to liver hypertrophy, liver cirrhosis, and even liver cancer, which seriously endangers human health. More importantly, due to the lack of effective prevention and control measures, HCV infection is still prevalent in the world. According to incomplete statistics, about 170 million people in the world are infected with HCV, and millions of infected people are added every year. Nearly 2.5% of the population (approximate...

AI Technical Summary

Problems solved by technology

The 3′UTR region is closely related to the replication and assembly of the virus, but it is worth pointing out that the 3′ end of the HCV genome sequence in GenBank generally ends at polyA, and researchers did not take into account when constructing the HCV genome cDNA The following 98 conserved nucleotide sequences

Although there are a few reports (Dash et at 1997, Am JPathology151: 363; Yoo et at 1995, J Yirol69: 32) after transfection of human liver cancer cell line Huh-7 or Hep G2 with HCV genome, the replication of HCV can be detected ( Nucleic acid detection by nested PCR), but neither can confirm the production of infectious virus particles in the cells, nor can it guarantee the long-term existence and high-level expression of HCV in the transfected cells

Therefore, there have been no literature reports on the establishment of a stable and effective in vitro culture system and experimental model of HCV in vitro, nor have there been reports on the cultivation of complete HCV viruses in vitro for scientific research and the sale of related system products.

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