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Fusion protein of divisive ranilla luciferase and HBC (hepatitis B core) and application of fusion protein

A technology of Renilla luciferase and fusion protein, which is applied in the field of fusion protein, Renilla luciferase and HBC fusion protein, can solve the problems of lack of drug screening cell model, difficult analysis of conditions and mechanisms, and achieves good application value. , the effect of preventing copying

Inactive Publication Date: 2016-12-07
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although prokaryotically expressed HBC dimers can be detected by HPLC, however, HBV dimers expressed in HCC cell lines are in a transitional state, so far there is no suitable detection method
This has resulted in two situations. First, in terms of basic research, it is difficult to analyze the conditions and mechanism of HBC dimer formation. Second, in terms of drug development, there is a lack of drug screening cell models targeting HBC dimer formation.

Method used

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  • Fusion protein of divisive ranilla luciferase and HBC (hepatitis B core) and application of fusion protein
  • Fusion protein of divisive ranilla luciferase and HBC (hepatitis B core) and application of fusion protein
  • Fusion protein of divisive ranilla luciferase and HBC (hepatitis B core) and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of plasmid RlucN-HBC

[0035] RlucN-HBC expresses a fusion protein of hepatitis B virus core protein and Renilla luciferase N-terminal 229 amino acids. The two are connected by a 94 amino acid G4S linker (named 94G4S linker). The amino acid sequence of the 94G4S linker It is: SSGS(GGGGS)×18.

[0036] The specific construction process of plasmid RlucN-HBC is as follows:

[0037] 1. Construction of transition vector plasmid GG1

[0038] Plasmid GG1 is a transitional vector convenient for subsequent cloning operations, and its structure is as follows figure 1 As shown, GG1 contains a 94G4S linker sequence, and upstream of the 94G4S sequence is a primer sequence that facilitates PCR amplification of 94G4S. The construction process is:

[0039] Using the plasmid GFP-92G4S-HBC (this plasmid and its construction method have been disclosed in the patent application CN 201510076239.1, which is the same as the GFP-92G4S-HBc in the patent application) as a template...

Embodiment 2

[0053] Example 2: Construction of plasmid RlucC-HBC

[0054] The plasmid RlucC-HBC expresses a fusion protein of the core protein of hepatitis B virus and the C-terminal 83 amino acids (amino acids 229-311) of Renilla luciferase. The two are connected by a 94G4S linker. The specific construction process is as follows:

[0055] Carry out PCR amplification, reaction system: template PLR-TK 10ng, primers FrlucCGG (10μM) and RrlucCGG (10μM) each 1μl, 2XPrimeSTAR HS Mix 25μl, sterilized ultrapure water to make up the volume to 50μl. Reaction conditions: 95°C pre-denaturation for 3 minutes; 94°C for 15s, 58°C for 15s, 72°C for 40s, 35 cycles. The amplified fragment was recovered with a gel recovery kit, and the recovered fragment was named frag6.

[0056] The three fragments of frag6 and frag4 and frag5 obtained in Example 1 are subjected to Golden gate ligation reaction. The reaction system:

[0057] BsmB I enzyme

0.75μl

Tango buffer

1μl

DTT

1μl

T7 ligase

0.25μl

ATP

1μl

...

Embodiment 3

[0060] Example 3: Construction of plasmids RlucN-dHBC and RlucC-dHBC

[0061] RlucN-dHBC and RlucC-dHBC are based on RlucN-HBC and RlucC-HBC respectively. The HBC sequence is removed and only RlucN-49G4S and RlucC-49G4S are retained. These two plasmids will be used as negative controls. The construction adopts a single segment Golden gate connection strategy, and the specific process is as follows:

[0062] Fragment amplification reaction system: template RlucN-HBC 10ng, primers Fsv4GG2 (10μM) and Rg4sGG2 (10μM) each 1μl, 2XPrimeSTAR HS Mix 25μl, sterilized ultrapure water to make up the volume to 50μl. Reaction conditions: 95°C pre-denaturation for 3 minutes; 94°C for 15s, 58°C for 15s, 72°C for 40s, 35 cycles. The amplified fragment was recovered with a gel recovery kit, and the recovered fragment was named frag7.

[0063] The obtained fragment frag7 does its own Golden gate connection reaction, and the reaction system:

[0064] BsmB I enzyme

0.75μl

Tango buffer

1μl

DTT ...

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Abstract

The invention discloses fusion protein of divisive ranilla luciferase and HBC (hepatitis B core). The fusion protein is obtained by joint connection of 1 to 229 amino acids of the N-terminal of the ranilla luciferase and core protein of hepatitis B virus (HBV), or obtained by joint connection of 229 to 311 amino acids of the N-terminal of the ranilla luciferase and the core protein of the HBV, the sequence of the amino acids is as shown in SEQ (sequence) ID (identity) No: 1 and No: 3 respectively, and nucleotide sequence encoding the two fusion protein is as shown in SEQ ID No: 2 and No: 4 respectively. The invention further discloses a carrier containing the nucleotide sequence as is shown in the SEQ ID No: 2 and No: 4. and discloses an application about whether the fusion protein as is shown in the SEQ ID No: 1 or the No: 3 is formed or not in indication of core protein dimers of the HBV. An importation foundation is laid for establishing cell models for drug screening in formation of the anti-HBC dimers.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and relates to a fusion protein, in particular to a fusion protein of split Renilla luciferase and HBC and applications thereof. Background technique [0002] Hepatitis B virus (HBV) infection is a serious public health problem. It covers a wide range. There are nearly 400 million chronically infected patients worldwide. Chronic HBV infection can lead to serious consequences such as liver cirrhosis and hepatocellular carcinoma, and deaths every year. Nearly a million. HBV is a spherical particle with a diameter of 42nm, with a double-shell structure. The outer shell is mainly composed of three surface proteins (HBs). The inner nucleocapsid is composed of core protein (HBC). 120 HBC dimers, each dimer is connected by HBC monomers through disulfide bonds. HBC is composed of 183 amino acids. In the three-dimensional structure, 4 alpha helices are formed (aa13-30, aa50-60-78, aa82-93-110, aa112-123-128)...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62G01N33/68
CPCC07K14/005C07K2319/61C12N2730/10122G01N33/6845
Inventor 胡接力黄爱龙甘春杨
Owner CHONGQING MEDICAL UNIVERSITY
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