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B hepatitis virus X protein transduction system expression vector and its construction

A technology of hepatitis B virus and protein transduction, applied in the direction of introducing foreign genetic material using vectors, biochemical equipment and methods, applications, etc., which can solve the problems of low transfection efficiency, non-repeatability, and difficult quantitative control

Inactive Publication Date: 2004-11-17
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods have achieved success to a certain extent, they have shortcomings such as difficult quantitative control, expensive equipment, and time-consuming and labor-intensive experiments.
At present, the research on the transformation and carcinogenic mechanism of HBx mostly adopts eukaryotic gene expression system to transfect cells to realize HBx gene introduction into cells. control, with randomness and non-repeatability

Method used

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  • B hepatitis virus X protein transduction system expression vector and its construction
  • B hepatitis virus X protein transduction system expression vector and its construction
  • B hepatitis virus X protein transduction system expression vector and its construction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Construction of the expression vector of the hepatitis B virus X protein transduction system

[0033] (1) Template: The eukaryotic gene expression recombinant plasmid pCMV-X (the recombinant plasmid of x-gene and plasmid pCMV) was selected as the template. pCMV-X is donated by Professor Graessmann A of Freie University in Germany or the hepatitis B virus genomic DNA extracted from the serum of hepatitis B patients or a plasmid carrying the X gene; the X gene sequence is the full length of the genotype (genotype) D subtype sequence, no mutation;

[0034] (2) PCR primer design: PCR primers are based on the full-length sequence of the X gene in Genbank (AB104894.1),

[0035] Displayed as follows:

[0036] 1 atggctgcta gggtgtgctg ccaactggat cctgcgcggg acgtcctttg tttacgtccc

[0037] 61 gtcggcgctg aatcctgcgg acgacccttc tcggggtcgc ttggggactct ctcgtcccct

[0038] 121 tctccgtctg ccgttccgac cgaccacggg gcgcacctct ctttacgcgg actccccgtc

[0039] 181 tgtgccttct cat...

Embodiment 2

[0050] Example 2 Expression and Identification of Hepatitis B Virus X Protein Transduction System

[0051] The recombinant plasmid pTAT-GFP-X was transformed into Escherichia coli BL21, and the expression of the fusion protein TAT-GFP-X was induced by IPTG (isopropylthiogalactoside). The protein products were electrophoresed and stained with Coomassie brilliant blue. The results (see FIG. 6A ) showed that a large amount of fusion protein was expressed in the recombinant clone, and the size was consistent with the expected fusion protein molecular weight (about 43kDa). The rabbit anti-HBx polyclonal antibody was used for Western blot identification, and the result (Fig. 6B) showed a specific band, which proved that the HBx protein could be expressed in a fusion form. A HBV X protein transduction system was successfully constructed, and the fusion protein TAT-GFP-X can be induced to express through the transduction system.

[0052] Figure 6 A: SDS-PAGE electrophoresis, Coomass...

Embodiment 3

[0053] Example 3 Application of Hepatitis B Virus X Protein Transduction System

[0054] The hepatitis B virus X protein transduction system TAT-GFP-X of above-mentioned construction is carried out according to the following method when in use: (1) the recombinant plasmid pTAT-GFP-X is transformed into E. Galactoside) induces the expression of the fusion protein TAT-GFP-X, uses the Ni-NTA affinity purification system to carry out protein purification, identifies by SDS-PAGE electrophoresis, and purifies the fusion protein TAT-GFP-X with a single band, uses rabbit Anti-HBx polyclonal antibody was used for Western blot to further identify the purified single band as the fusion protein TAT-GFP-X. After the purified fusion protein is sterilized by filtration and quantified, it can be used in quantitative protein X transduction experiments of cells. The specific steps are as follows: Dissolve a certain dose of TAT-GFP-X fusion protein in the buffer system to prepare a mother solut...

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Abstract

The invention discloses a type B hepatitis virus X protein transduction system expression vector and its construction, by constructing restructured plasmid pTAT-GFP-X through the X gene of the human hepatitis B virus and pTAT-GFP vector, wherein the constructed pTAT-GFP-X restructured plasmid can express the fusion protein containing X protein of hepatitis B viruse, and a vast amount of expression fusion proteins can be evoked from isopropylthiog-alactoside (IPTG), purification can be carried out through 6 histidines (His6), the TAT polypeptide has the structure capable of traversing cell membrane.

Description

technical field [0001] The invention relates to a viral protein transduction system expression vector and its construction. Background technique [0002] Hepatitis B virus X protein (HBx) plays an important role in cell transformation and the occurrence and development of liver cancer, but its specific transformation and carcinogenesis mechanism is still unclear. Transferring HBx or its gene into eukaryotic cells is an effective method and means for qualitative and quantitative research on cell transformation and carcinogenic mechanism of HBx. In the field of biological research, mammalian cells are often treated with protein import by transfection of eukaryotic gene expression vectors, microinjection or fusion of peptide analogs. Although these methods have achieved success to a certain extent, they have shortcomings such as difficult quantitative control, expensive equipment, and time-consuming and labor-intensive experiments. At present, the research on the transformati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/51C12N15/62C12N15/63C12Q1/68
Inventor 张晓东叶丽虹东楠王洪辉
Owner NANKAI UNIV
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