Fusion protein related to HBV, preparing method thereof and application thereof

A technology of fusion protein and composition, applied in the field of biomedicine

Active Publication Date: 2016-03-02
SHANGHAI SIXTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently in the art, there is still a lack of effective techniques for inducing the body to eliminate HBV in the body

Method used

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  • Fusion protein related to HBV, preparing method thereof and application thereof
  • Fusion protein related to HBV, preparing method thereof and application thereof
  • Fusion protein related to HBV, preparing method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0199] Construction and expression of embodiment 1CTP-Ub-HbcAg (CUH) and Ub-HbcAg-CTP (UHC) prokaryotic expression vector

[0200] 1. Gene synthesis

[0201] 1.1 PCR amplification was carried out after whole gene synthesis of CTP-Ub-HBcAg, Ub-HBcAg-CTP, Ub-HBcAg and CTP-HbcAg sequences.

[0202] According to the sequences of CTP-Ub-HBcAg, Ub-HBcAg-CTP, Ub-HBcAg and CTP-HBcAg, primers for whole gene synthesis were designed, and the synthesized fragments were named CUH, UHC, UH and CH, respectively. The primer synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd., and the primer sequences are as follows. Considering the construction of recombinant plasmids when designing primers, BamHI and XhoI restriction sites were introduced at both ends of the primers.

[0203] The attached primer sequences are shown in the table below:

[0204]

[0205]

[0206] 1.2 Gene synthesis

[0207] PCR amplification for gene synthesis

[0208] Synthesis of CTP-Ub-HBcAg (CUH): prim...

Embodiment 2

[0246] Example 2 In Vitro Activity Detection

[0247] method:

[0248] 1. Isolate and culture myeloid-derived DCs from inbred Balb / c mice in vitro, add rGM-CSF and rIL-4 and culture them for 5 days, then add LPS to induce DC maturation.

[0249] Different groups of fusion proteins were added to the cell culture medium, and the distribution and localization of immunofluorescence in the cells were observed under a laser confocal microscope, and the fluorescence intensity was quantitatively analyzed.

[0250] The transduction efficiency of Ub-HBcAg-CTP was detected by Western blot to observe the difference of HBcAg expression in different groups of cells. Antibodies used in Western blot were anti-HBcAg antibody (primary antibody) and goat anti-mouse secondary antibody (purchased from Wuhan Boster Company).

[0251] The expression of DC surface molecules was measured by flow cytometry, and fluorescently labeled with FITC-labeled mouse monoclonal antibodies CD11c, CD80, CD83, CD8...

Embodiment 3

[0263] Example 3 In vivo activity detection

[0264] method:

[0265] 1. In vivo activity experiment of BALB / c mice

[0266] BALB / c mice were randomly divided into experimental group Ub-HBcAg-CTP (20 μg), control group CTP-HBcAg (20 μg), HBcAg-Ub (20 μg), HBcAg (20 μg) and blank group (normal saline), after muscle immunization Mice, once a week, 3 times in total, 7 days after the last immunization, the following tests were performed:

[0267] Detection of Cytokines in T Lymphocytes by Flow Cytometry (FCM)

[0268] Enzyme-linked immunosorbent assay (ELISA) was used to detect cytokines secreted by T lymphocytes;

[0269] Cell counting kit (CCK-8) was used to detect the proliferative activity of T lymphocytes.

[0270] 2. In vivo activity experiment of HBV transgenic mice

[0271] HBV transgenic mice (purchased from the Liver Disease Center of the 458th Hospital of the Chinese People's Liberation Army, the HBV gene is integrated in the genome of the mouse, and the characteri...

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Abstract

The invention provides a fusion protein related to HBV, a preparing method thereof and an application thereof. Particularly, a prokaryotic expression carrier related to HbcAg is built and expressed through the genetic engineering technology, and the obtained fusion protein can remarkably induce an HBV-specificity CTL reaction in vitro and vivo.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to a fusion protein related to HBV, its preparation method and application. Background technique [0002] Human hepatitis B virus (human hepatitis B virus, HBV) infection is a global public health problem. According to the statistics of the World Health Organization (World Health Organization, WHO), more than 2 billion people have been infected with HBV worldwide, of which 350 million are positive Suffering from chronic HBV infection (WHO, 2000). my country is a high-incidence area of ​​hepatitis B. The national viral hepatitis serum epidemiological survey data in 2006 showed that the positive rate of hepatitis B surface antigen (HBsAg) in the whole population was 7.18 %. Based on this, it is estimated that about 93 million people in my country are HBV carriers, of which 30 million are patients with chronic hepatitis B. Studies have also shown that chronic HBV infecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/29A61K47/48A61K48/00A61P31/20A61K47/42
Inventor 汤正好臧国庆余永胜陈小华胡建军李丹潘庆春宋玲玲卓萌戴胜兰
Owner SHANGHAI SIXTH PEOPLES HOSPITAL
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