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Hepatitis B virus multi-epitope fusion protein and preparation method and application thereof

A technology of hepatitis B virus and fusion protein, which is applied in the field of fusion protein, can solve the problems of poor immunogenicity, difficulty in adapting to long-term treatment of patients with chronic HBV infection, multiple immunizations, and single epitope, so as to enhance the immune effect and solve multiple problems. Second immune tolerance, strong immunogenic effect

Active Publication Date: 2011-09-28
ARMY MEDICAL UNIV
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Problems solved by technology

However, these vaccines are not perfect. For example, most peptide vaccines are composed of a single epitope or multiple epitopes restricted by a single antigen and a single MHC in series, which have the disadvantages of single epitope, host restriction, and poor immunogenicity; protein vaccines can be multiple However, due to the poor immunogenicity of a single protein epitope, it cannot effectively stimulate specific CTL responses in vivo; DNA vaccines can effectively stimulate specific CTL responses in vivo, but due to the non-specific transfection of plasmid DNA, it is not effective for Transfection of somatic cells can lead to T cell tolerance to the antigen, and there is a problem of poor multiple immunization effects, making it difficult to adapt to the requirements of long-term treatment and multiple immunizations for patients with chronic HBV infection

Method used

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  • Hepatitis B virus multi-epitope fusion protein and preparation method and application thereof
  • Hepatitis B virus multi-epitope fusion protein and preparation method and application thereof
  • Hepatitis B virus multi-epitope fusion protein and preparation method and application thereof

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Embodiment 1

[0028] Embodiment 1, the design and preparation of HBV multi-epitope fusion protein

[0029] 1. Design of HBV multi-epitope fusion protein

[0030] First, use the HBV antigen supertype epitope database established by the Institute of Immunology of the Chinese People’s Liberation Army to screen the supertype epitopes that meet the following criteria: ①The antigen is HBsAg, HBcAg or pol; ②MHC is restricted to A2, A3 supertype; ③ The length of the epitope was 9 peptides or 10 peptides; ④ The ability of inducing specific CTL to secrete cytokine IFN-γ in vitro was higher than that of the control epitope HBcAg18-27. As a result, the following four supertype epitopes were screened out, namely:

[0031] HBsAg313-321 (amino acid sequence is IPIPSSWAF), MHC restricted to A3 supertype (including A0301, A1101, A3301);

[0032] HBsAg335-343 (amino acid sequence is WLSLLVPFV), MHC restricted to A2 supertype (including A0201, A0202, A0203, A0206 and A6802);

[0033] Pol150-159 (amino ...

Embodiment 2

[0044] Example 2, the immune protection of HBV multi-epitope fusion protein

[0045] F1 HBV generation after hybridization of immunized A2, A3 transgenic mice (purchased from Jackson Laboratory) and HBV transgenic mice (purchased from Guangzhou Air Force Hospital) + A2 + , HBV + A3 + 60 transgenic C57 mice were divided into 6 groups on average: HBV multi-epitope fusion protein immunization group, HBc particle-HBV multi-epitope fusion peptide mixed immunization control group, HBc particle immunization control group, HBV multi-epitope fusion peptide immunization control group group, non-treatment control group, and placebo control group. The immunization method is as follows: HBV multi-epitope fusion protein immunization group: HBV multi-epitope fusion protein 100 μg / time / animal, 1 time / week; HBc particles-HBV multi-epitope fusion peptide mixed immunization control group: HBc particles and HBV multi-epitope fusion peptide Mixture of fusion peptides 100 μg / time / mouse, 1 tim...

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Abstract

The invention relates to a hepatitis B virus multi-epitope fusion protein and a preparation method and application thereof. The fusion protein is obtained by inserting hepatitis B virus multi-epitope fusion peptide (with the sequence shown as SEQIDNo.1) formed by serially connecting HBsAg313-321, HBsAg335-343, Pol150-159, Pol455-463 and Padre epitopes through connecting peptide between amino acidat the 78th position and amino acid at the 79th position of a hepatitis B virus core protein; and the preparation method comprises the following steps of: constructing a hepatitis B virus multi-epitope fusion protein expression plasmid pET28-HBc-HP; performing isopropyl thiogalactoside (IPTG) inducing expression by using an Escherichia coli expression system; and purifying by using affinity chromatography. The fusion protein carries a plurality of supertype epitopes of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and polymerase, is viral particles, has the advantages of strong immunogenicity, wide applicable range and the like, and can be used for preparing therapeutic hepatitis B vaccines.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a fusion protein, in particular to a hepatitis B virus (Hepatitis B Virus, HBV) multi-epitope fusion protein, a preparation method of the fusion protein and its application in the field of pharmacy . Background technique [0002] HBV is one of the hepatitis viruses that seriously endanger human health and is an important pathogenic factor of liver diseases. About 30 million people in our country are patients with chronic HBV infection, and nearly 500,000 patients die from it every year. Patients with chronic HBV infection mainly present with liver damage and metabolic disorders associated with steatosis and fibrosis, and patients with long-term infection may develop liver cirrhosis and hepatocellular carcinoma. At present, the treatment of chronic HBV infection is mainly based on antiviral drugs and cytokines, supplemented by traditional Chinese medicine. Although it has achieved certai...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/51C12N15/63A61K39/29A61P1/16A61P31/20
Inventor 刘东王莉李霞吴玉章
Owner ARMY MEDICAL UNIV
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