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Artificial transcription factor of hepatitis B virus (HBx) genes and application of artificial transcription factor

A hepatitis B virus, gene transcription technology, applied in the field of molecular biology, can solve the problems of time-consuming and laborious, and achieve the effect of high inhibitory activity

Active Publication Date: 2012-12-12
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the regulation of gene expression is an extremely complex and delicate process. The specificity of the binding of artificial transcription factors to DNA sequences, the interaction between subunits and domains of transcription factors will affect the effect of transcription. Therefore, artificial transcription There are many uncontrollable factors affecting the regulation of genes. Finding artificial transcription factors with strong specificity and good regulatory effect is not only a very time-consuming and laborious task, but also a challenging task.

Method used

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  • Artificial transcription factor of hepatitis B virus (HBx) genes and application of artificial transcription factor
  • Artificial transcription factor of hepatitis B virus (HBx) genes and application of artificial transcription factor
  • Artificial transcription factor of hepatitis B virus (HBx) genes and application of artificial transcription factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1, construction and screening of phage display library

[0044] 1. Construction of library

[0045] According to the literature [Mark I, Yen C. Rapid, high-throughput engineering of sequence-specific zinc finger DNA-binding proteins. Methods Enzymol, 2001, 340: 593-609], 18 common sequences were designed, including randomized sites 108 sequences. Phosphate addition, annealing, and ligation of the zinc finger fragments of the zinc finger library Lib12 and Lib23 are routine operations, carried out in accordance with the Molecular Cloning Experiment Guide (Second Edition).

[0046] design primers

[0047] Lib-top: 5'-TCGCGGCCCAGCCGGCCATGGCGGAAGAGA-3'(30nt)

[0048] Lib-end: 5'-TGTAGCGGCCGCCTTCTGTCTTAAATG-3'(27nt),

[0049] The ligation products of the zinc finger fragments of Lib12 and Lib23 were amplified by PCR, and restriction sites of SfiI and NotI were introduced at both ends of the fragments.

[0050] The conditions of PCR are: using conventional Taq enz...

Embodiment 2

[0108] Example 2, Affinity and specificity identification of phage display zinc finger library

[0109] 1. The affinity of zinc fingers screened by ELISA detection

[0110] The test procedure is the same as part vi of Example 1. Such as image 3 As shown, three zinc finger clones U5 and U37 have good affinity for their screening target sequence GCCAAGTGT (U sequence), while three zinc finger clones D13 and D46 have good affinity for their screening target sequence GCTGACGCA (D sequence). Affinity.

[0111] 2. Detection of specificity of zinc finger clones by single base mutation test of target sequence

[0112] Synthesize 9 pairs of biotin-labeled new target sequences, each pair of sequences contains a base conversion relative to the original 9bp target sequence ( or ) mutation. The nine pairs of mutant sequences and the original target sequence were respectively wrapped in a streptavidin plate, and the difference in the affinity of each sequence to the rescue supernat...

Embodiment 3

[0160] Example 3, construction of six zinc finger proteins

[0161] To ensure specificity, artificial transcription factors should only act on their own target sequences when regulating gene expression. Since the human genome contains about 3×10 9 bp, so usually a sequence of 17 bp (417 possible combinations) or longer can guarantee that it has only a single copy in the human genome. As mentioned above, each zinc finger unit usually recognizes a 3bp sequence, so the DNA binding domain of an artificial transcription factor should consist of six or more zinc finger units. At present, most of the structures obtained through library screening are three-zinc-finger structures, so it is often necessary to connect multiple zinc-finger units in series through linkers.

[0162] The three zinc finger protein D13 for the D sequence is fused with the three zinc finger protein U5 for the U sequence through the linker peptide shown in SEQ ID NO20 to construct the six zinc finger protein U...

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Abstract

The invention discloses an artificial transcription factor capable of specifically combining a C2H2 trizinc finger protein and a six-zinc finger protein in a hepatitis B virus (HBx) gene transcription regulation domain and fusing an effect function domain capable of inhibiting gene transcription. The invention further discloses an application of the artificial transcription factor to the preparation of drugs for treating HBx infectious diseases. The transcription factor has an obvious inhibiting effect on the transcription of HBx genes and the growth of Hep3B cells integrated with target genes HBx.

Description

technical field [0001] The invention relates to a zinc finger protein and its application in transcriptional regulation target gene, which belongs to the field of molecular biology. Background technique [0002] Primary hepatocellular carcinoma (HCC) is one of the most common cancers in the world, and chronic hepatitis B virus (HBV) infection is one of the most important risk factors for HCC. Studies have shown that HCC in HBV carriers The incidence rate is 5-15 times higher than that of non-carriers. About 85-90% of HBV-positive HCC cases have HBV integrated fragments found in liver cells, and X gene is the most frequently integrated HBV fragment in HCC patients. Gene X (hereinafter referred to as HBx) is an ORF of HBV, and its protein product is called HbxAg, which usually consists of 154 amino acids. At present, it is found that HBx will interact with some factors in cells after HBV infects the host, and may play a regulatory role in the process of HBV-induced HCC (prim...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K19/00C12N9/22C12N9/10C12N9/90A61K38/16A61K38/46A61K38/45A61K38/52A61P31/20
Inventor 陈薇赵兴卉黄培堂朱旭东周晓巍徐俊杰付玲
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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