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Method for evaluating X protein binding molecule of human hepatitis C virus

A hepatitis B virus, binding molecule technology, applied in the fields of biotechnology and genetic engineering, can solve the problems of difficult high-throughput detection research, time-consuming and labor-intensive, and high technical requirements for operators, achieving signal sensitivity, meeting screening and evaluation requirements Requirements, the effect of convenient detection

Active Publication Date: 2018-07-03
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the main method to study the function of HBx is to conduct relevant experiments by transfecting the overexpression plasmid of HBx protein, and due to the limitation of high-efficiency antibodies and detection reagents, usually add Tag tag or GFP protein to the N-terminal or C-terminal of X protein; HBx protein The detection of HBx protein is mainly by lysing the cells expressing HBx protein (expressed by the transfected plasmid carrying HBx gene), extracting the cell extract, and using the corresponding antibody to perform immunoblotting to detect the HBx protein itself or the binding protein of the HBx protein. The detection method is time-consuming and labor-intensive, and has high technical requirements for operators, so it is difficult to be used in high-throughput detection research

Method used

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  • Method for evaluating X protein binding molecule of human hepatitis C virus
  • Method for evaluating X protein binding molecule of human hepatitis C virus
  • Method for evaluating X protein binding molecule of human hepatitis C virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Construction of recombinant fusion protein expression vector

[0043] Construct pSecTag2A-HBx-Nluc expression vector:

[0044] Use the primer pair HBx_F1 / HBx_R1 to amplify the HBx gene sequence, and recover the PCR reaction fragment; use the primer Nluc_F1 / Nluc_R1 to amplify the Nluc gene sequence, and recover the PCR reaction fragment; use the primer HBx_F1 / Nluc_R1 to perform Overlap PCR on the HBx and Nluc gene fragments, and get The fragment of the PCR product was cloned into the secretory eukaryotic expression vector pSecTag2A (purchased from Invitrogen, the vector has a nucleic acid sequence encoding the Igκ signal peptide), to obtain the expression vector pSecTag2A-HBx-Nluc of the fusion gene HBx-Nluc (such as figure 1 above);

[0045] Construct pSecTag2A-Nluc-HBx expression vector:

[0046] Use primers Nluc_F2 / Nluc_R2 to amplify the Nluc gene sequence, and recover the PCR reaction fragment; use the primer pair HBx_F2 / HBx_R2 to amplify the HBx gene...

Embodiment 2

[0047] Example 2: Expression and detection of fusion protein

[0048] The fusion protein expression plasmid was transiently transfected into Huh7 cells, and after a certain period of time, the expression of the fusion protein was detected using the specific antibody of HBx protein;

[0049] Western blot results show that the fusion protein can be recognized by the specific antibody of HBx protein, and its molecular weight is about 35kDa (such as figure 2 Shown in A), the results of immunofluorescence showed that the fusion protein was distributed in both the nucleus and the cytoplasm (such as figure 2 Shown in B), as time goes by, the activity of the luciferase detected in the supernatant gradually increases, and reaches a relatively high expression level in 48 hours (as figure 2 C shown).

Embodiment 3

[0050] Embodiment 3: RNA interference suppression experiment

[0051] Use shRNA targeting HBx gene to inhibit the expression of fusion protein in cells (such as image 3 shown), the positive control showed that the shRNA targeting the HBx gene could knock down the expression of the HBx gene (such as image 3 Shown in A); this shRNA can also knock down the expression of fusion protein (such as image 3 Shown in B); along with the reduction of fusion protein expression, the Nluc luciferase activity in the cell supernatant correspondingly significantly reduces (as image 3 C shown).

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Abstract

The invention belongs to the field of biotechnology and genetic engineering, and relates to a method for evaluating an X protein (HBx protein) binding molecule of human hepatitis C virus. The method provided by the invention comprises the following steps: expressing a secreting type fusion protein containing the HBx protein and luciferase NanoLuciferase (Nluc) in a cell; and quantifying the levelof the secreted HBx protein by detecting the activity of luciferase of the fusion protein. The method can be used for screening and identifying the HBx protein binding molecule, provides a support forresearching an HBx protein targeted drug, and has good application prospect.

Description

technical field [0001] The invention relates to the fields of biotechnology and genetic engineering, and discloses a method for evaluating human hepatitis B virus X protein (HBx protein) binding molecules. The invention also discloses a construction method of a secreted recombinant fusion protein containing HBx protein and luciferase NanoLuciferase (Nluc). The technical gist of the present invention is to express a secreted recombinant fusion protein containing HBx protein and Nluc luciferase in cells, and quantify the level of secreted HBx protein by detecting the luciferase activity of the secreted fusion protein. It can be used for rapid screening and identification of binding molecules of HBx protein, as well as for the development of targeted drugs for HBx protein. Background technique [0002] The prior art discloses that hepatitis B virus (hepatitis B virus, HBV) infection is one of the serious public health problems in the world. At present, there are 350 million HB...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C07K19/00
CPCC12N15/62C12N15/85C07K14/005C07K2319/61C12N2730/10122C12N2800/107
Inventor 谢幼华谷陈建刘晶陶帅潘少坤
Owner FUDAN UNIV
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