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Preparation method of HBx protein derived from Hepatitis B Virus HBV

A protein and mercaptoethanol technology, applied in the biological field, can solve the problems of inability to obtain high-purity, stable and uniform protein samples, low solubility, and failure to remove nucleic acids.

Pending Publication Date: 2020-07-21
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The protein can spontaneously polymerize and has strong nucleic acid binding ability, which makes it difficult to obtain uniform and soluble HBx protein samples in various expression systems
Highly pure, stable and uniform protein samples cannot be obtained by using the reported recombinant expression or denatured methods of fusion proteins such as Trx and MBP
The shortcoming of the HBx albumen that obtains before is: the one, it is still low solubility state, and the highest concentration can only reach about 1mg / ml in the bibliographical report; 280nm absorbance ratio) close to or greater than 1, much higher than the normal ratio of pure protein (about 0.57)
For the full-length HBx protein containing all amino acids, there is no publicly reported method to obtain a stable, uniform, highly folded, and soluble protein sample

Method used

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  • Preparation method of HBx protein derived from Hepatitis B Virus HBV
  • Preparation method of HBx protein derived from Hepatitis B Virus HBV
  • Preparation method of HBx protein derived from Hepatitis B Virus HBV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Solubilization and purification of HBx inclusion bodies

[0045] In the present invention, the full-length sequence of HBx is cloned on the pet15b expression vector, the endonuclease NcoI and XhoI restriction sites are selected, 6×His at the N-terminal of the vector is retained, and 6 His can also be added at the C-terminal of the vector. Recombinant proteins with only N-terminal 6×His and both N and C-terminal 6×His can be used for subsequent solubilization and purification.

[0046] In the present invention, the prokaryotic cell expression system of Escherichia coli is preferably used (but other expression systems are not excluded, such as expression in other bacteria or other eukaryotic cells); the above-mentioned proteins are expressed in the form of inclusion body proteins , the His tag is preferred (but other tags, such as MBP, GST or SUMO tags are not excluded).

[0047] Specifically, the present invention transforms the plasmid containing the HBx sequ...

Embodiment 2

[0056] Example 2 Preparation of HBx monoclonal antibody

[0057] Using the HBx sample obtained by the above purification method as an antigen, the HBx monoclonal antibody can be obtained by using the usual monoclonal antibody preparation method. E.g:

[0058] The HBx sample obtained by the above purification method is used as an antigen and injected into mice to prepare mouse-derived HBx monoclonal antibody, and obtain a hybridoma cell line capable of stably producing mouse-derived HBx monoclonal antibody.

[0059] Using the HBx obtained by the above purification method as an antigen, injecting other animals than mice can prepare monoclonal antibodies from other animals.

[0060] Humanized HBx monoclonal antibodies can also be obtained by using the above-mentioned animal-derived (such as mouse-derived) monoclonal antibodies using common antibody humanization methods.

[0061] The sequence used in the present invention is as follows:

[0062] SEQ.ID.NO1:

[0063] MAARVCCQLD...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a dissolving and purifying method of an HBx protein inclusion body derived from Hepatitis B Virus HBV. The preparation method comprises the following steps: expressing HBx by using a recombinant expression system; collecting cells containing the HBx protein which has been subjected to recombinant expression, breaking the cells by using a lysis buffer solution under a cooling condition, and collecting a part containing an HBx protein inclusion body; adding a dissolution buffer solution into the obtained part containing the HBx protein inclusion body for resuspension and denaturation; performing renaturationon on the product, removing nucleic acid, performing purification, and obtaining HBx protein; whereinthe steps are completed under the environmental condition of less than or equal to 10 DEG C. By using the method provided by the invention, a stable and uniform monomer HBx sample can be obtained, andthe concentration can reach 15mg / ml. The protein sample can be used as a scientific research reagent, and a monoclonal antibody of HBx can be prepared by using the protein sample.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for solubilizing and purifying inclusion bodies of HBx protein derived from hepatitis B virus HBV. Background technique [0002] The HBx protein in hepatitis B virus (HBV virus) is a key protein that replicates in the human body after virus infection, causes hepatitis and liver cancer, and is an important target protein for related diseases caused by HBV infection. [0003] The HBx protein is about 16.6kDa in size and consists of 154 amino acid residues. At present, there is still a lack of basic understanding of the protein's structure and function. The protein can spontaneously polymerize and has a strong nucleic acid binding ability, which makes it difficult to obtain uniform and soluble HBx protein samples in various expression systems. High-purity, stable and uniform protein samples cannot be obtained by using the recombinant expression or denaturation meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/02C12N15/70C07K1/22C07K1/113
CPCC07K14/005C12N15/70C12N2730/10151
Inventor 胡小健李继喜王森苏晓琴李龙
Owner FUDAN UNIV
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