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Efficiently-replicated human hepatitis B virus recombinant vector and application thereof

A technology of hepatitis B virus and recombinant vector, which is applied in the fields of genetic engineering, gene therapy and biomedicine, which can solve problems such as difficulties and limited capacity of viral nucleocapsid, and achieve the effect of efficient replication

Active Publication Date: 2014-04-02
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the genome of hepatitis B virus is very small, only 3.2kb, the viral genes and genes and regulatory regions overlap each other, and the capacity of the viral nucleocapsid is extremely limited (experiments have proved that if the genomic DNA is greater than 3.5kb, it cannot be detected. Therefore, it is very difficult to artificially modify the genome of hepatitis B virus so that it can accommodate a certain length of foreign gene without affecting the efficient replication of the virus. Some international attempts have failed (Chaisomchit S, Tyrrell D, Chang L, 1997. Gene Therapy. 4:1330-1340)

Method used

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  • Efficiently-replicated human hepatitis B virus recombinant vector and application thereof
  • Efficiently-replicated human hepatitis B virus recombinant vector and application thereof
  • Efficiently-replicated human hepatitis B virus recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Recombinant human hepatitis B virus vector and recombinant hepatitis B virus have efficient replication ability

[0042] Below with the expression plasmid PHY-5c3c ( image 3 ) as an example to illustrate the construction method of the viral vector. With the plasmid CMV-1.1HBV containing 1.1 copies of the wild-type hepatitis B virus genome ( image 3 ) as a template, design corresponding primers according to the upstream and downstream sequences of the SP15-SP143 segment of the hepatitis B virus polymerase spacer, and amplify the DNA fragment of the plasmid sequence lacking the above segment by reverse PCR method. Plasmid, namely PHY-5c3c.

[0043] Containing 1.1 copies of the expression plasmid PHY-5c3c of the recombinant human hepatitis B virus vector genome, the expression plasmid PHY-5c3c-Tat of the Tat protein gene inserted at the deletion site of the virus vector, and the expression plasmid of the red fluorescent protein gene inserted at the deletion ...

Embodiment 2

[0044] Example 2: Recombinant hepatitis B virus with Tat or red fluorescent protein gene can express foreign gene

[0045] The expression plasmid PHY-5c3c of the recombinant human hepatitis B virus vector genome and the expression plasmid PHY-5c3c-Tat of the recombinant hepatitis B virus genome with Tat gene and the luciferase expression plasmid LTR-luc promoted by the LTR promoter were co-transfected into liver cancer cells Huh7 cells were harvested 48 hours after transfection, the expression of luciferase was detected, and the liver cancer cell line Huh7 cells were co-transfected with pcDNA3 empty plasmid and pcDNA3-Tat and LTR-luc as negative and positive controls; the results are shown in the attached Figure 5 As shown, the Tat protein in PHY-5c3c-Tat can be effectively expressed; the expression plasmid PHY-5c3c-red containing 1.1 copies of the recombinant hepatitis B virus genome with the red fluorescent protein gene was transfected into liver cancer cell line Huh7 cells,...

Embodiment 3

[0046] Example 3: Detecting the Integrity of Recombinant Virus Particles

[0047]The expression plasmid PHY-5c3c of the recombinant human hepatitis B virus vector genome, the expression plasmid PHY-5c3c-Tat of the recombinant hepatitis B virus genome with the Tat protein gene, and the hepatitis B surface protein expression plasmid sp-s were used to transfect liver cancer cell line Huh7 cells, Cell supernatants were collected 72 hours after transfection, and the pull down experiment was performed with an antibody against PreS1 (located at the amino terminal of the large surface protein), and nucleic acid hybridization was used to detect whether the pull down recombinant hepatitis B virus contained viral DNA. The supernatant of cells transfected with PHY-5c3c and PHY-5c3c-Tat was used as a negative control, and the supernatant of cells transfected with PWT was used as a positive control. Such as Figure 7 As shown, whether the recombinant virus is co-transfected with sp-s or no...

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Abstract

The invention belongs to the field of medicine and gene engineering, and relates to a recombinant vector based on human hepatitis B viruses; the virus genome has partially or all deleted hepatitis B virus polymerase spacers relative to wild-type hepatitis B virus vectors. The invention also provides a preparation method of the recombinant vector and an application in gene therapy of targeting livers or hepatocytes. The human hepatitis B virus recombinant vector of the invention can enable the expression of inserted exogenous genes and the efficient replication of recombinant hepatitis B viruses, and can form complete recombinant hepatitis B virus particles with the supply of surface protein, which shows the application prospects that the human hepatitis B virus recombinant vector of the invention can be used as a liver-specific gene introduction vector.

Description

technical field [0001] The invention relates to the fields of genetic engineering, gene therapy and biomedicine, in particular to a highly efficient replicating recombinant human hepatitis B virus vector and its application to the liver or liver cell-specific introduction of exogenous genes. Background technique [0002] Liver diseases such as chronic viral hepatitis, liver cirrhosis, liver cancer, liver disease caused by metabolic disorders, toxic liver disease caused by drugs and other reasons, autoimmune liver disease, etc. are common clinical diseases, which are extremely harmful to the health of the body. It is a very effective treatment method, and new treatment methods or drugs are urgently needed, and liver or hepatic cell-specific gene therapy methods have potential drug development value. [0003] The most critical and difficult problem of gene therapy is how to specifically and efficiently introduce exogenous genes into specific types of target cells in specific t...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N15/11C12N5/10C12N15/64A61K48/00A61P35/00
Inventor 谢幼华洪冉翟建伟刘晶张继明李新艳刘伟
Owner FUDAN UNIV
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