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Replicative HBV (Hepatitis B Virus) vector carrying foreign gene and recombinant HBV generated after transfection and corresponding preparation method and application

An exogenous gene and replication technology, applied in the field of biomedical engineering, can solve the problems of non-replicating HBV vector application, low replication level, loss of infectivity, etc.

Active Publication Date: 2014-08-13
BEIQIUEN INT PEACE HOSPITAL P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen from the figure that the HBV replication level after HBVtat vector transfected cells is too low compared with the wild type, and cannot be used as a replicating HBV vector
Although the article does not state the infectivity of recombinant HBV, it is conceivable that due to the absence of the large outer membrane protein, this recombinant HBV must lose its infectivity

Method used

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  • Replicative HBV (Hepatitis B Virus) vector carrying foreign gene and recombinant HBV generated after transfection and corresponding preparation method and application
  • Replicative HBV (Hepatitis B Virus) vector carrying foreign gene and recombinant HBV generated after transfection and corresponding preparation method and application
  • Replicative HBV (Hepatitis B Virus) vector carrying foreign gene and recombinant HBV generated after transfection and corresponding preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment １

[0099] Example 1 Replication-type HBV vector carrying foreign genes and its preparation method

[0100] Two replicating HBV vectors, pCH-BsdR and pCH-hrGFP, were constructed to insert foreign genes. Both vectors use cytomegalovirus (CMV) promoter to initiate transcription, contain the whole HBV genome, and express blasticidin respectively. Resistance gene (BsdR) and humanized Renilla green fluorescent protein (hrGFP) gene.

[0101] Such as image 3 As shown, the left side of the figure is the wild-type HBV vector pCH-9 / 3093, with CMV promoter, prokaryotic replication initiation site (Ori), prokaryotic screening marker ampicillin resistance gene (Amp), containing 1.05 times the HBV genome, The full length is 6336 bp. After the vector transfects cells, it can transcribe the same genomic RNA and various subgenomic RNAs as wild-type HBV, and express various HBV proteins, complete the full cycle of HBV replication, and package complete, infectious cells. Wild-type HBV virions....

Embodiment 2

[0124] Example 2 The inspection of the HBV vector carrying foreign gene

[0125] 1. Two tandem Rbm3 IRES on HBV pregenomic RNA can effectively initiate protein translation

[0126] At present, no studies have reported the translation initiation efficiency of Rbm3 IRES in human hepatocytes. In order to test the ability of Rbm3 IRES to guide translation initiation on the RNA strand similar to HBV pregenomic RNA (HBV pgRNA), and to prove whether Rbm3 IRES is applicable For the construction of bicistronic and tricistronic vectors, two series of 8 types of plasmids were constructed using Rbm3 IRES, which were transfected into HepG2 and Huh7 cells derived from liver cancer cells, and the exogenous fluorescent groups guided by Rbm3 IRES were detected. The expression level of the cluster was used to verify the feasibility of using Rbm3 IRES to construct a replicative HBV vector and use it in hepatocytes. At the same time, it was compared with the HBV vector using EMCV IRES to compar...

Embodiment 3

[0246] Example 3 Recombinant HBV and its preparation method after a replicative HBV vector carrying an exogenous gene is transfected into a cell

[0247] A recombinant HBV carrying an exogenous gene, the recombinant HBV is the recombinant HBV collected in the cell culture supernatant after the HBV vector described in Example 2 is transfected into liver cancer cells, the recombinant HBV not only retains the ability to replicate and infect Can express foreign genes.

[0248] This embodiment also provides a method for preparing the above-mentioned recombinant HBV carrying the foreign gene, which is carried out according to the following sequence of steps:

[0249] (21) Construction of HBV vectors carrying foreign genes;

[0250] (22) Use the vector prepared in step (21) to transfect the cell line derived from liver cancer, and add 1%-2% dimethyl sulfoxide to the cell culture medium, which can significantly improve the replication level of recombinant HBV and the final Yield; ...

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Abstract

The invention discloses a replication-type HBV vector carrying foreign genes, recombinant HBV produced after transfecting cells with the vector, and a corresponding preparation method and application. Molecular cloning technology separates the overlapping C gene and P gene on the HBV genome, each forming a complete open reading frame, inserting a protein translation initiation sequence or protease cleavage site in between, respectively guiding the expression of the foreign gene and the P gene; carrying the foreign gene The replicative HBV vector transiently transfects liver cancer cells to secrete recombinant HBV; after the HBV vector prepared by the present invention transfects cells, the recombinant HBV produced can express foreign genes while still retaining the ability to replicate and infect; The invention is suitable for constructing animal models of HBV chronic infection, constructing cell models of stable and autonomous replication of HBV cccDNA, constructing traceable HBV virus strains, used to study the molecular mechanism of HBV infection, replication, packaging and other links, and screening new anti-HBV drugs .

Description

technical field [0001] The invention belongs to the field of biomedical engineering, and relates to a viral vector, in particular to a replicating HBV vector carrying foreign genes, recombinant HBV produced after its transfection, and a corresponding preparation method and application. Background technique [0002] Worldwide, more than 400 million people are chronically infected with hepatitis B virus (HBV). This population is at greatly increased risk of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Existing anti-HBV drugs include two categories of interferon and nucleoside analogues. However, these two types of drugs have unsatisfactory curative effects, and the side effects of interferon are relatively large, and nucleoside analogs can easily induce drug-resistant mutations in HBV. A major obstacle to the development of new anti-HBV drugs is the lack of a good experimental research platform. [0003] HBV specifically infects liver cells and has a very narrow...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/86C12R1/93
Inventor 孙殿兴程欣王梓华武丽李东康富标
Owner BEIQIUEN INT PEACE HOSPITAL P L A
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