Novel anti-HBV endogenous protein HNF1alpha and application thereof

A protein and protein technology, applied in the direction of application, peptide/protein composition, peptide source, etc., can solve the problem of not finding hepatitis B

Active Publication Date: 2013-11-27
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, humans have not found an effective drug that can comp

Method used

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  • Novel anti-HBV endogenous protein HNF1alpha and application thereof
  • Novel anti-HBV endogenous protein HNF1alpha and application thereof
  • Novel anti-HBV endogenous protein HNF1alpha and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Overexpression of HNF1α to detect the effect of HNF1α on HBV replication

[0037] 1. Construction of HNF1α eukaryotic expression vector

[0038] (1) Total RNA of Huh7 was extracted by Trizol method and reverse transcribed into cDNA.

[0039] (2) Using cDNA as a template, the HNF1α gene was amplified with primers HNF1αP1 and HNF1αP2 (see Table 1).

[0040] The amino acid sequence of HNF1α is shown in SEQ ID No.1, and the amplified HNF1α gene sequence is shown in SEQ ID No.2.

[0041] PCR program: 95°C 5min; (95°C 30s, 60°C 30s, 72°C 2min) 30cycles; 72°C 7min.

[0042] (3) Perform 1% agarose gel electrophoresis on the PCR product, and after the correct size is identified, cut the gel and recover.

[0043] (4) Ligate the recovered product with the pMD18-T vector to obtain a recombinant plasmid, and transform the recombinant plasmid into Escherichia coli DH5α.

[0044] (5) Screen the positive clones with the ampicillin plate, pick a single clone, extract the ...

Embodiment 2

[0072] Example 2. Inhibition of the expression of HNF1α and detection of its effect on HBV replication

[0073] 1. Construction of pSilencer-HNF1α

[0074] (1) Synthesize the primers siHNF1αP1 and siHNF1αP2 (see Table 1) of the HNF1α inhibitory vector, and anneal the primers.

[0075] (2) Digest the pSilencer2.1-U6hygro (hereinafter referred to as pSilencer) vector with BamHI and HindIII to obtain a large fragment of the vector, and connect the annealed primers and the large fragment of the vector to obtain a recombinant plasmid.

[0076] (3) The recombinant plasmid was transformed into Escherichia coli DH5α, positive clones were screened with ampicillin, the plasmid was extracted after 12 hours of liquid culture, and the sequence was determined after identification by BamHI and HindIII digestion, and the sequenced vector was named pSilencer-HNF1α.

[0077] The sequencing results of the inhibitory sequence in pSilencer-HNF1α are as follows: image 3 As shown in A, pSilencer-...

Embodiment 3

[0098] Example 3, Interaction Analysis of HNF1α and Enhancer I (EnhancerI, EnhI) of HBV

[0099] 1. Construction of pGL3-EnhI

[0100] (1) The six regulatory elements S1, S2, CORE, X promoter, enhancer I and enhancer II of HBV were analyzed using MatInspector. The analysis results showed that enhancer I (including the X promoter region of HBV) contained HNF1α binding site.

[0101] (2) Using pHBV1.2 as a template, using EnhancerI P1 and EnhancerI P2 as primers (see Table 1 for the sequence) to amplify HBV enhancer I, perform 1% agarose electrophoresis on the amplified product, and recover a band of about 411bp from the gel . The sequence of the amplified enhancer I is shown in SEQ ID No.3.

[0102] (3) The PCR amplification product was connected to the pMD18-T vector to obtain the recombinant plasmid pMD18-T-EnhI. The recombinant plasmid was transformed into Escherichia coli DH5α, positive clones were screened with ampicillin plate, the plasmid was extracted after 12 hours...

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Abstract

The invention discloses a novel anti-HBV endogenous protein HNF1alpha and an application thereof. The amino acid sequence of HNF1alpha is represented by SEQ ID No.1. HNF1alpha can inhibit liver cells from secreting and expressing s antigen (HBsAg) and e antigen (HBeAg), and can reduce HBV DNA load. The HNF1alpha can perform the effect of inhibiting HBV expression through combining with a specific locus of an HBV enhancer I. HNF1alpha can be developed into an effective anti-HBV medicine. Also, a basis for researching and developing anti-HBV medicines is established.

Description

technical field [0001] The invention relates to a novel anti-HBV endogenous protein HNF1α and its application. Background technique [0002] Hepatitis B virus (HBV) infection is an important problem affecting health worldwide, and clinical HBV infection can cause acute or chronic hepatitis B. Worldwide, the number of chronically infected people exceeds 350 million, accounting for 5% of the global population. People with chronically infected people have a high risk of liver cirrhosis and hepatocellular carcinoma. Effective treatment of the hepatitis B virus can stop the further development of liver inflammation. At present, anti-hepatitis drugs are divided into several categories: nucleoside analogs, interferons and immunomodulators (Zhou Xia, Lu Qiujun. Research progress on the mechanism of action and targets of anti-hepatitis B virus drugs. Foreign Medical Pharmacy Volume , 2005, 32(3):184-188.). So far, humans have not found an effective drug that can completely elimina...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/85C12N5/10A61K38/17A61K48/00A61P31/20A61P1/16A61P35/00C12Q1/68
Inventor 周育森李军锋代晓朋张伟孙世惠寇志华赵光宇于虹郭彦
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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