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Heat shock protein 65- multiple epitope hepatitis B virus core antigen recombinant protein (HSP65-HBcAg)

A technology of hepatitis B virus and heat shock protein, applied in the field of genetic engineering recombinant fusion protein, can solve the problem of ineffective activation of CTL

Inactive Publication Date: 2010-10-13
BEIJING HYDVAX BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after the exogenous protein antigen is immunized to the human body, it is usually taken up and processed by antigen-presenting cells, enters the MHC class II presentation pathway, and stimulates the humoral immune response (Heikema A, Agsteribbe E, Wilschut J, Huckriede A. Generation of heat shock protein-based vaccines by intracellular loading of gp96 with antigenic peptides. Immunol Lett 1997 Jun 1; 57(1-3): 69-74), but cannot effectively activate CTL

Method used

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  • Heat shock protein 65- multiple epitope hepatitis B virus core antigen recombinant protein (HSP65-HBcAg)
  • Heat shock protein 65- multiple epitope hepatitis B virus core antigen recombinant protein (HSP65-HBcAg)
  • Heat shock protein 65- multiple epitope hepatitis B virus core antigen recombinant protein (HSP65-HBcAg)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 obtains the coding gene of 65KD heat shock protein (HSP65)

[0064]1. BCG: from Changchun Institute of Biological Products. The Sutong potato medium is used to cultivate BCG at a temperature of 37-39°C, and the grown BCG presents dry wrinkled light yellow pellicle. The pellicles were collected from which BCG genomic DNA was extracted.

[0065] 2. Extraction of BCG genomic DNA: the method refers to the book Molecular Cloning (J.Sambrook, Isolation of high-molecular-weight DNA from mammalian cells, 9.16-9.22, ColdSpring Harbor Laboratory Press, Molecular cloning, 1989).

[0066] 3. Isolation of HSP65 structural gene: The HSP65 structural gene was isolated from BCG by PCR method. The 5' end primer and the 3' end primer used are respectively shown in SEQ ID NO: 6 and SEQ ID NO: 7.

[0067] PCR operating procedure

[0068] Add the following reagents to a 500 μl microcentrifuge tube:

[0069] Template cDNA (mmol / L) 5μl

[0070] 10×PCR buffer (containing mag...

Embodiment 2

[0079] Example 2 Obtaining of Multi-epitope Hepatitis B Virus Core Antigen Gene

[0080] The multi-epitope HBV core antigen gene was synthesized by two rounds of PCR.

[0081] 1. The first round of PCR: the used primer 1 and primer 1' are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The product obtained from the first round of PCR is shown in SEQ ID NO:11.

[0082] 2. The second round of PCR: the primers 2 and 2' used are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively. The product obtained by the second round of PCR is shown as SEQ ID NO: 14, which represents the multi-epitope gene of the hepatitis B virus core antigen.

[0083] The PCR reaction conditions were: 94°C, 30"; 55°C, 1'; 72°C, 4', after 30 cycles, 72°C was extended for 10 minutes.

[0084] The PCR map is attached to the instructions figure 2 shown.

Embodiment 3

[0085] Construction of embodiment 3HSP65-HBcAg fusion gene

[0086] 1. The heat shock protein 65 (HSP65) coding gene was digested with NcoI and EcoRI, and the multi-epitope hepatitis B virus core antigen gene was digested with EcoRI and HindIII at 37° C. for 2 hours. The digested products were separated by agarose gel electrophoresis. Electrophoresis conditions are: 1% agarose gel, 1×TAE buffer, 150-200mA, electrophoresis for 0.5-1 hour.

[0087] 20×TAE buffer: 0.8mol / L Tris base

[0088] 0.4mol / L NaOAc

[0089] 0.04mol / L Na 2 EDTA

[0090] Adjust the pH to 8.3 with glacial acetic acid.

[0091] 2. Observe under ultraviolet light and cut out the agarose gel containing the DNA band, freeze at -70°C for 15 minutes, and then melt the gel in a water bath at 65°C; add an equal volume of TE-saturated phenol, vigorously Shake for 30 seconds, then freeze at -70°C for 15 minutes; after thawing at room temperature, centrifuge at 12,000 rpm for 5 minu...

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Abstract

The invention relates to a recombinant fusion protein HSP65-HbcAg, which is fused from heat shock protein 65 and multi-epitope hepatitis B virus HbcAg, in the recombinant fusion protein, the multi-epitope hepatitis B virus HbcAg comprises 5 different antigen epitopes through interconnection by mathematical combination modes. the recombinant fusion protein can make hepatitis B virus HbcAg specificcell toxic T lymphocyte CTL eradicate the cells infected by hepatitis B virus, thus achieving the goal of treating and / or preventing hepatitis B. The invention also provides the nucleic acid sequenceencoding the recombination fusion protein.

Description

technical field [0001] The invention relates to a gene engineering recombinant fusion protein, in particular to a recombinant fusion protein for preventing and / or treating human hepatitis B virus hepatitis. The present invention also relates to the gene encoding the recombinant fusion protein. Background technique [0002] Hepatitis B virus is a worldwide disease caused by the hepatitis B virus, and the incidence rate is higher in developing countries. According to statistics, there are more than 280 million asymptomatic carriers (HBsAg carriers) in the world, and my country accounts for more than 100 million. Among asymptomatic infected persons, about 1 / 3 of them have clinical manifestations of liver damage. It is characterized by slow onset, and subclinical and chronic types are more common. Anicteric HBsAg persistently positive patients tend to become chronic. The disease is mainly transmitted through blood and close contact in daily life, and another mode of transmis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/51C07K14/00C07K14/435C07K14/02A61K38/16A61P31/20
Inventor 王丽颖万敏邵明玉于永利
Owner BEIJING HYDVAX BIOTECH
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