High-transduction-efficiency hepatophilic cell hepatitis C virus pseudo-particle and envelope protein coded sequence thereof

A hepatitis C and liver cell technology, applied in the field of pseudovirion particles and its preparation, can solve the problems of low virus yield and transduction efficiency

Inactive Publication Date: 2011-11-09
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the HCVpp preparation methods currently established in all laboratories have a common problem, that is, the virus yield and transduction efficiency are very low, and the virus titers in the culture supernatant of 293T cells are all lower than 105 FFU (focus forming unit) / ml. This largely limits the application of HCVpp, so the development of HCV-mimetic virus particles with high titer, high transduction efficiency and high hepatocyte tropism is of great significance in HCV research and biotechnology application

Method used

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  • High-transduction-efficiency hepatophilic cell hepatitis C virus pseudo-particle and envelope protein coded sequence thereof
  • High-transduction-efficiency hepatophilic cell hepatitis C virus pseudo-particle and envelope protein coded sequence thereof
  • High-transduction-efficiency hepatophilic cell hepatitis C virus pseudo-particle and envelope protein coded sequence thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Cloning of HCV envelope protein full-length gene

[0021] 1. Extraction of HCV nucleic acid

[0022] Viral nucleic acid RNA was extracted from the sera of 50 HCV RNA-positive patients, respectively, using the QIAamp Ultraseus virus kit from QIAGEN Company.

[0023] Specific method: Take 1ml of serum to a 2ml EP tube and equilibrate the temperature to 15°C-25°C, add 800ul Buffer AC, add 5.6ulcarrier RNA to the lid of the EP tube, then vortex for 10s to mix; incubate at room temperature for 10 minutes, then centrifuge at 3200rpm for 3min , discard all the supernatant; add 300ul Buffer AR (preheated at 60°C) and 20ul proteinase K, and vortex to dissolve the precipitate; vortex once for 5 seconds during 10 minutes in a water bath at 40°C, and transfer it all into the QIAamp Spin Column 6000rpm Centrifuge for 1min to discard the waste liquid; add 500ul Buffer AW1 and centrifuge at 7500rpm for 1min to discard the waste liquid, add 500ul Buffer AW2 and centrifug...

Embodiment 2

[0040] Example 2: Screening of functional HCV envelope protein coding sequences

[0041] 1. Construction of HCV envelope protein expression plasmid

[0042] The correct HCV envelope protein gene identified by enzyme digestion and sequencing was amplified by PCR. The primer sequences were designed and synthesized according to the 5' and 3' end sequences obtained by sequencing, and the enzyme cutting site Nhe was added to the 5' and 3' ends respectively. I and EcoRI. The PCR amplified product was digested with NheI and EcoRI, inserted into the plasmid expression vector pCI-neo (promega product), identified by enzyme digestion and then sequenced. The correct plasmid was used to make HCVpp.

[0043] specific method:

[0044] A restriction endonuclease digestion: the identified correct clone with the HCV envelope protein coding sequence and the pCI-neo expression plasmid were digested with NheI and EcoRI restriction endonuclease respectively, and the enzyme digestion system was:...

Embodiment 3

[0054] Embodiment 3: Optimization of HCV envelope protein gene

[0055] 1. Our previous studies have shown that the basic amino acid residues in the HCV envelope protein HVR1 have an important impact on the infectivity of HCVpp, especially if the 8th and 12th amino acid residues are basic amino acids, they can significantly enhance HCVpp infectivity. Based on these findings, we mutated the 8th valine residue (V) and the 12th threonine residue (T) of the C50 strain HCV envelope protein gene encoding HVR1 into histidine residues individually or simultaneously. Base, using Stratagene kit (QuikChange TM Site-Directed Mutagenesis Kit) for mutation. The mutants were named as C50-V8H, C50-T12H, and C50-VT-2H, respectively.

[0056] 2. Our previous studies also showed that amino acid residues 16-24 in the HCV envelope protein HVR1 sterically hinder the binding of the HCV envelope protein to the SR-BI receptor, and deletion of this peptide can significantly enhance the binding of H...

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Abstract

The invention relates to the technical field of biological medicine engineering. In the invention, a hepatitis C virus envelope protein gene with high transduction efficiency and high hepatic cell tropism is obtained by screening with a genetic mutation method. By performing cotransfection on an expression plasmid of the gene and an HIV (Human Immunodeficiency Virus)-based slow virus packaging plasmid, a hepatitis C virus pseudo-particle with the infective titer of 1*107 FFU / ml can be obtained. The infective titer of the hepatitis C virus pseudo-particle obtained with the method is 100 times higher than that of hepatitis C virus pseudo-particles obtained with other methods. Referring to specific infection of hepatic cells, the hepatitis C virus pseudo-particle can be applied to the research of the infection mechanism of hepatitis C, the evaluation of a neutralizing antibody and the screening of small molecular medicaments, can be possibly taken as a hepatic cell targeting gene therapycarrier, and has an important application prospect.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, and relates to a pseudovirion particle containing hepatitis C envelope protein with high transduction efficiency and high hepatocyte tropism and a preparation method thereof. Background technique [0002] Hepatitis C virus (HCV) is a hepatotropic single-stranded positive-sense RNA virus, which belongs to the Flavivirus genus. The mechanism of the virus's hepatotropic properties is still unclear. At present, CD81, the scavenger receptor SR-BI, and claudin 1 and occludin, members of the human tetraspanin family, have been identified as receptors that HCV infection must depend on, but these four molecules are also expressed in other tissue cells. Therefore, it cannot perfectly explain the hepatotropic properties of HCV. The in vitro cell culture of HCV is very difficult. At present, it is not possible to infect the target cells in vitro with the serum of infected persons so as to ach...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K48/00
Inventor 王岳谭文杰赵平
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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